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Sample records for platelet collagen receptors

  1. Role of nitric oxide synthase in collagen-platelet interaction: involvement of platelet nonintegrin collagen receptor nitrotyrosylation.

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    Chiang, T M; Cole, F; Woo-Rasberry, V; Kang, E S

    2001-05-15

    Platelets possess the endothelial isoform of nitric oxide synthase (eNOS), which plays an important role in platelet function. Other laboratories, including ours, have reported that nitric oxide (NO) is released upon exposure of platelets to collagen, but the mechanism of the interaction is not yet established. The objective of this study is to examine the possible role of nonintegrin receptor nitrotyrosylation on collagen-induced platelet aggregation. Results of the study show that two platelet proteins with M(r) of 65- and 23-kDa proteins are nitrotyrosylated in a time-dependent manner after the addition of type I collagen. The M(r) 65-kDa protein is identified as the platelet receptor for type I collagen. The recombinant protein of the platelet receptor for type I collagen can also be nitrotyrosylated. The nitrotyrosylated recombinant protein loses its ability to inhibit type I collagen-induced platelet aggregation. In addition, the polyclonal anti-65 kDa immunoprecipitates eNOS suggesting that the platelet nonintegrin receptor for type I collagen is closely linked to the eNOS. These results demonstrate that the inhibitory effect of NO on collagen-induced platelet aggregation may be mediated by the nitrotyrosylation of the 65-kDa receptor.

  2. The Toxicity of a Chemically Synthesized Peptide Derived from Non-Integrin Platelet Collagen Receptors

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    Thomas M. Chiang

    2008-01-01

    Full Text Available A chemically synthesized peptide derived from platelet non-integrin collagen receptor has been shown to be an effective agent for inhibiting collagen-induced platelet aggregation and adhesion of washed radiolabeled platelets onto natural matrices and collagen coated microtiter plates. In order to be a therapeutic agent, we have used a cell culturing system and an animal model to test its cytotoxicities. In cell culture experiments, the peptide is not toxic to MEG-01, a megakaryoblastic cell line. Prior to performing experiments in rats, the existence of both platelet type I and type III collagen receptors and its functional roles in rat platelets had to be established. In this investigation, we report that rat platelets contain both receptors and the cHyB peptide inhibits both type I and type III collagen-induced rat platelet aggregation. In addition, analysis of the rat sera collected at various time intervals following an injection of cHyB into the rat-tail vein, did not show an increase in the activity of key enzymes which indicate tissue and/or organ damage. These results suggest that the cHyB peptide is safe and its development into a potential therapeutic agent for inhibiting thrombi formation is possible.

  3. The Toxicity of a Chemically Synthesized Peptide Derived from Non-Integrin Platelet Collagen Receptors

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    Thomas M. Chiang

    2008-08-01

    Full Text Available A chemically synthesized peptide derived from platelet non-integrin collagen receptor has been shown to be an effective agent for inhibiting collagen-induced platelet aggregation and adhesion of washed radiolabeled platelets onto natural matrices and collagen coated microtiter plates. In order to be a therapeutic agent, we have used a cell culturing system and an animal model to test its cytotoxicities. In cell culture experiments, the peptide is not toxic to MEG-01, a megakaryoblastic cell line. Prior to performing experiments in rats, the existence of both platelet type I and type III collagen receptors and its functional roles in rat platelets had to be established. In this investigation, we report that rat platelets contain both receptors and the cHyB peptide inhibits both type I and type III collagen-induced rat platelet aggregation. In addition, analysis of the rat sera collected at various time intervals following an injection of cHyB into the rat-tail vein, did not show an increase in the activity of key enzymes which indicate tissue and/or organ damage. These results suggest that the cHyB peptide is safe and its development into a potential therapeutic agent for inhibiting thrombi formation is possible.

  4. Structural Basis for Platelet Collagen Responses by the Immune-type Receptor Glycoprotein VI

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    Horii,K.; Kahn, M.; Herr, A.

    2006-01-01

    Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immunetype receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

  5. Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets.

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    Smith, Christopher W; Thomas, Steven G; Raslan, Zaher; Patel, Pushpa; Byrne, Maxwell; Lordkipanidzé, Marie; Bem, Danai; Meyaard, Linde; Senis, Yotis A; Watson, Steve P; Mazharian, Alexandra

    2017-05-01

    Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif-containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif-containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1-deficient mice. Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1-deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI-specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI-FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein-coupled receptors. © 2017 American Heart Association, Inc.

  6. Non-invasive molecular imaging of fibrosis using a collagen-targeted peptidomimetic of the platelet collagen receptor glycoprotein VI.

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    Julien Muzard

    Full Text Available BACKGROUND: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI, the main platelet receptor for collagens I and III. METHODOLOGY/PRINCIPAL FINDINGS: An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10(-7 M in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI. Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis. CONCLUSION/SIGNIFICANCE: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.

  7. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets.

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    Tsuji, M; Ezumi, Y; Arai, M; Takayama, H

    1997-09-19

    The mechanism by which occupancy of collagen receptors is coupled to platelet activation has been uncertain. Our group previously demonstrated that glycoprotein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet interaction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR gamma-chain and GPVI in human platelets. Our present study revealed the following. FcR gamma-chain was physically and stably associated with GPVI in human platelets; both FcR gamma-chain and GPVI were proportionally absent in GPVI-deficient platelets; GPVI cross-linking or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR gamma-chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR gamma-chain is a collagen receptor featuring the signaling through immune receptors.

  8. Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen*

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    Herr, Andrew B.; Farndale, Richard W.

    2009-01-01

    Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α2β1, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of ...

  9. Synthesis of huaicarbon A/B and their activating effects on platelet glycoprotein VI receptor to mediate collagen-induced platelet aggregation

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    Yu, Hongli; Chen, Yeqing; Wu, Hao; Wang, Kuilong; Liu, Liping; Zhang, Xingde

    2017-01-01

    Quercetin and rhamnose were efficiently converted into huaicarbon A/B by heating at 250°C for 10-15 min or at 200°C for 25-30 min. With the optimum molar ratio of quercetin/rhamnose (1:3), huaicarbon A and B yields reached 25% and 16% respectively after heating at 250°C, with 55% quercetin conversion. Huaicarbon A/B both promoted washed platelet aggregation dose-dependently, which was antagonized by an inhibitor of glycoprotein VI (GPVI) receptor. Similarly, they both promoted collagen-induced platelet aggregation in platelet-rich plasma in dose-dependent manners. According to the S type dose-response model, EC50 values of huaicarbon A and huaicarbon B were calculated as 33.48 μM and 48.73 μM respectively. They induced intracellular Ca2+ accumulation that was specifically blocked by GPVI antagonist. Huaicarbon A/B enhanced intracellular Ca2+ accumulation and facilitated collagen-induced platelet aggregation, which were blocked by GPVI antagonist. They were conducive to collagen-induced platelet aggregation by activating platelet GPVI receptor. PMID:28337278

  10. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.

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    Ezumi, Y; Shindoh, K; Tsuji, M; Takayama, H

    1998-07-20

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

  11. Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

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    Ezumi, Yasuharu; Shindoh, Keisuke; Tsuji, Masaaki; Takayama, Hiroshi

    1998-01-01

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex. PMID:9670039

  12. Molecular cloning, genomic structure, chromosomal localization, and alternative splice forms of the platelet collagen receptor glycoprotein VI.

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    Ezumi, Y; Uchiyama, T; Takayama, H

    2000-10-14

    Glycoprotein VI (GPVI) is the major collagen receptor underlying platelet activation. We cloned the full-length cDNA for GPVI (GPVI-1) and its two isoforms (GPVI-2 and -3) from phorbol-ester-stimulated CMK cells. The GPVI-1 cDNA was identical in the coding region with the cDNA that has recently been reported to belong to the immunoglobulin superfamily. The GPVI gene consisted of 8 exons spanning over 23 kbp and was mapped on the chromosome 19q13. 4. The promoter of GPVI gene lacked TATA and CAAT boxes and had multiple transcription start sites like other megakaryocytic genes. When COS-7 cells were cotransfected with the GPVI isoforms and Fc receptor gamma chain, Fc receptor gamma chain was associated with GPVI-1 and -2 but did not affect the GPVI expression levels. GPVI-1 and -2 could bind the collagen-related peptide, which exhibits triple-helical and polymeric structure of collagen to activate platelets via GPVI. Copyright 2000 Academic Press.

  13. α2β1 integrin, GPVI receptor, and common FcRγ chain on mouse platelets mediate distinct responses to collagen in models of thrombosis.

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    Robin J Marjoram

    Full Text Available Platelets express the α2β1 integrin and the glycoprotein VI (GPVI/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ-/- or the complex was depleted. The development of α2β1-/- and GPVI-/- mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro.To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1-/-, FcRγ-/-, and GPVI-/- mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ-/- platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI-/- and wild type platelets. The difference between FcRγ-/- and GPVI-/- platelet phosphotyrosine levels correlated with the in vivo thrombosis findings.Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet

  14. Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.

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    Ezumi, Yasuharu; Kodama, Kumi; Uchiyama, Takashi; Takayama, Hiroshi

    2002-05-01

    The platelet collagen receptor glycoprotein (GP) VI-Fc receptor gamma-chain (FcRgamma) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcRgamma complex signaling. Methyl-beta-cyclodextrin (MbetaCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI-FcRgamma complex. The GPVI-FcRgamma complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MbetaCD treatment. Thus, we provide the first evidence that the GPVI-FcRgamma complex is constitutively and functionally associated with membrane rafts.

  15. Surface proteome analysis identifies platelet derived growth factor receptor-alpha as a critical mediator of transforming growth factor-beta-induced collagen secretion.

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    Heinzelmann, Katharina; Noskovičová, Nina; Merl-Pham, Juliane; Preissler, Gerhard; Winter, Hauke; Lindner, Michael; Hatz, Rudolf; Hauck, Stefanie M; Behr, Jürgen; Eickelberg, Oliver

    2016-05-01

    Fibroblasts are extracellular matrix-producing cells in the lung. Fibroblast activation by transforming growth factor-beta leads to myofibroblast-differentiation and increased extracellular matrix deposition, a hallmark of pulmonary fibrosis. While fibroblast function with respect to migration, invasion, and extracellular matrix deposition has been well-explored, little is known about the surface proteome of lung fibroblasts in general and its specific response to fibrogenic growth factors, in particular transforming growth factor-beta. We thus performed a cell-surface proteome analysis of primary human lung fibroblasts in presence/absence of transforming growth factor-beta, followed by characterization of our findings using FACS analysis, Western blot, and siRNA-mediated knockdown experiments. We identified 213 surface proteins significantly regulated by transforming growth factor-beta, platelet derived growth factor receptor-alpha being one of the top down-regulated proteins. Transforming growth factor beta-induced downregulation of platelet derived growth factor receptor-alpha induced upregulation of platelet derived growth factor receptor-beta expression and phosphorylation of Akt, a downstream target of platelet derived growth factor signaling. Importantly, collagen type V expression and secretion was strongly increased after forced knockdown of platelet derived growth factor receptor-alpha, an effect that was potentiated by transforming growth factor-beta. We therefore show previously underappreciated cross-talk of transforming growth factor-beta and platelet derived growth factor signaling in human lung fibroblasts, resulting in increased extracellular matrix deposition in a platelet derived growth factor receptor-alpha dependent manner. These findings are of particular importance for the treatment of lung fibrosis patients with high pulmonary transforming growth factor-beta activity.

  16. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

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    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA) State Univ. of New York, Buffalo (USA))

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  17. Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets.

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    Briddon, S J; Watson, S P

    1999-02-15

    The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cgamma2 through a pathway that is dependent on the Fc receptor gamma (FcR gamma) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR gamma chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin alpha2beta1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR gamma chain. This was not true of the other platelet Src-family kinases. The association between the FcR gamma chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR gamma chain phosphorylation.

  18. Platelet affinity for burro aorta collagen

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    Schneider, M.D.

    1977-10-01

    Despite ingenious concepts, there are no unequivocal clues as to what, when, and how some undefined biochemical factor(s) or constituent(s) that localizes in the arterial wall can precipitate a thromboatheromatous lesion or arterial disease. The present study focused on the extraction, partial purification, and characterization of a collagen-active platelet stimulator from the aortas of aged burros. The aggregator moiety in the aorta extracts invariably had a higher affinity for platelets in citrated platelet-rich plasma of human beings than for platelets of homologous burros. The platelet-aggregating factor(s) in the aorta extract was retained by incubation with ..cap alpha..-chymotrypsin. Platelet-aggregating activity was rapidly abolished after incubation with collagenase, as determined by platelet-aggregometry tests. Evidence based on light microscope and polysaccharide histochemical reactions indicates a probability that the intracellular amorphous matrix (PAS-positive) and filamentous components (PTAH-positive) expelled from smooth muscle cells disrupted during homogenization of the aorta may be a principal source of a precursor collagen species which is a potent inducer of platelet aggregation.

  19. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

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    Nieswandt, B; Brakebusch, C; Bergmeier, W

    2001-01-01

    Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating...... subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed......, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von...

  20. Epithelial sodium channel modulates platelet collagen activation.

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    Cerecedo, Doris; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Benítez-Cardoza, Claudia; Ortega, Arturo

    2014-03-01

    Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation.

  1. Effect of platelet age on adhesiveness to collagen and platelet surface charge

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    Castellan, R.M.; Steiner, M.

    1976-11-30

    Adhesion to collagen was investigated as a function of platelet age in rat platelets. Platelet adherence was measured using EDTA-containing platelet- rich plasma which was added to preparations of collagen fibers clamped between magnetic stirrers by recording changes in light transmission. The plot of light transmission versus logarithm of time was linear and allowed calculation of a slope factor which related to the rate of adherence. Neither the amount of collagen nor the platelet count were limiting in the test. Young platelet populations (less than or equal to 1 day old) were obtained during the recovery phase from immune induced thrombocytopenia. Old platelet populations were prepared by blocking thrombopoiesis with cyclophosphamide. Young platelets did not differ significantly from randomly aged platelets in this function. The electrophoretic mobility of platelets was not affected by their age.

  2. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

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    Keith B Neeves

    Full Text Available Microfluidic flow assays (MFA that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T, the rate of platelet accumulation (V(PLT, and platelet surface coverage (SC. A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT and SC at all wall shear rates. A longer Lag(T for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104 and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

  3. Simplagrin, a platelet aggregation inhibitor from Simulium nigrimanum salivary glands specifically binds to the Von Willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo.

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    Andrezza C Chagas

    2014-06-01

    Full Text Available Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum, named Simplagrin.Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI with high affinity (2-15 nM, and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2β1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K(D 11 nM similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease.Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.

  4. Interaction of platelets with collagen substrate: role of platelet prostaglandin endoperoxides and thromboxane A/sub 2/

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    Mazurov, A.B.; Leitin, V.L.; Repin, V.S.

    1985-04-01

    In this investigation, the role of PG-endoperoxides and TA/sub 2/ in interaction of platelets and a collagen-coated surface was studied. For this purpose, the effects of exogenous AA, of U46619, a stable analog of PG endoperoxides, which simulates the action of PG endoperoxides and TA/sub 2/ on platelets at the receptor level, and of aspirin, a cyclooxygenase inhibitor, on platelet-surface interaction were investigated. The quantity of TA/sub 2/ synthesized in the platelets was determined by measuring accumulation of its stable product TB/sub 2/. After adhesion of the platelets the incubation medium was removed, nonadherent platelets were destroyed by freezing and thawing, and TB/sub 2/ was determined by radioimmunoassay.

  5. Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan.

    Directory of Open Access Journals (Sweden)

    Peng Jiang

    Full Text Available Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II type I receptor (AT1R antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2 receptor (TP and the glycoprotein VI (GPVI. Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg . mL(-1 and 10 μg . mL(-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25 and non-treated (n=30 patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy.ClinicalTrials.gov NCT00763893.

  6. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

    NARCIS (Netherlands)

    Tomlinson, M. G.; Calaminus, S. D.; Berlanga, O.; Bori-Sanz, T.; Meyaard, L.; Watson, S. P.; Auger, J.M.

    2007-01-01

    Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to

  7. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

    NARCIS (Netherlands)

    Tomlinson, M. G.; Calaminus, S. D.; Berlanga, O.; Bori-Sanz, T.; Meyaard, L.; Watson, S. P.; Auger, J.M.

    2007-01-01

    Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to

  8. Cordycepin-Enriched WIB801C from Cordyceps militaris Inhibits Collagen-Induced [Ca2+]i Mobilization via cAMP-Dependent Phosphorylation of Inositol 1, 4, 5-Trisphosphate Receptor in Human Platelets

    Science.gov (United States)

    Lee, Dong-Ha; Kim, Hyun-Hong; Cho, Hyun-Jeong; Yu, Young-Bin; Kang, Hyo-Chan; Kim, Jong-Lae; Lee, Jong-Jin; Park, Hwa-Jin

    2014-01-01

    In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca2+]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca2+]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca2+]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca2+-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease. PMID:25009703

  9. Platelets stimulate fibroblast-mediated contraction of collagen gels

    Directory of Open Access Journals (Sweden)

    Lundahl Joachim

    2003-10-01

    Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

  10. Burro aortic collagen: composition and characteristics of interaction with platelets

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.D. (Comparative Animal Research Lab., Oak Ridge, TN); Huang, W.O.W.; Cross, F.H.; Lin, K.T.D.

    1979-03-01

    A collagenous protein(s) from the aortas of three aged burros (Equus asinus) was isolated, using an acid (83.5 mM glacial acetic acid) extraction technique and subsequent pepsin digestion of the extracts with extensive dialysis. This protein(s) was then precipitated by adding solid NaCl (w/v, 9.45 g/100 ml) to the dialyzed aortic tissue extracts. The precipitate was collected by ultracentrifugation, concentrated by dissolving in one-fourth the original volume of acetic acid, and again extensively dialyzed. Values for amino acids in residues/1,000 indicated that the extractable aortic material(s) contained substantial amounts of proline, hydroxyproline, lysine, hydroxylysine, cysteine, and all other amino acid residues usually found in collagen. Examination by electrophoresis in sodium dodecyl sulfate polyacrylamide-gel indicated the presence of 5 major polypeptide subunits of the molecular weight range of 116,000 to 300,000. A powerful inducer of platelet aggregation was found in the acid solubilized, pepsin-digested aortic extracts. Human platelets were sensitive to the agent(s) down to 0.1 to 0.2 ..mu..g of aortic solids added to 0.45 ml of platelet-rich plasma. Platelets from goats and sheep were sensitive down to additions of 0.2 ..mu..g of solids, burro platelets to 0.5 to 1 ..mu..g, bovine platelets to 1 to 2 ..mu..g, and swine platelets to 2 to 3 ..mu..g. Thus, this powerful burro aortic collagen-containing material(s) may help to distinguish minor modifications in functional characteristics of platelets from persons or animals suffering from hemostatic or thrombotic disorders.

  11. Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito.

    Science.gov (United States)

    Yoshida, Shigeto; Sudo, Toshiki; Niimi, Masashi; Tao, Lian; Sun, Bing; Kambayashi, Junichi; Watanabe, Hiroyuki; Luo, Enjie; Matsuoka, Hiroyuki

    2008-02-15

    During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.

  12. Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions.

    Science.gov (United States)

    Farndale, Richard W; Lisman, Ton; Bihan, Dominique; Hamaia, Samir; Smerling, Christiane S; Pugh, Nicholas; Konitsiotis, Antonios; Leitinger, Birgit; de Groot, Philip G; Jarvis, Gavin E; Raynal, Nicolas

    2008-04-01

    Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

  13. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI.

    Science.gov (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Arai, M; Yamamoto, N; Takahashi, H; Okuma, M

    1997-01-03

    Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha2 beta1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha2 beta1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha2 beta1. In response to collagen, these platelets exhibited alpha2 beta1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.

  14. Salvianolic Acid B inhibits platelet adhesion under conditions of flow by a mechanism involving the collagen receptor alpha 2 beta 1

    NARCIS (Netherlands)

    Wu, Ya Ping; Zhao, Xiao Min; Pan, Shao Dong; Guo, De An; Wei, Ran; Han, Ji Ju; Kainoh, Mie; Xia, Zuo Li; de Groot, Philip G.; Lisman, Ton

    2008-01-01

    Salvianolic acid B (SAB) is a component of Danshen, a herb widely used in Chinese medicine, and was previously shown to exert a number of biological activities including inhibition of platelet function, but the exact mechanisms involved are unclear. SAB dose-dependently inhibited platelet deposition

  15. Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

    Science.gov (United States)

    Wang, Su Bin; Jang, Ji Yong; Chae, Yun Hee; Min, Ji Hyun; Baek, Jin Young; Kim, Myunghee; Park, Yunjeong; Hwang, Gwi Seo; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-06-01

    Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.

  16. SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

    Science.gov (United States)

    Walsh, Tony G; Harper, Matthew T; Poole, Alastair W

    2015-01-01

    Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway.

  17. Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Gaudette, D.C.; Holub, B.J. (Univ. of Guelph, Ontario (Canada))

    1990-05-15

    The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapendaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using (3H)inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 micrograms/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-biphosphate (PIP2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 microM) significantly attenuated the collagen-induced increased PIP and PIP2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty microM DHA was less effective at attenuating the increased PIP and PIP2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A2.

  18. Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release.

    Science.gov (United States)

    Kalinowski, Leszek; Matys, Tomasz; Chabielska, Ewa; Buczko, Włodzimierz; Malinski, Tadeusz

    2002-10-01

    This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

  19. Platelet adhesion and plasma protein adsorption control of collagen surfaces by He{sup +} ion implantation

    Energy Technology Data Exchange (ETDEWEB)

    Kurotobi, K. E-mail: kurotobi@postman.riken.go.jp; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M

    2003-05-01

    He{sup +} ion implanted collagen-coated tubes with a fluence of 1 x 10{sup 14} ions/cm{sup 2} were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2}. Platelet adhesion (using platelet rich plasma) was inhibited on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Platelet activation with washed platelets was observed on untreated collagen and He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was inhibited with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He{sup +} ion implanted collagen over a fluence of 1 x 10{sup 13} ions/cm{sup 2}. On the 1 x 10{sup 14} ions/cm{sup 2} implanted collagen, no platelet activation was observed due to the influence of plasma proteins. >From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and that plasma protein adsorption took an important role repairing the graft surface.

  20. Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

    Directory of Open Access Journals (Sweden)

    Véronique Ollivier

    Full Text Available Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI, we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

  1. Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

    Science.gov (United States)

    Clemetson, Kenneth J

    2015-01-01

    Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.

  2. Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

    Science.gov (United States)

    Kim, Daehwan; You, Eunae; Min, Na Young; Lee, Kwang-Ho; Kim, Hyoung Kyu; Rhee, Sangmyung

    2013-05-01

    The collagen matrix constitutes the primary extracellular matrix (ECM) portion of mammalian connective tissues in which the interaction of the cell and the surrounding collagen fibers has a significant impact on cell and tissue physiology, including morphogenesis, development and motility. Discoidin domain receptors (DDR1 and DDR2) have been identified as the receptor tyrosine kinases that are activated upon collagen binding. However, there is a lack of evidence regarding the effect of DDRs on the mechanical interaction between fibroblasts and ECM. In this study, we demonstrated that one of the major phosphotyrosine proteins in human fibroblasts during 3D collagen matrix polymerization is DDR2. Treatment of fibroblasts in 3D collagen matrices with platelet-derived growth factor (PDFG) has been shown to increase DDR2 phosphorylation. Silencing of DDR2 with siRNA in fibroblasts significantly reduced the number of dendritic extensions regardless of whether cells were cultured in the collagen or fibronectin 3D matrices. Decreasing dendritic extensions in DDR2-silenced cells has also been shown to decrease the ability of fibroblast entanglement to collagen fibrils in 3D collagen matrices. Finally, we also showed that the silencing of DDR2 decreased the cell migration in 3D nested collagen matrices but had no effect on 3D floating matrix contraction. Collectively, these results suggest that DDR2 functioning is required for the membrane dynamics to control the mechanical attachment of fibroblasts to the 3D collagen matrices in an integrin-independent manner.

  3. Abnormal whole blood thrombi in humans with inherited platelet receptor defects.

    Directory of Open Access Journals (Sweden)

    Francis J Castellino

    Full Text Available To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS and Glanzmann's Thrombasthenia (GT. We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

  4. Abnormal whole blood thrombi in humans with inherited platelet receptor defects.

    Science.gov (United States)

    Castellino, Francis J; Liang, Zhong; Davis, Patrick K; Balsara, Rashna D; Musunuru, Harsha; Donahue, Deborah L; Smith, Denise L; Sandoval-Cooper, Mayra J; Ploplis, Victoria A; Walsh, Mark

    2012-01-01

    To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

  5. Extract of feverfew inhibits interactions of human platelets with collagen substrates

    Energy Technology Data Exchange (ETDEWEB)

    Loesche, W.M.; Mazurov, A.V.; Heptinstall, S.; Groenewegen, W.A.; Repin, V.S.; Till, U.

    1987-12-01

    The interaction of platelets with surfaces coated with collagens of type III (C III) or IV (C IV) has been studied by measuring the deposition of /sup 51/Cr-labeled platelets and by scanning electron microscopy (SEM). Experiments were performed using platelet-rich plasma (PRP) and suspensions of gel-filtered platelets (GFP). Platelets were deposited on C III mainly as surface-bound aggregates. In contrast they were deposited on C IV mainly as spread forms of individual cells. Formation of aggregates on C III was more extensive for PRP than for GFP; in contrast platelet spreading on C IV was more extensive for GFP than for PRP. The effects of an extract of the plant feverfew on platelet-collagen interactions were determined. Feverfew extract inhibited the deposition of /sup 51/Cr-labeled platelets on both C III and C IV in a dose-dependent way. Similar concentrations of extract were needed to inhibit the formation of surface-bound aggregates and to inhibit platelet spreading in both PRP and GFP.

  6. Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

    Science.gov (United States)

    Guglielmini, Giuseppe; Appolloni, Viviana; Momi, Stefania; De Groot, Philip G; Battiston, Monica; De Marco, Luigi; Falcinelli, Emanuela; Gresele, Paolo

    2016-01-01

    Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, pMMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, pMMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.

  7. Drug effects on platelet adherence to collagen and damaged vessel walls.

    Science.gov (United States)

    Packham, M A; Cazenave, J P; Kinlough-Rathbone, R L; Mustard, J F

    1978-01-01

    The interaction of platelets with damaged vessel walls leads to the formation of platelet-fibrin thrombi and may also contribute to the development of atherosclerotic lesions because platelets adherent to exposed collagen release a mitogen that stimulates smooth muscle cell proliferation. The first step in thrombus formation, platelet adherence to an injured vessel wall, can be studied quantitatively by the use of platelets labeled with 51chromium. In these investigations, rabbit aortas were damaged by passage of a balloon catheter and segments of the aortas were everted on probes that were rotated in platelet suspensions. Collagen-coated glass cylinders were also used. Adherence was measured in a medium containing approximately physiologic concentrations of calcium, magnesium, protein and red blood cells. Conditions of testing influence the effect of non-steroidal anti-inflammatory drugs, sulfinpyrazone, and dipyridamole on platelet adherence. Aspirin and sulfinpyrazone were not inhibitory when tested in a medium with a 40% hematocrit; this indicates that products formed by platelets from arachidonate probably do not play a major part in the adherence of the first layer of platelets to the surface, although they may be involved in thrombus formation. Indomethacin, dipyridamole, prostaglandin E1, methylprednisolone and penicillin G and related antibiotics did inhibit platelet adherence although the concentrations required were higher than would likely be achieved in vivo upon administration to human patients. None of the non-steroidal anti-inflammatory drugs inhibited the release of granule contents from adherent platelets. Pretreatment of the damaged vessel wall with aspirin increased platelet adherence, presumably because it prevented the formation of PGI2 by the vessel wall. Platelet adherence to undamaged or damaged vessel walls was enhanced by prior exposure of the wall to thrombin. Platelet reactions with aggregating agents and platelet survival can be

  8. Inhibition of glutamate receptors reduces the homocysteine-induced whole blood platelet aggregation but does not affect superoxide anion generation or platelet membrane fluidization.

    Science.gov (United States)

    Karolczak, Kamil; Pieniazek, Anna; Watala, Cezary

    2017-01-01

    Homocysteine (Hcy) is an excitotoxic amino acid. It is potentially possible to prevent Hcy-induced toxicity, including haemostatic impairments, by antagonizing glutaminergic receptors. Using impedance aggregometry with arachidonate and collagen as platelet agonists, we tested whether the blockade of platelet NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors with their inhibitors: MK-801 (dizocilpine hydrogen maleate, [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), CNQX (7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile) and UBP-302 (2-{[3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxo-3,6-dihydropyrimidin 1(2H)-yl]methyl}benzoic acid) may hamper Hcy-dependent platelet aggregation. All the tested compounds significantly inhibited Hcy-augmented aggregation of blood platelets stimulated either with arachidonate or collagen. Hcy stimulated the generation of superoxide anion in whole blood samples in a concentration-dependent manner; however, this process appeared as independent on ionotropic glutamate receptors, as well as on NADPH oxidase and protein kinase C, and was not apparently associated with the extent of either arachidonate- or collagen-dependent platelet aggregation. Moreover, Hcy acted as a significant fluidizer of surface (more hydrophilic) and inner (more hydrophobic) regions of platelet membrane lipid bilayer, when used at the concentration range from 10 to 50 µmol/l. However, this effect was independent on the Hcy action through glutamate ionotropic receptors, since there was no effects of MK-801, CNQX or UBP-302 on Hcy-mediated membrane fluidization. In conclusion, Hcy-induced changes in whole blood platelet aggregation are mediated through the ionotopic excitotoxic receptors, although the detailed mechanisms underlying such interactions remain to be elucidated.

  9. Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

    Directory of Open Access Journals (Sweden)

    Tso-Hsiao Chen

    Full Text Available The platelet-derived soluble CD40L (sCD40L release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB, has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ. We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2 expression/activity and reactive oxygen species (ROS formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

  10. Characterization of human platelet binding of recombinant T cell receptor ligand

    Directory of Open Access Journals (Sweden)

    Meza-Romero Roberto

    2010-11-01

    Full Text Available Abstract Background Recombinant T cell receptor ligands (RTLs are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS. RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE. The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. Methods We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. Results Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. Conclusions Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

  11. G6f-like is an ITAM-containing collagen receptor in thrombocytes.

    Directory of Open Access Journals (Sweden)

    Craig E Hughes

    Full Text Available Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM. Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.

  12. Regulation of fibrinogen receptor expression on human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Shattil, S.J.; Motulsky, H.J.; Insel, P.A.; Brass, L.F.

    1986-03-01

    Platelet aggregation requires the binding of fibrinogen to specific receptors on the plasma membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The authors have developed a monoclonal anti-IIb-IIIa antibody (PAC-1) that binds only to stimulated platelets and only in the presence of Ca. In order to better understand the steps leading to platelet aggregation, the authors used radiolabeled PAC-1 and fibrinogen to examine the effect of the ..cap alpha../sub 2/-adrenergic agonist, epinephrine, on the expression and function of the fibrinogen receptor. The addition of epinephrine to unstirred platelets caused and immediate increase in PAC-1 and fibrinogen binding that was associated with platelet aggregation once the platelets were stirred. Even after prolonged incubation of the platelets with epinephrine, fibrinogen receptor expression could be reversed by adding EGTA, PGl/sub 2/, or the ..cap alpha../sub 2/-adrenergic antagonist, phentolamine. When unstirred platelets were exposed to epinephrine for more than 10 min, the extent of aggregation caused by subsequent stirring was decreased by 70%. Surprisingly, these desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due to a decrease in fibrinogen receptor expression or function. These studies demonstrate that: (1) fibrinogen receptor expression is dependent on extracellular CA; (2) induction of the fibrinogen receptor by epinephrine requires the continued presence of the agonist; and (3) prolonged stimulation of the platelet by epinephrine can lead to a reduced aggregation response by a mechanism that does not involve a loss of either fibrinogen recepor expression or fibrinogen binding.

  13. LIPOPOLYSACCHARIDE INDUCES EXPOSURE OF FIBRINOGEN RECEPTORS ON HUMAN PLATELETS

    Institute of Scientific and Technical Information of China (English)

    于希春; 吴其夏

    1995-01-01

    The effect of lipopolysaccharide (LPS) on the exposure of platelet fibrinogen receptors was investigated.The results showed that:1)LPS increased the binding of fibrinogen-gold complexes to platelets and the labels were primarily limited to shape-changed platelets;2)LPS caused a dose-dependent rise in intracellular Ca2+ concentration in platelets;3)LPS induced the activation of platelet protein kinase C(PKC) and the phosphorylation of glycoprotein llla (GP llla) which was inhibited by H-7.All these results suggest that stimulation of platelets with LPS causes a conformational change in glycoprotein llb/Illa (GPllb/llla) through platelet shape change and/or phosphorylation of GPllla via PKC,which serves to expose the fibrinogen binding sites of GPllb/llla on human platelets.

  14. Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study

    OpenAIRE

    Hubbard, G; Wolffram, S; Vos, de, W.M.; Bovy, A.G.; Gibbins, J.; Lovegrove, J

    2006-01-01

    Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertak...

  15. The collagen receptor uPARAP/Endo180

    DEFF Research Database (Denmark)

    Engelholm, Lars H; Ingvarsen, Signe; Jürgensen, Henrik J

    2009-01-01

    The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind...... and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem...... in collagen breakdown seems to be involved in invasive tumor growth....

  16. The collagen receptor uPARAP/Endo180

    DEFF Research Database (Denmark)

    Engelholm, Lars H; Ingvarsen, Signe; Jürgensen, Henrik J

    2009-01-01

    The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind...... and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem...... in collagen breakdown seems to be involved in invasive tumor growth....

  17. The collagen receptor uPARAP/Endo180

    DEFF Research Database (Denmark)

    Engelholm, Lars H; Ingvarsen, Signe; Jürgensen, Henrik J

    2009-01-01

    The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind...... and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem...... in collagen breakdown seems to be involved in invasive tumor growth Udgivelsesdato: 2009...

  18. The expression levels of platelet adhesive receptors in PRP derived platelet concentrates during storage

    Directory of Open Access Journals (Sweden)

    Fatemeh Nassaji

    2016-04-01

    Full Text Available Background: Major platelet adhesive receptors that contribute significantly to thrombus formation include platelet receptor glycoprotein Ibα (GPIbα of the GPIb-IX-V complex and platelet glycoprotein VI (GPVI. GPIbα plays a crucial role in platelet tethering to sub-endothelial matrix, which initiates thrombus formation at arterial shear rates, whereas GPVI is critically involved in platelets firm adhesion to the site of injury regardless of shear condition. During storage, platelets experience some changes that deleteriously affect the expression levels of platelet receptors, which in turn can alter platelet functional behaviors. Considering the important roles of GPIbα and GPVI in platelet adhesion, it seems that any dramatic changes in the expression levels of these receptors can influence adhesive function of transfused platelets. Thereby examining GPIbα and GPVI expression during the storage of platelet concentrates may provide some useful information about the functional quality of these products after transfusion. Methods: In our experimental study, 5 PRP-platelet concentrates were randomly obtained from Iranian Blood Transfusion Organization (IBTO. All the platelet products met the standard quality assessment based on AABB (American Association of Blood Banks guidelines. Washed platelets were subjected to flowcytometry analysis for the evaluation of GPIbα and GPVI receptor expression in day 1, 3 and 5 after storage. Data were presented as mean fluorescence intensity (MFI and analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. Results: The GPIbα expression on first day (MFI=86±5.9 was reduced three days after storage (MFI= 69±6.9. The expression levels continued to reduce until day 5 in which GPIbα expression was markedly decreased to (MFI= 61±7.7 (P= 0.0094. GPVI expression on the days 1, 3 and 5 after storage were 20.6±3.3, 24±2.5 and 14±4.9, respectively. The results showed a significant decrease of

  19. Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts.

    Science.gov (United States)

    Lu, Jiamei; Zhu, Yanting; Feng, Wei; Pan, Yilin; Li, Shaojun; Han, Dong; Liu, Lu; Xie, Xinming; Wang, Guizuo; Li, Manxiang

    2014-09-01

    Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13- induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.

  20. Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

    Indian Academy of Sciences (India)

    Jiamei Lu; Yanting Zhu; Wei Feng; Yilin Pan; Shaojun Li; Dong Han; Lu Liu; Xinming Xie; Guizuo Wang; Manxiang Li

    2014-09-01

    Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.

  1. Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase.

    Science.gov (United States)

    Blair, Price; Rex, Sybille; Vitseva, Olga; Beaulieu, Lea; Tanriverdi, Kahraman; Chakrabarti, Subrata; Hayashi, Chie; Genco, Caroline A; Iafrati, Mark; Freedman, Jane E

    2009-02-13

    Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam(3)CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin alpha(IIb)beta(3), generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam(3)CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

  2. Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis.

    Science.gov (United States)

    Daga, Shruti; Shepherd, James G; Callaghan, J Garreth S; Hung, Rachel K Y; Dawson, Dana K; Padfield, Gareth J; Hey, Shi Y; Cartwright, Robyn A; Newby, David E; Fitzgerald, J Ross

    2011-03-01

    Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis.

  3. Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

    Science.gov (United States)

    Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

    2011-01-01

    Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

  4. Identification of the platelet ADP receptor targeted by antithrombotic drugs.

    Science.gov (United States)

    Hollopeter, G; Jantzen, H M; Vincent, D; Li, G; England, L; Ramakrishnan, V; Yang, R B; Nurden, P; Nurden, A; Julius, D; Conley, P B

    2001-01-11

    Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

  5. Genetic variation responsible for mouse strain differences in integrin {alpha}{sub 2} expression is associated with altered platelet responses to collagen

    Energy Technology Data Exchange (ETDEWEB)

    Li, Tong-Tong; Larrucea, Susana; Souza, Shiloe; Leal, Suzanne M.; Lopez, Jose A.; Rubin, Edward M.; Nieswandt, Bernhard; Bray, Paul F.

    2003-11-01

    Formation of a thrombus at the site of an injured vessel requires the coordinated action of critical platelet plasma membrane adhesion molecules. The most important initial contact of platelets with the exposed endothelial collagen and von Willebrand factor (VWF) involves the binding of glycoprotein (GP) Ib{alpha} to immobilized VWF. The VWF-GPIb{alpha} interaction is ''fast-on'' and relatively ''fast-off,'' and results in a rolling of platelets along the exposed subendothelium. This slowing of the platelets allows binding of the activating collagen-receptor, GPVI, to its ligand, resulting in activation of platelet integrins and subsequent firm adhesion, where the reactions between receptor and ligand are relatively ''slow-on'' but irreversible. The binding of integrin {alpha}{sub 2} {beta}{sub 1} underlying firm adhesion. Intracellular signaling between and through these adhesive receptors plays a crucial role in platelet adhesion and aggregation. The importance of the GPIb-IX-V and {alpha}{sub IIb} {beta}{sub 3} in normal hemostasis is under scored by the bleeding diatheses that have been reported in patients with quantitative or qualitative deficiencies of the genes that encode them. Mouse models are now commonplace for studying hemostasis and thrombosis, and important insights pertaining to the major platelet adhesive receptors have been gleaned from mouse studies involving targeted disruptions of the genes for GPIb{alpha}, GPVI, and integrin chains 2,9,10 1,4 IIb 11 and 3.12 A variety of different mouse strains have been used to assess hemostasis. For example, the FVB strain is typically used for transgenic experiments, the 129/Sv strain is used to derive embryonic stem (ES) cells, and the C57 strain is used for uniform background breeding studies. Different strains may exhibit different levels of gene expression, a feature that has been used to elucidate crucial gene regions regulating transcription

  6. Losartan inhibits the adhesion of rat platelets to fibrillar collagen--a potential role of nitric oxide and prostanoids.

    Science.gov (United States)

    Matys, T; Chabielska, E; Pawlak, R; Kucharewicz, I; Buczko, W

    2000-12-01

    The aim of the study was to evaluate the effect of losartan on rat platelet adhesion to fibrillar collagen. Washed platelets were counted before and after 15 minutes incubation with collagen (50 microg/ml) and the percentage of adhering platelets was calculated as the index of their adhesion. When the platelets were incubated with collagen 40.8 +/- 0.3% of the platelets adhered. Losartan produced a dose dependent decrease in a number of adhering platelets both when the drug was administered to the animals ex vivo at doses of 3, 10 and 30 mg/kg (p < 0.01-0.001) or was added to the preparation of washed platelets in vitro in concentrations of 10(-8)-10(-5) M (p < 0.01-0.001). In the next step of the study we assessed the influence of L-NAME (10 mg/kg ex vivo, 30 microM in vitro) and indomethacin (2.5 mg/kg ex vivo, 30 microM in vitro) on the antiadhesive effect of losartan (10 mg/kg ex vivo, 10(-6) M in vitro). Blockade of nitric oxide synthase with L-NAME partially reversed the antiadhesive effect of losartan both ex vivo and in vitro. Indomethacin diminished the inhibitory effect of losartan on platelet adhesion when administered ex vivo, but it failed to modify this parameter when added to the suspension of platelets in vitro. In conclusion, losartan reduces platelet adhesion to fibrillar collagen in a dose-dependent manner. The observed action of losartan seems to be mediated mainly by endothelium- and platelet-derived nitric oxide.

  7. The discoidin domain receptor DDR2 is a receptor for type X collagen.

    Science.gov (United States)

    Leitinger, Birgit; Kwan, Alvin P L

    2006-08-01

    During endochondral ossification, collagen X is deposited in the hypertrophic zone of the growth plate. Our previous results have shown that collagen X is capable of interacting directly with chondrocytes, primarily via integrin alpha2beta1. In this study, we determined whether collagen X could also interact with the non-integrin collagen receptors, discoidin domain receptors (DDRs), DDR1 or DDR2. The widely expressed DDRs are receptor tyrosine kinases that are activated by a number of different collagen types. Collagen X was found to be a much better ligand for DDR2 than for DDR1. Collagen X bound to the DDR2 extracellular domain with high affinity and stimulated DDR2 autophosphorylation, the first step in transmembrane signalling. Expression of DDR2 in the epiphyseal plate was confirmed by RT-PCR and immunohistochemistry. The spatial expression of DDR2 in the hypertrophic zone of the growth plate is consistent with a physiological interaction of DDR2 with collagen X. Surprisingly, the discoidin domain of DDR2, which fully contains the binding sites for the fibrillar collagens I and II, was not sufficient for collagen X binding. The nature of the DDR2 binding site(s) within collagen X was further analysed. In addition to a collagenous domain, collagen X contains a C-terminal NC1 domain. DDR2 was found to recognise the triple-helical region of collagen X as well as the NC1 domain. Binding to the collagenous region was dependent on the triple-helical conformation. DDR2 autophosphorylation was induced by the collagen X triple-helical region but not the NC1 domain, indicating that the triple-helical region of collagen X contains a specific DDR2 binding site that is capable of receptor activation. Our study is the first to describe a non-fibrillar collagen ligand for DDR2 and will form the basis for further studies into the biological function of collagen X during endochondral ossification.

  8. The Role of Inflammation in Regulating Platelet Production and Function: Toll-like Receptors in Platelets and Megakaryocytes

    OpenAIRE

    Beaulieu, Lea M.; Freedman, Jane E.

    2009-01-01

    Platelets have been extensively studied as hemostatic regulators, stopping uncontrolled flow of blood from an injured vessel and allowing for repair. However, multiple studies have shown that platelets can interact with bacterial proteins, particularly seen during sepsis and inflammation. Immune cells recognize pathogens through Toll-like Receptors (TLRs). These same receptors allow platelets to recognize bacterial proteins and regulate platelet immunity and function. This review examines the...

  9. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho

    2017-02-01

    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  10. Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

    Science.gov (United States)

    Smith, D F; Kosow, D P; Wu, C; Jamieson, G A

    1977-08-11

    A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.

  11. Collagen quantification in rabbit dermal wounds treated with heterologous platelet-rich plasma gel

    Directory of Open Access Journals (Sweden)

    Maria Elisa Marin Marques

    2017-03-01

    Full Text Available Platelet-rich plasma (PRP has been extensively studied as a biomaterial for wound treatment, and the heterologous PRP is usefulin the event that obtaining the patient’s own blood is impossible. This study aimed to evaluate and compare wound healing in rabbits and quantify the collagen in experimentally induced wounds in a control group and in a group treated with heterologous PRP gel. We hypothesize that this gelis capable of promoting proper healing with no adverse reactions, increased collagen content. The clinical aspects of coloring, edema, hyperemia, exudation, crust, granulation, pain sensitivity, and retraction index of the wounds were measuredon days 7, 14, and 17 after the injury. Collagen quantification by Picrosirius staining and evaluation under polarized light was performed on the 17th day. Crust was present in both groups at all evaluated time points, with the absence of other clinical signs. The wound contraction rate and collagen quantity did not differ between groups. In conclusion, the suggested hypothesis was partially confirmed; the heterologous PRP gel was unable to increase the amount of collagen and accelerate the wound healing process, however, wound healing was efficient and similar in both groups and there was no local adverse reaction. Thus, despite the scarcity of studies in the literature, the heterologous PRP gel is an effective alternative treatment for wounds in the absence of other sources of PRP.

  12. Platelet P2 receptors: from curiosity to clinical targets.

    Science.gov (United States)

    Cusack, N J; Hourani, S M

    2000-07-01

    Adenosine 5'-diphosphate (ADP) is a paracrine mediator that activates human blood platelets, causing them to become adhesive and thereby contributing to their role in hemostasis. The actions of ADP were initially thought to be mediated by a unique ADP receptor termed P2(T) found only on platelets and antagonized by ATP, but it appears that at least two P2Y receptor subtypes are involved, a P2Y(1) receptor linked in some way to control of intracellular-free calcium levels and another P2Y receptor linked via an inhibitory G protein to adenylate cyclase. In addition, the presence of excitatory P2X(1) receptors that mediate the influx of monovalent and divalent cations in response to both ADP and ATP has been demonstrated. The precise contribution that each of these P2 receptors make to the overall phenomena associated with platelet aggregation, adhesion and hemostasis is yet to be defined. Antithrombotic agents that interfere with the actions of ADP are marketed, and P2 receptor antagonists are entering clinical trials for acute treatments of thrombosis. This review seeks to summarize the present state of knowledge of platelet P2 receptor pharmacology and therapeutics.

  13. Injection temperature significantly affects in vitro and in vivo performance of collagen-platelet scaffolds.

    Science.gov (United States)

    Palmer, M P; Abreu, E L; Mastrangelo, A; Murray, M M

    2009-07-01

    Collagen-platelet composites have recently been successfully used as scaffolds to stimulate anterior cruciate ligament (ACL) wound healing in large animal models. These materials are typically kept on ice until use to prevent premature gelation; however, with surgical use, placement of a cold solution then requires up to an hour while the solution comes to body temperature (at which point gelation occurs). Bringing the solution to a higher temperature before injection would likely decrease this intra-operative wait; however, the effects of this on composite performance are not known. The hypothesis tested here was that increasing the temperature of the gel at the time of injection would significantly decrease the time to gelation, but would not significantly alter the mechanical properties of the composite or its ability to support functional tissue repair. Primary outcome measures included the maximum elastic modulus (stiffness) of the composite in vitro and the in vivo yield load of an ACL transection treated with an injected collagen-platelet composite. In vitro findings were that injection temperatures over 30 degrees C resulted in a faster visco-elastic transition; however, the warmed composites had a 50% decrease in their maximum elastic modulus. In vivo studies found that warming the gels prior to injection also resulted in a decrease in the yield load of the healing ACL at 14 weeks. These studies suggest that increasing injection temperature of collagen-platelet composites results in a decrease in performance of the composite in vitro and in the strength of the healing ligament in vivo and this technique should be used only with great caution.

  14. Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study

    NARCIS (Netherlands)

    Hubbard, G.; Wolffram, S.; Vos, de C.H.; Bovy, A.G.; Gibbins, J.; Lovegrove, J.

    2006-01-01

    Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate t

  15. Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

    Science.gov (United States)

    Bonacina, F.; Barbieri, S.S.; Cutuli, L.; Amadio, P.; Doni, A.; Sironi, M.; Tartari, S.; Mantovani, A.; Bottazzi, B.; Garlanda, C.; Tremoli, E.; Catapano, A.L.; Norata, G.D.

    2016-01-01

    Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cell's derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis. PMID:26976330

  16. An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

    Institute of Scientific and Technical Information of China (English)

    程远国; 吴厚永; 李德昌

    1999-01-01

    Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC50 was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca2+ level of platelets in response to collagen were com

  17. von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen

    NARCIS (Netherlands)

    Bonnefoy, A.; Romijn, R. A.; Vandervoort, P. A. H.; Van Rompaey, I.; Vermylen, J.; Hoylaerts, M. F.

    2006-01-01

    Background: Binding of von Willebrand factor (VWF) to platelet GPIb alpha and to collagen is attributed to VWF A1 and A3 domains, respectively. Objectives: Using VWF, VWF lacking A1 (Delta A1-VWF) or A3 (Delta A3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residu

  18. Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

    OpenAIRE

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W.; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the...

  19. Megakaryocytes and platelets express nicotinic acetylcholine receptors but nicotine does not affect megakaryopoiesis or platelet function.

    Science.gov (United States)

    Schedel, Angelika; Kaiser, Kerstin; Uhlig, Stefanie; Lorenz, Florian; Sarin, Anip; Starigk, Julian; Hassmann, Dennis; Bieback, Karen; Bugert, Peter

    2016-01-01

    In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function and in vitro differentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in an ex vivo model using megakaryocytic cells differentiated from cord blood derived CD34(+) cells (CBMK) and an in vivo model using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRβ2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume--MPV and platelet distribution width--PDW) were determined as indicators for changes of in vivo megakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry. CHRNA7, CHRNA4 and CHRNB2 gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRβ2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in our ex vivo and in vivo models. Thus, the functional role of the nAChR in these cells remains open.

  20. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

    Science.gov (United States)

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

  1. Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide

    Directory of Open Access Journals (Sweden)

    Guilai Liu

    2015-11-01

    Full Text Available Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i, which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP, which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml and CRP (5ug/ml within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM. However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM. Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

  2. A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

    Science.gov (United States)

    Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

    2016-08-01

    A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.

  3. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI.

    Science.gov (United States)

    Gardiner, Elizabeth E; Andrews, Robert K

    2014-04-01

    Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count.

  4. The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review)

    DEFF Research Database (Denmark)

    Carlsen Melander, Eva Maria; Jürgensen, Henrik J; Madsen, Daniel H

    2015-01-01

    as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important......The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen...

  5. An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.

    Science.gov (United States)

    Coxon, Carmen H; Sadler, Amanda J; Huo, Jiandong; Campbell, R Duncan

    2012-01-01

    Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50) for both CRP and collagen.

  6. An investigation of hierachical protein recruitment to the inhibitory platelet receptor, G6B-b.

    Directory of Open Access Journals (Sweden)

    Carmen H Coxon

    Full Text Available Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM and an immunoreceptor tyrosine-based switch motif (ITSM. The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC(50 for both CRP and collagen.

  7. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    Science.gov (United States)

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  8. A novel collagen/platelet-rich plasma (COL/PRP) scaffold: preparation and growth factor release analysis.

    Science.gov (United States)

    Zhang, Xiujie; Wang, Jingwei; Ren, Mingguang; Li, Lifeng; Wang, Qingwen; Hou, Xiaohua

    2016-06-01

    Platelet-rich plasma (PRP) has been widely used in clinical practice for more than 20 years because it causes the release of many growth factors. However, the burst release pattern and short release period of PRP have become obstacles to its application. An optimal controllable release system is an urgent need for researchers. This study investigated whether collagen/PRP (COL/PRP) scaffolds can serve as a vehicle for the controllable release of growth factors. We fabricated a novel scaffold that integrates PRP activated by thrombin or collagen into type I collagen. The mechanical properties, cytotoxicity, and transforming growth factor β1 (TGF-β1), platelet derived growth factor (PDGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) content were evaluated. Our results demonstrate that the COL/PRP scaffolds were not cytotoxic to L-929 fibroblasts. The PDGF and FGF content in the thrombin group was at a higher level and lasted for a long period of time. Collagen and thrombin played the same role in the release of TGF-β1 and VEGF. These data suggest that the novel COL/PRP scaffolds provide a carrier for the controllable release of growth factors and may be used in tissue- regenerative therapies.

  9. Cystamine immobilization on TiO 2 film surfaces and the influence on inhibition of collagen-induced platelet activation

    Science.gov (United States)

    Zhou, Yujuan; Weng, Yajun; Zhang, Liping; Jing, Fengjuan; Huang, Nan; Chen, Junying

    2011-12-01

    Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO 2 films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO 2 films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

  10. Platelets

    Science.gov (United States)

    ... tiny fraction of the blood volume. The principal function of platelets is to prevent bleeding. Red blood cells are ... forming a long string. This illustrates the basic function of platelets, to stick to any foreign surface and then ...

  11. High Residual Collagen-Induced Platelet Reactivity Predicts Development of Restenosis in the Superficial Femoral Artery After Percutaneous Transluminal Angioplasty in Claudicant Patients

    Energy Technology Data Exchange (ETDEWEB)

    Gary, Thomas, E-mail: thomas.gary@medunigraz.at [Medical University of Graz, Division of Angiology, Department of Internal Medicine (Austria); Prüller, Florian, E-mail: florian.prueller@klinikum-graz.at; Raggam, Reinhard, E-mail: reinhard.raggam@klinikum-graz.at [Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics (Austria); Mahla, Elisabeth, E-mail: elisabeth.mahla@medunigraz.at [Medical University of Graz, Department of Anesthesiology and Intensive Care Medicine (Austria); Eller, Philipp, E-mail: philipp.eller@medunigraz.at; Hafner, Franz, E-mail: franz.hafner@klinikum-graz.at; Brodmann, Marianne, E-mail: marianne.brodmann@medunigraz.at [Medical University of Graz, Division of Angiology, Department of Internal Medicine (Austria)

    2016-02-15

    PurposeAlthough platelet reactivity is routinely inhibited with aspirin after percutaneous angioplasty (PTA) in peripheral arteries, the restenosis rate in the superficial femoral artery (SFA) is high. Interaction of activated platelets and the endothelium in the region of intervention could be one reason for this as collagen in the subendothelium activates platelets.Materials and MethodsA prospective study evaluating on-site platelet reactivity during PTA and its influence on the development of restenosis with a total of 30 patients scheduled for PTA of the SFA. Arterial blood was taken from the PTA site after SFA; platelet function was evaluated with light transmission aggregometry. After 3, 6, 12, and 24 months, duplex sonography was performed and the restenosis rate evaluated.ResultsEight out of 30 patients developed a hemodynamically relevant restenosis (>50 % lumen narrowing) in the PTA region during the 24-month follow-up period. High residual collagen-induced platelet reactivity defined as AUC >30 was a significant predictor for the development of restenosis [adjusted odds ratio 11.8 (9.4, 14.2); P = .04].ConclusionsHigh residual collagen-induced platelet reactivity at the interventional site predicts development of restenosis after PTA of the SFA. Platelet function testing may be useful for identifying patients at risk.

  12. Bone neoformation in defects treated with platelet-rich fibrin membrane versus collagen membrane: a histomorphometric study in rabbit femurs.

    Directory of Open Access Journals (Sweden)

    Edwin Meza

    2015-02-01

    Full Text Available The aim of the present research was to compare bone neoformation in bone defects treated with platelet-rich fibrin (PRF and collagen membrane (CM at 3 and 5 weeks. For this purpose, two bone defects with a width of 4 mm and depth of 6 mm were created in the left distal femur diaphysis of New Zealand rabbits (n=12. The subjects were randomly allocated into two groups. One of the defects was covered with a platelet-rich fibrin membrane (Centrifuged resorbable autologous blood biopolymer without biochemical modification or a collagen membrane (gold standard - Neo Mem. The second defect was left uncovered (NC. The rabbits were sacrificed after 3 and 5 weeks (3 rabbits per period. The femur was completely removed and processed histomophometrically. The bone neformation analysis was performed using a differential point-counting method. Data was statistically analyzed (ANOVA, Tukey. The histomorphometric results showed that bone neformation in the defects treated with PRF at 3 weeks was equivalent to the CM (p<0.05. After 5 weeks, bone neformation obtained with PRF was higher than the control group and lower compared with the CM (p<0.05. The conclusion of the present study is that bone neformation in defects treated with PRF showed lower histomorphometric results compared with the one obtained with the collagen membrane and higher when compared with the control defects.

  13. Collagen Type I as a Ligand for Receptor-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Iris Boraschi-Diaz

    2017-05-01

    Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  14. Thrombin Receptor-Activating Protein (TRAP-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1 from Platelets in DM Patients

    Directory of Open Access Journals (Sweden)

    Haruhiko Tokuda

    2016-05-01

    Full Text Available It is generally known that heat shock protein 27 (HSP27 is phosphorylated through p38 mitogen-activated protein (MAP kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP on the release of HSP27 in platelets in type 2 diabetes mellitus (DM patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78 were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC of small aggregates (9–25 µm induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm, large aggregates (50–70 µm and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

  15. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius

    2013-01-01

    of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies......Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent...... advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently...

  16. Investigation of platelet function and platelet disorders using flow cytometry.

    Science.gov (United States)

    Rubak, Peter; Nissen, Peter H; Kristensen, Steen D; Hvas, Anne-Mette

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  17. Platelets possess functional TGF-beta receptors and Smad2 protein.

    Science.gov (United States)

    Lev, P R; Salim, J P; Marta, R F; Osorio, M J Mela; Goette, N P; Molinas, F C

    2007-02-01

    TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

  18. Sulfonamide inhibitors of α2β1 integrin reveal the essential role of collagen receptors in in vivo models of inflammation.

    Science.gov (United States)

    Nissinen, Liisa; Ojala, Marika; Langen, Barbara; Dost, Rita; Pihlavisto, Marjo; Käpylä, Jarmo; Marjamäki, Anne; Heino, Jyrki

    2015-06-01

    Small molecule inhibitors of α2β1 integrin, a major cellular collagen receptor, have been reported to inhibit platelet function, kidney injury, and angiogenesis. Since α2β1 integrin is abundantly expressed on various inflammation-associated cells, we tested whether recently developed α2β1 blocking sulfonamides have anti-inflammatory properties. Integrin α2β1 inhibitors were shown to reduce the signs of inflammation in arachidonic acid-induced ear edema, PAF stimulated air pouch, ovalbumin-induced skin hypersensitivity, adjuvant arthritis, and collagen-induced arthritis. Thus, these sulfonamides are potential drugs for acute and allergic inflammation, hypersensitivity, and arthritis. One sulfonamide with potent anti-inflammatory activity has previously been reported to be selective for activated integrins, but not to inhibit platelet function. Thus, the experiments also revealed fundamental differences in the action of nonactivated and activated α2β1 integrins in inflammation when compared to thrombosis.

  19. [Platelets "Toll-like receptor" engagement stimulates the release of immunomodulating molecules].

    Science.gov (United States)

    Cognasse, F; Hamzeh-Cognasse, H; Garraud, O

    2008-09-01

    Platelets are nonnucleated cellular elements that play a role in the process of haemostasis, and also in various ways in innate immunity and in inflammation. Platelets also contain numerous secretory products and can exert critical roles in several aspects of haemostasis. In addition, they house and secrete a variety of cytokines, chemokines and associated molecules which behave as ligands for receptors/counterparts displayed by endothelial cells lining tissue vessels and most leukocyte subsets. These latter studies show that platelets have an important role in innate as well as adaptive immunity; thus platelets can take part in an immune directive response. Moreover, platelets display receptors for several types of cytokines/chemokines along with FcgammaRII receptors. Finally, platelets not only express a variety of Toll-like receptors, with recently identified functions or not as-yet fully identified, but have also been demonstrated to express the key tandem pair of inflammatory and antigen presentation molecules (CD40 and CD40-ligand/CD154), this latter function making them the major purveyors of soluble CD40L in the plasma. It appears that platelets may be regarded as one of the neglected components of immune cell regulators, and platelets contribute to some interesting aspects in bridging innate and adaptive immunity. We propose that platelets discriminate danger signals and adapt the subsequent responses, with polarized cytokine secretion. Platelets may recognize several types of infectious pathogens and limit microbial colonization by sequestering these pathogens and releasing immunomodulatory factors. This review allows us to re-explore indications that platelets exert direct anti-infection immunity and we will present experimentally-driven arguments in favour of a role of platelet TLR in regulating certain immune activities.

  20. EDTA dependent pseudothrombocytopenia caused by antibodies against the cytoadhesive receptor of platelet gpIIB-IIIA.

    OpenAIRE

    1994-01-01

    AIMS--To clarify the mechanisms involved in the development of EDTA dependent pseudothrombocytopenia, particularly the platelet receptors. METHODS--Platelets were measured in 33 patients with pseudothrombocytopenia, using different anticoagulants to collect blood samples (direct test). The results were compared with the counts obtained by adding patients' serum or immunoglobulins to normal blood samples (indirect test). The role of platelet function was explored using ASA, PGE1, and apyrase a...

  1. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  2. The Effect of Platelet-Rich Fibrin, Calcium Sulfate Hemihydrate, Platelet-Rich Plasma and Resorbable Collagen on Soft Tissue Closure of Extraction Sites

    Directory of Open Access Journals (Sweden)

    Lisa M. Yerke

    2017-05-01

    Full Text Available Rapid and complete soft tissue healing after tooth extraction minimizes surgical complications and facilitates subsequent implant placement. We used four treatment methods and assessed changes in soft tissue socket closure following tooth extraction in humans. The effects of platelet-rich fibrin-calcium sulfate hemihydrate (PRF-CSH, platelet-rich plasma-calcium sulfate hemihydrate (PRP-CSH, a resorbable collagen dressing (RCD, and no grafting material were compared in a randomized, controlled pilot study with a blinded parallel design (N = 23. Patients with a hopeless tooth scheduled for extraction were randomly assigned to one of the four treatment groups. Socket measurements were obtained immediately after extraction and treatment, as well as after 21 days. There was a significant decrease in the total epithelialized external surface area of the extraction sockets in each group at all time points. However, there were no significant differences in soft tissue closure (p > 0.05 at any time point and PRF-CSH or PRP-CSH did not provide any additional benefit to enhance the soft tissue closure of extraction sockets compared with either RCD or sites without graft.

  3. Histologic Evidence of New Collagen Formulation Using Platelet Rich Plasma in Skin Rejuvenation: A Prospective Controlled Clinical Study

    Science.gov (United States)

    Abuaf, Ozlem Karabudak; Baloglu, Hüseyin; Bilgili, Memet Ersan; Simsek, Hasan Aktug; Dogan, Bilal

    2016-01-01

    Background Platelet-rich plasma (PRP) is an autologous concentration of human platelets contained in a small volume of plasma and has recently been shown to accelerate rejuvenate aging skin by various growth factors and cell adhesion molecules. Objective This study was conducted to evaluate the efficacy and safety of intradermal injection of PRP in the human facial rejuvenation. Methods This study was a prospective, single-center, single-dose, open-label, non-randomized controlled clinical study. PRP injected to the upper site of this right infra-auricular area and all face. Saline was injected to the left infra-auricular area. Histopathological examinations were performed before PRP treatment, 28 days after the PRP, and saline (control) treatments. Results Twenty women ranging in age from 40 to 49 years (mean age, 43.65±2.43 years) were enrolled in the study. The mean optical densities (MODs) of collagen in the pre-treatment, control, and PRP-treated area were measured. They were 539±93.2, 787±134.15, 1,019±178, respectively. In the MOD of PRP, 89.05 percent improvement was found when MOD of PRP was compared with MOD of pre-treatment. The mean MOD of collagen fibers was clearly highest on the PRP side (p<0.001). The PRP-to-saline improvement ratio (89.05% to 46.01%) was 1.93:1. No serious side effects were detected. Conclusion PRP increases dermal collagen levels not only by growth factors, but also by skin needling (the mesotherapy technique 'point by point'). PRP application could be considered as an effective (even a single application) and safety procedure for facial skin rejuvenation. PMID:27904271

  4. Synthesis, development, characterization and effectiveness of bovine pure platelet gel-collagen-polydioxanone bioactive graft on tendon healing

    Science.gov (United States)

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid

    2015-01-01

    Bovine platelet gel (BPG) is an accessible and cost-effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI-PDS were served as controls for the CI-PDS-BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI-PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals’ serum PDGF level, CI-PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI-PDS-BPG may be a valuable bioscaffold in tendon reconstructive surgery. PMID:25702535

  5. Platelet-rich plasma diminishes calvarial bone repair associated with alterations in collagen matrix composition and elevated CD34+ cell prevalence.

    Science.gov (United States)

    Giovanini, Allan Fernando; Deliberador, Tatiana Miranda; Gonzaga, Carla Castiglia; de Oliveira Filho, Marco Antonio; Göhringer, Isabella; Kuczera, Juliane; Zielak, João Cesar; de Andrade Urban, Cícero

    2010-06-01

    The interaction between platelets and both type I and III collagens plays an important role in modulating platelet adhesion and aggregation, also contributing to the chemotaxis of CD34+ cells. The interaction with type III collagen can maintain high levels of collagen and alter the biology of bone repair when the PRP is used. The aim of this study was to evaluate the effect of platelet-rich plasma (PRP) and autograft on the presence of type III and type I collagens, the ratio between them, as well as the presence of CD34+ progenitor cells, while comparing these results by means of a histomorphometric analysis of the bone tissue. Four bone defects (8.0mm in diameter and 2.0mm in depth) were produced on the calvarium of 23 rabbits. The surgical defects were treated with either autogenous bone grafts, autogenous bone grafts with PRP and PRP alone. Animals were euthanized at 2, 4 or 6 weeks post-surgery. Histological, histomorphometric and immunohistochemical analyses were performed to assess repair time, as well as the expression of type I and III collagens, and number of progenitor CD34+ cells. Data were analyzed using the ANOVA and Student-Newman-Keuls test (alpha=5%). An enlarged granulation and medullary tissue areas in the PRP groups were observed. The use of PRP in this study hindered bone deposition, also enhanced type III to type I collagen ratio and the chemotaxis of CD34+ progenitor cells, similarly to a thrombogenic effect.

  6. Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

    Science.gov (United States)

    Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

    2017-08-29

    Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y12 receptor expression in megakaryocytes. Platelet P2Y12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y12 expression correlates with ADP-induced platelet aggregation (r=0.89, Pdiabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, Pdiabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, Pdiabetes mellitus. Platelet P2Y12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

  7. [ROLE α2-ADRENERGIC RECEPTORS IN REGULATION PLATELET REACTIVITY IN THE ELDERLY AT CHRONIC OBSTRUCTIVE PYELONEPHRITIS].

    Science.gov (United States)

    Barinov, E F

    2016-01-01

    Objective of the research was to determine involvement of platelets and the role of adrenaline in chronic inflammation maintaining and the initiation of acute inflammatory response in elderly patients with chronic obstructive pyelonephritis against this background. The study includes 60 patients with chronic obstructive pyelonephritis (COPN), which are distributed into two groups: basic - 22 elderly patients (age 73±1,5 years) and the comparison group - 38 middle-aged patients (52,5±2,4 years). The study excluded patients who took antiplatelet drugs and non-selective blockers of α adrenergic receptors at least 1 week before the study. Analysis of platelets adrenoreactivity in vitro was carried out at the time of hospitalization before the start of conservative therapy. Platelet-rich plasma was isolated from peripheral blood by centrifuging. ADP and epinephrine were used in the effective (EC50) and sub-threshold (EC10) concentrations to stimulate platelets. The formation of platelet-leukocyte aggregates was reproduced in vitro upon incubation of stimulated platelets (at a concentration of adrenaline EC50) and intact leukocytes isolated from patient peripheral blood. The study of platelet reactivity revealed that in elderly patients acute inflammatory response realization (relapse of COPN) is against optimal functioning of platelets α2 adrenergic receptors. Significant increase in the number of platelet-leukocyte aggregates is possible. Remission of COPN (the presence of chronic inflammation) in the examined patients of various ages was associated with platelet hypoadrenoreactivity. Increased platelet adrenoreactivity during transition from remission to relapse of COPN in the elderly patients is possible if adequate synthesis of ADP in platelets and its secretion from dense granules are preserved. The observed interaction of adrenaline and ADP with stimulated platelet hyporesponsiveness probably ensures adaptive response aimed at acute inflammatory response in

  8. Platelet membrane glycoproteins and their function: an overview.

    Science.gov (United States)

    Kunicki, T J

    1989-07-01

    The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.

  9. Usefulness of the INNOVANCE PFA P2Y test cartridge for the detection of patients with congenital defects of the platelet P2Y₁₂ receptor for adenosine diphosphate.

    Science.gov (United States)

    Scavone, Mariangela; Germanovich, Ksenia; Femia, Eti A; Cattaneo, Marco

    2014-02-01

    The platelet function analyzer (PFA)-100 is used in clinical practice to screen patients with bleeding diathesis and suspected defects of primary hemostasis. A new cartridge, INNOVANCE PFA P2Y, has been specifically developed to monitor patients' response to drugs inhibiting the platelet P2Y₁₂ receptor for ADP. In this study, we compared the ability of INNOVANCE PFA P2Y to detect congenital defects of the platelet P2Y₁₂ receptor to that of standard cartridge formulations currently in clinical use. We studied two patients with severe P2Y₁₂ deficiency, one patient with heterozygous P2Y₁₂ deficiency and one with dysfunctional P2Y₁₂ receptor. Closure times were measured using 3 cartridges: collagen/ADP, collagen/epinephrine, and INNOVANCE PFA P2Y. The results obtained in the four patients with P2Y₁₂ defects were compared to those obtained for 20 healthy controls. In 2 patients with severe P2Y₁₂ deficiency, closure times of INNOVANCE PFA P2Y and collagen/ADP cartridges were >300 s, while those of collagen/epinephrine cartridge were variable (186s and >300 s). In the patient with dysfunctional P2Y₁₂, closure time of INNOVANCE PFA P2Y was >300 s, while closure times of collagen/ADP and collagen/epinephrine were normal. Closure times of all cartridges were normal in the patient with heterozygous P2Y₁₂ deficiency. Our study provides the first evidence that INNOVANCE PFA P2Y cartridge is sensitive to congenital severe and moderate defects of the platelet P2Y₁₂ receptors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. P2Y{sub 12} Platelet Receptors: Importance in Percutaneous Coronary Intervention

    Energy Technology Data Exchange (ETDEWEB)

    Falcão, Felipe Jose de Andrade, E-mail: felipejaf@gmail.com [Universidade Federal de São Paulo - Escola Paulista de Medicina, São Paulo, SP (Brazil); Carvalho, Leonardo; Chan, Mark [National University of Singapore - Yong Loo Lin School of Medicina (Singapore); Alves, Cláudia Maria Rodrigues; Carvalho, Antônio Carlos Camargo; Caixeta, Adriano Mendes [Universidade Federal de São Paulo - Escola Paulista de Medicina, São Paulo, SP (Brazil)

    2013-09-15

    Apart from their role in hemostasis and thrombosis, platelets are involved in many other biological processes such as wound healing and angiogenesis. Percutaneous coronary intervention is a highly thrombogenic procedure inducing platelets and monocytes activation through endothelial trauma and contact activation by intravascular devices. Platelet P2Y{sub 12} receptor activation by adenosine diphosphate facilitates non-ADP agonist-mediated platelet aggregation, dense granule secretion, procoagulant activity, and the phosphorylation of several intraplatelet proteins, making it an ideal drug target. However, not all compounds that target the P2Y{sub 12} receptor have similar efficacy and safety profiles. Despite targeting the same receptor, the unique pharmacologic properties of each of these P2Y{sub 12} receptor-directed compounds can lead to very different clinical effects.

  11. Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis

    DEFF Research Database (Denmark)

    Jürgensen, Henrik J; Johansson, Kristina; Madsen, Daniel H

    2014-01-01

    Members of the well-conserved mannose receptor (MR) protein family have been functionally implicated in diverse biological and pathological processes. Importantly, a proposed common function is the internalization of collagen for intracellular degradation occurring during bone development, cancer...... invasion, and fibrosis protection. This functional relationship is suggested by a common endocytic capability and a candidate collagen-binding domain. Here we conducted a comparative investigation of each member's ability to facilitate intracellular collagen degradation. As expected, the family members u......PARAP/Endo180 and MR bound collagens in a purified system and internalized collagens for degradation in cellular settings. In contrast, the remaining family members, PLA2R and DEC-205, showed no collagen binding activity and were unable to mediate collagen internalization. To pinpoint the structural elements...

  12. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    Science.gov (United States)

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  13. A role for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in collagen biosynthesis in mouse skin

    Science.gov (United States)

    Markiewicz, Margaret; Znoyko, Sergey; Stawski, Lukasz; Ghatnekar, Angela; Gilkeson, Gary; Trojanowska, Maria

    2012-01-01

    Hormonal regulation of the dermal collagenous extracellular matrix plays a key role in maintaining proper tissue homeostasis, however the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking ERα or ERβ. Collagen content was significantly increased in the skin of ΕRα-/- mice as measured by acetic acid extraction and the hydroxyproline assay and correlated with the decreased levels of MMP-15 and elevated collagen production by ΕRα-/- fibroblasts. In contrast, collagen content was decreased in the skin of ERβ-/- mice despite markedly increased collagen production by ERβ-/- fibroblasts. However, expression of several matrix metalloproteinases (MMPs), including MMP-8 and -15 was significantly elevated suggesting increased degradation of dermal collagen. Furthermore, ERβ-/- mice were characterized by significantly reduced levels of small leucine proteoglycans (SLRPs), lumican and decorin, leading to the defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα-/- mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of lumican and decorin. Together, these results demonstrate distinct functions of estrogen receptors in the regulation of collagen biosynthesis in mouse skin in vivo. PMID:22895361

  14. Human platelets express Toll-like receptor 3 and respond to poly I:C.

    Science.gov (United States)

    Anabel, Antonio-Santos; Eduardo, Pérez-Campos; Pedro Antonio, Hernández-Cruz; Carlos, Solórzano-Mata; Juana, Narváez-Morales; Honorio, Torres-Aguilar; Nicolás, Villegas-Sepúlveda; Sergio Roberto, Aguilar-Ruiz

    2014-12-01

    Platelets functions in hemostasis have been widely studied. Currently, growing evidence shows that platelets have also a role in the immune innate response. Recently, protein expression of Toll-like receptors (TLR's) 2, 4, 7, 8, and 9, and the presence of TLRs 1 and 6 mRNA in human platelets was described. Up to now the functionality of TLR-2, 4 and 9 in human platelets has been demonstrated. Due to the relevance of TLRs functions to PAMPS (pathogen-associated molecular patterns) recognizing, we evaluated the presence of TLR3 in human platelets founding low percentages of platelets expressing surface or intracellular TLR3 protein. The activation with thrombin induced an increase in the percentage of platelets expressing surface TLR3 and higher levels of TLR3 expression in the whole population. Human platelets responded to poly I:C by increasing [Ca(2+)]i, the percentages of cells expressing TLR4 and CD62P, and by releasing CXCL4 and IL-1β in comparison to unstimulated platelets. These results demonstrate that human platelets express TLR3 and are capable of responding to poly I:C, suggesting that these cells might influence the immune innate response when detecting viral dsRNA. Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  15. Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in diabetic patients: the PLavix Use for Treatment Of Diabetes (PLUTO-Diabetes) trial.

    Science.gov (United States)

    Serebruany, Victor L; Malinin, Alex I; Pokov, Alex; Barsness, Gregory; Hanley, Dan F

    2008-01-01

    Clopidogrel is widely used in diabetic patients after vascular events; however, the ability of this thienopyridine to yield additional antiplatelet protection on top of aspirin has never been explored in a controlled study with comprehensive assessment of platelet activity. The objective of this study was to compare the antiplatelet profiles of clopidogrel + aspirin in combination (C + ASA) versus aspirin alone (ASA) in patients with type 2 diabetes mellitus. Seventy patients with documented diabetes already treated with antecedent aspirin were randomly assigned to receive C + ASA or ASA in the PLUTO-Diabetes trial. Platelet studies included adenosine diphosphate-, collagen-, and arachidonic acid-induced aggregometry; PFA-100 (Dade-Behring, Miami, FL) and Ultegra (Accumetrics, San Diego, CA) analyzers; and expression of 6 major receptors by flow cytometry at baseline and at day 30 after randomization. There were no differences in the baseline clinical and platelet characteristics between the C + ASA and ASA groups, or subsequent significant changes in platelet biomarkers in the ASA group, except for diminished collagen-induced aggregation (P = .02). In contrast, when compared with the ASA group, therapy with C + ASA resulted in significant inhibition of platelet activity assessed by adenosine diphosphate aggregation (P = .0001); closure time prolongation (P = .0003) and reduction of platelet activation units with Ultegra (P = .0001); and expression of platelet/endothelial cell adhesion molecule 1 (P = .002), glycoprotein IIb/IIIa antigen (P = .0002), and activity (P = .0001). Treatment with C + ASA for 1 month provides significantly greater inhibition of platelet activity than ASA alone in diabetic patients in this small randomized trial. However, despite dual antiplatelet regimen, diabetic patients exhibit high residual activity of some platelet biomarkers, including unaffected protease-activated receptor 1 receptor expression.

  16. Cell-collagen interactions : the use of peptide Toolkits to investigate collagen-receptor interactions

    NARCIS (Netherlands)

    Farndale, Richard W.; Lisman, Ton; Bihan, Dominique; Hamaia, Samir; Smerling, Christiane S.; Pugh, Nicholas; Konitsiotis, Antonios; Leitinger, Birgit; de Groot, Philip G.; Jarvis, Gavin E.; Raynal, Nicolas

    2008-01-01

    Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptide

  17. Platelet Adhesion to Podoplanin Under Flow is Mediated by the Receptor CLEC-2 and Stabilised by Src/Syk-Dependent Platelet Signalling

    Science.gov (United States)

    Pollitt, Alice Y.; Lowe, Kate; Latif, Arusa; Nash, Gerard B.

    2015-01-01

    Summary Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice. PMID:25694214

  18. The collagen receptor DDR2 is expressed during early cardiac development.

    Science.gov (United States)

    Goldsmith, Edie C; Zhang, Xiadong; Watson, James; Hastings, Josh; Potts, Jay D

    2010-05-01

    Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase which has been shown to regulate cell migration upon binding its ligand, collagen. Expression studies determined that DDR2 mRNA and protein are present in the atrioventricular canal during epithelial-mesenchymal transformation (EMT) and the receptor is expressed in both activated endothelial and migrating mesenchymal cells in vivo.

  19. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    Science.gov (United States)

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  20. Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

    Science.gov (United States)

    Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid

    2016-05-03

    Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

  1. IVBT-documented platelet function correlates with flow cytometric data.

    Science.gov (United States)

    Hoffmann, J; Bonacker, G; Kretschmer, V; Schulzki, T; Heimanns, J

    1996-12-01

    Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (fibrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate time (ST). The thrombocytes with the most impaired function (OT > or = 485 s/ST > 30 min) had significantly less platelet fluorescence when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets/microliters a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the IVBT-documented platelet function clearly corresponded to an increased expression of the collagen receptor and the thrombin receptor of platelets.

  2. Lysyl oxidase is associated with increased thrombosis and platelet reactivity.

    Science.gov (United States)

    Matsuura, Shinobu; Mi, Rongjuan; Koupenova, Milka; Eliades, Alexia; Patterson, Shenia; Toselli, Paul; Thon, Jonathan; Italiano, Joseph E; Trackman, Philip C; Papadantonakis, Nikolaos; Ravid, Katya

    2016-03-17

    Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2β1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

  3. Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2.

    Science.gov (United States)

    Leitinger, Birgit

    2003-05-09

    The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.

  4. The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses.

    Science.gov (United States)

    Moscardó, A; Vallés, J; Latorre, A; Santos, M T

    2014-08-25

    We have investigated the presence of thromboxane A2 (TXA2) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA2 receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA2 analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as αIIbβ3 integrin activation and P-selectin exposure. Raft disruption also inhibited TXA2-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y12 receptor. An important proportion of TXA2 receptor (40%) was colocalized at lipid rafts. The presence of the TXA2 receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA2.

  5. The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors

    Directory of Open Access Journals (Sweden)

    Gianni Francesco Guidetti

    2012-01-01

    Full Text Available Integrins and other families of cell adhesion receptors are responsible for platelet adhesion and aggregation, which are essential steps for physiological haemostasis, as well as for the development of thrombosis. The modulation of platelet adhesive properties is the result of a complex pattern of inside-out and outside-in signaling pathways, in which the members of the Rap family of small GTPases are bidirectionally involved. This paper focuses on the regulation of the main Rap GTPase expressed in circulating platelets, Rap1b, downstream of adhesion receptors, and summarizes the most recent achievements in the investigation of the function of this protein as regulator of platelet adhesion and thrombus formation.

  6. Phagocytosis of Collagen Fibrils by Fibroblasts In Vivo is Independent of the uPARAP/Endo180 Receptor

    DEFF Research Database (Denmark)

    Sprangers, Sara; Behrendt, Niels; Engelholm, Lars

    2017-01-01

    As a crucial step in ECM remodeling, collagen degradation occurs through different processes, including both extracellular and intracellular degradation. The extracellular pathways of collagen degradation require secretion of collagenolytic proteases, whereas intracellular collagen degradation...... occurs in the lysosomal compartment after uptake, involving either pre-cleaved or intact fibrillar collagen. The endocytic collagen receptor uPARAP/Endo180 plays an important role in internalization of large collagen degradation products, whereas its role in the phagocytosis of fibrillar collagen has...... been debated. In fact, the role of this receptor in regular collagen phagocytosis in vivo has not been established. In this study, we have studied the role of uPARAP in the phagocytosis of collagen fibrils in vivo by analyzing different connective tissues of mice lacking uPARAP. Using transmission...

  7. HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi.

    Science.gov (United States)

    Ahrens, Ingo; Chen, Yung-Chih; Topcic, Danijal; Bode, Michael; Haenel, David; Hagemeyer, Christoph E; Seeba, Hannah; Duerschmied, Daniel; Bassler, Nicole; Jandeleit-Dahm, Karin A; Sweet, Matthew J; Agrotis, Alex; Bobik, Alex; Peter, Karlheinz

    2015-11-01

    High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

  8. Role of platelet-derived growth factor/platelet-derived growth factor receptor axis in the trafficking of circulating fibrocytes in pulmonary fibrosis.

    Science.gov (United States)

    Aono, Yoshinori; Kishi, Masami; Yokota, Yuki; Azuma, Momoyo; Kinoshita, Katsuhiro; Takezaki, Akio; Sato, Seidai; Kawano, Hiroshi; Kishi, Jun; Goto, Hisatsugu; Uehara, Hisanori; Izumi, Keisuke; Nishioka, Yasuhiko

    2014-12-01

    Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

  9. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

    DEFF Research Database (Denmark)

    Bergmeier, W; Bouvard, D; Eble, J A

    2001-01-01

    Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the v...

  10. The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review).

    Science.gov (United States)

    Melander, Maria C; Jürgensen, Henrik J; Madsen, Daniel H; Engelholm, Lars H; Behrendt, Niels

    2015-10-01

    The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important pathological functions of uPARAP/Endo180 have been identified in various cancers and in several fibrotic conditions. With a particular focus on matrix turnover in cancer, this review presents the necessary background for understanding the function of uPARAP/Endo180 at the molecular and cellular level, followed by an in-depth survey of the available knowledge of the expression and role of this receptor in various types of cancer and other degenerative diseases.

  11. Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

    Science.gov (United States)

    Gündüz, Dursun; Tanislav, Christian; Sedding, Daniel; Parahuleva, Mariana; Santoso, Sentot; Troidl, Christian; Hamm, Christian W.; Aslam, Muhammad

    2017-01-01

    Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

  12. OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice

    DEFF Research Database (Denmark)

    Barrow, Alexander David; Raynal, Nicolas; Levin Andersen, Thomas

    2011-01-01

    -activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined. Osteoclast-associated receptor (OSCAR) was proposed to be a potent FcRγ-associated costimulatory receptor expressed......, we found that OSCAR binds to specific motifs within fibrillar collagens in the ECM that become revealed on nonquiescent bone surfaces in which osteoclasts undergo maturation and terminal differentiation in vivo. OSCAR promoted osteoclastogenesis in vivo, and OSCAR binding to its collagen motif led...... to signaling that increased numbers of osteoclasts in culture. Thus, our results suggest that ITAM-containing receptors can respond to exposed ligands in collagen, leading to the functional differentiation of leukocytes, which provides what we believe to be a new concept for ITAM regulation of cytokine...

  13. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    Energy Technology Data Exchange (ETDEWEB)

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-07-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

  14. TRA-418, a novel compound having both thromboxane A(2) receptor antagonistic and prostaglandin I(2) receptor agonistic activities: its antiplatelet effects in human and animal platelets.

    Science.gov (United States)

    Yamada, N; Miyamoto, M; Isogaya, M; Suzuki, M; Ikezawa, S; Ohno, M; Otake, A; Umemura, K

    2003-08-01

    TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 micro g kg min-1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.

  15. A critical role for the transient receptor potential channel type 6 in human platelet activation.

    Directory of Open Access Journals (Sweden)

    Hari Priya Vemana

    Full Text Available While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6 mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules, integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.

  16. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors

    OpenAIRE

    Jones, M.L.; Norman, J E; Morgan, N. V.; Mundell, S J; Lordkipanidze, M.; Lowe, G. C.; Daly, M E; Simpson, M.A.; Drake, S.; Watson, S P; Mumford, A D; UKGAPPS,

    2016-01-01

    Platelet responses to activating agonists are influenced by common\\ud population variants within or near G protein-coupled receptor (GPCR)\\ud genes that affect receptor activity. However, the impact of rare GPCR\\ud gene variants is unknown. We describe the rare single nucleotide variants\\ud (SNVs) in the coding and splice regions of 18 GPCR genes in\\ud 7,595 exomes from the 1,000-genomes and Exome Sequencing\\ud Project databases and in 31 cases with inherited platelet function disorders\\ud (I...

  17. Human thromboxane A2 receptor genetic variants: in silico, in vitro and "in platelet" analysis.

    Directory of Open Access Journals (Sweden)

    Scott Gleim

    Full Text Available Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity or promote (hyperactivity vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C(68S, V(80E, E(94V, A(160T, V(176E, and V(217I for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C(68S to normal (V(217I, with most variants demonstrating functional alteration in binding, expression or activation (V(80E, E(94V, A(160T, and V(176E. In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.

  18. Exogenous L-arginine and HDL can alter LDL and ox-LDL-mediated platelet activation: using platelet P-selectin receptor numbers.

    Science.gov (United States)

    Sener, Azize; Enc, Elif; Ozsavci, Derya; Vanizor-Kural, Birgul; Yanikkaya-Demirel, Gulderen; Oba, Rabia; Uras, Fikriye; Demir, Muzaffer

    2011-01-01

    The aim of this study is to investigate the effects of exogenous L-arginine and HDL on LDL and oxidized LDL (ox-LDL)-mediated platelet activation. Adenosine diphosphate (ADP)-activated platelets have been incubated with lipoproteins with or without L-arginine. P-selectin receptor numbers per platelet have been measured by flow cytometry. After incubation with only L-arginine (without lipoproteins), platelet nitric oxide (NO) levels and P-selectin receptor numbers significantly increased compared to the controls (P LDL or ox-LDL, receptor numbers of P-selectin significantly increased (P LDL or L-arginine + ox-LDL decreased compared to the levels in platelets treated with only LDL or ox-LDL (P HDL to L-arginine + ox-LDL caused significant reduction in P-selectin receptor numbers as in the control values (P LDL or ox-LDL on platelet P-selectin receptor numbers and HDL also strengthens this effect of L-arginine.

  19. Function of eltrombopag-induced platelets compared to platelets from control patients with immune thrombocytopenia.

    Science.gov (United States)

    Haselboeck, Johanna; Kaider, Alexandra; Pabinger, Ingrid; Panzer, Simon

    2013-04-01

    Data on the in vivo function of platelets induced by the thrombopoietin receptor agonist eltrombopag are scarce. To assess a possible influence of eltrombopag we compared platelet function of eltrombopag-treated immune thrombocytopenia (ITP) patients (group 1; n=10) after treatment response to that from control ITP patients (group 2; n=12). We further analysed platelet function at baseline and after one, three, and four weeks of eltrombopag treatment and estimated daily changes of platelet function during the eltrombopag-induced platelet rise. The formation of platelet-monocyte aggregates (PMA), P-selectin expression [MFI], and platelet adhesion under high shear conditions (surface coverage, SC) in vivo and after in vitro addition of agonists (ADP, TRAP-6, Collagen) were similar between both groups after response to eltrombopag treatment. Only TRAP-6 induced a lower SC in the eltrombopag group (p=0.03). All platelet function parameters except for Collagen-induced P-selectin expression changed significantly during treatment with eltrombopag. PMA, naïve and after addition of ADP or TRAP-6 increased with increasing platelet counts. P-selectin expression decreased, when measured without and upon addition of ADP, increased in the presence of TRAP-6, and remained unchanged after addition of Collagen. SC increased during the eltrombopag-induced platelet rise. All significant changes of platelet function correlated to changes in platelet counts. Two patients developed venous thromboses during eltrombopag treatment, but no association with any distinct single platelet function parameter or combinations thereof was identifiable. Thus, eltrombopag-induced platelets function similar to those from control ITP patients without discernible increased hyper-reactivity.

  20. Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices

    Directory of Open Access Journals (Sweden)

    Ruiz Pedro A

    2012-02-01

    Full Text Available Abstract Background The two discoidin domain receptors (DDRs, DDR1 and DDR2 are receptor tyrosine kinases (RTKs with the unique ability among RTKs to respond to collagen. We have previously shown that collagen I induces DDR1 and matrix metalloproteinase (MMP-10 expression through DDR2 activation and a Janus kinase (JAK2 and extracellular signal-regulated kinase (ERK1/2-mediated mechanism in primary human lung fibroblasts suggesting that these signaling pathways play a role in fibroblast function. Fibroblasts can traverse basement membrane barriers during development, wound healing and pathological conditions such as cancer and fibrosis by activating tissue-invasive programs, the identity of which remain largely undefined. In the present work, we investigated the role of DDRs and DDR-associated signal transduction in these processes. Results Transwell migration experiments showed that normal human lung fibroblast (NHLF transmigration through collagen I-coated inserts is mediated by DDR2 and the DDR2-associated signaling kinases JAK2 and ERK1/2, but not DDR1. Additionally, experiments with specific small interfering (siRNAs revealed that collagen I-induced expression of MMP-10 and MMP-2 is DDR2 but not DDR1 dependent in NHLFs. Our data showed that collagen I increases NHLF migration through collagen IV, the main component of basement membranes. Furthermore, basal and collagen I-induced NHLF migration through collagen IV-coated inserts was both DDR2 and DDR1 dependent. Finally, DDR2, but not DDR1 was shown to be involved in fibroblast proliferation. Conclusions Our results suggest a mechanism by which the presence of collagen I in situations of excessive matrix deposition could induce fibroblast migration through basement membranes through DDR2 activation and subsequent DDR1 and MMP-2 gene expression. This work provides new insights into the role of DDRs in fibroblast function.

  1. The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2.

    Science.gov (United States)

    Leitinger, Birgit; Steplewski, Andrzej; Fertala, Andrzej

    2004-12-03

    The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.

  2. Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo

    DEFF Research Database (Denmark)

    Kuijpers, Marijke J E; Schulte, Valerie; Bergmeier, Wolfgang

    2003-01-01

    Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated...... in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function...... in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI...

  3. TRA-418, a thromboxane A2 receptor antagonist and prostacyclin receptor agonist, inhibits platelet-leukocyte interaction in human whole blood.

    Science.gov (United States)

    Miyamoto, Mitsuko; Ohno, Michihiro; Yamada, Naohiro; Ohtake, Atsushi; Matsushita, Teruo

    2010-10-01

    TRA-418, a compound with both thromboxane A2 receptor (TP receptor) antagonistic and prostacyclin receptor (IP receptor) agonistic activities, was synthesised in our laboratory as a new antithrombotic agent. In this study, we examined the effects of TRA-418 on platelet-leukocyte interactions in human whole blood. Platelet-leukocyte interactions were induced by U-46619 in the presence of epinephrine (U-46619 + epinephrine) or with thrombin receptor agonist peptide 1-6 (TRAP). Platelet-leukocyte interactions were assessed by flow cytometry, with examination of both platelet-neutrophil and platelet-monocyte complexes. In a control experiment, the TP receptor antagonist SQ-29548 significantly inhibited the induction of platelet-leukocyte complexes by the combination of U-46619 and epinephrine, but not TRAP-induced formation of platelet-leukocyte complexes. Conversely, the IP receptor agonist beraprost sodium inhibited platelet-leukocyte complex formation induced by both methods, although the IC50 values of beraprost sodium for U-46619 + epinephrine were at least 10-fold greater than for TRAP. Under such conditions, TRA-418 inhibited both U-46619 + epinephrine-induced and TRAP-induced platelet-leukocyte complex formation in a concentration-dependent manner, in a similar range. These results suggest that TRA-418 exerts its inhibitory effects on platelet-leukocyte interactions by acting as a TP receptor antagonist as well as an IP receptor agonist in an additive or synergistic manner. These inhibitory effects of TRA-418 on formation of platelet-leukocyte complexes suggest the compound is beneficial effects as an antithrombotic agent.

  4. Pharmacological Characterization of Inositol 1,4,5-tris Phosphate Receptors in Human Platelet Membranes

    Directory of Open Access Journals (Sweden)

    Yogesh Dwivedi

    2009-01-01

    Full Text Available The phosphatidylinositol (PI hydrolysis signaling system has been shown to be altered in platelets of depressed and schizophrenic subjects. Inositol (1,4,5 trisphosphate (Ins(1,4,5P3, an integral component of the PI signaling system, mobilizes Ca2+ by activating Ins(1,4,5P3 receptors. To eventually investigate the role of Ins(1,4,5P3 receptors in depression and other mental disorders, we characterized [H3]Ins(1,4,5P3 binding sites in crude platelet membranes prepared from small amounts of blood obtained from healthy human control subjects. We found a single, saturable binding site for [H3]Ins(1,4,5P3 to crude platelet membranes, which is time dependent and modulated by pH, inositol phosphates, and heparin. Since cyclic adenosine monophosphate (cAMP and Ca2+ have been shown to be important modulators in Ins(1,4,5P3 receptors, in the present study we also determined the effects of various concentrations of CaCI2 and forskolin on Ins(1,4,5P3 binding to platelet membranes. CaCI2 modulated [3H]Ins(1,4,5P3 binding sites in a biphasic manner: at lower concentrations it inhibited [3H]Ins(1,4,5P3 binding, whereas at higher concentrations, it stimulated [3H]Ins(1,4,5P3 binding. On the other hand, forskolin inhibited [3H]Ins(1,4,5P3 binding. Our results thus suggest that the pharmacological characteristics of [3H]Ins(1,4,5P3 binding to crude platelet membranes are similar to that of Ins(1,4,5P3 receptors; and that both Ca2+ and cAMP modulate [3H]Ins(1,4,5P3 binding in crude platelet membranes.

  5. The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen/LAIR-1 inhibitory immune interaction

    NARCIS (Netherlands)

    Lebbink, R.J.|info:eu-repo/dai/nl/318915006; van den Berg, M.C.; de Ruiter, T.; Raynal, N.; van Roon, J.A.G.|info:eu-repo/dai/nl/158985826; Lenting, P.J.; Jin, B.; Meyaard, L.|info:eu-repo/dai/nl/13444972X

    2008-01-01

    Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human

  6. Cystamine immobilization on TiO{sub 2} film surfaces and the influence on inhibition of collagen-induced platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Yujuan [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Weng Yajun, E-mail: wengyj7032@sohu.com [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhang Liping; Jing Fengjuan; Huang Nan [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Chen Junying, E-mail: chenjy@263.net [Key Lab. of Advanced Technology for Materials of Chinese Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2011-12-15

    Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO{sub 2} films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO{sub 2} films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

  7. SIRT1 prevents pulmonary thrombus formation induced by arachidonic acid via downregulation of PAF receptor expression in platelets.

    Science.gov (United States)

    Kim, Yun Hak; Bae, Jin Ung; Kim, In Suk; Chang, Chulhun L; Oh, Sae Ock; Kim, Chi Dae

    2016-12-01

    SIRT1, a class III histone deacetylase, is critically involved in cellular response to stress and modulates cardiovascular risk factors. However, its role in thrombus formation is largely unknown. Thus, this study investigated the effect of SIRT1 on pulmonary thrombus formation, and then identified its role in the modulation of platelet aggregation. In isolated human platelets, cell aggregation was increased by various platelet activators, such as platelet activating factor (PAF), arachidonic acid (AA), ADP, and thrombin. AA- and PAF-mediated platelet aggregations were suppressed by WEB2086, a PAF receptor (PAFR) antagonist. Pulmonary thrombus formation induced by PAF or AA was also attenuated by WEB2086, suggesting that PAFR plays a key role in AA-induced platelet aggregation. In platelets isolated from SIRT1-TG mice as well as in platelets treated with resveratrol or reSIRT1, PAFR expression was decreased, whereas this expressional downregulation by SIRT1 activators was inhibited in platelets treated with MG132 (a proteasome inhibitor) or NH4Cl (a lysosome inhibitor). Furthermore, platelet aggregation induced by AA was markedly attenuated by resveratrol and reSIRT1. Likewise, the increased pulmonary thrombus formation in mice treated with AA was also attenuated by SIRT1 activators. In line with these results, pulmonary thrombus formation was markedly attenuated in SIRT1-TG mice. Taken together, this study showed that SIRT1 downregulates PAFR expression on platelets via proteasomal and lysosomal pathways, and that this downregulation inhibits platelet aggregation in vitro and pulmonary thrombus formation in vivo.

  8. Current Role of Platelet Glycoprotein Ⅱ b/Ⅲ a Receptor Inhibitors in Clinic-al Trials of Cardiovascular Diseases

    Institute of Scientific and Technical Information of China (English)

    SHEN Di

    2001-01-01

    @@Thrombosis formation on disrupted atherosclerotic plaque is the most common acuse of cardiovascular dis eases, in the pathophysiology, increased platelet reactiv ity is a descriptor of the risk of cardiovascular events in healthy persons and in patients with overt coronary artery disease. Regardless of the stimulus for activation platelet-platelet interation and thrombus formation is ul timately regulated through the GP Ⅱ b/Ⅲ a receptor complex

  9. Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe

    2007-01-01

    The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...... in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis...

  10. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.

    Science.gov (United States)

    Rigg, Rachel A; Healy, Laura D; Nowak, Marie S; Mallet, Jérémy; Thierheimer, Marisa L D; Pang, Jiaqing; McCarty, Owen J T; Aslan, Joseph E

    2016-04-01

    Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.

  11. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  12. Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture.

    Science.gov (United States)

    Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

    2011-03-01

    The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

  13. Platelet Function Tests in Bleeding Disorders.

    Science.gov (United States)

    Lassila, Riitta

    2016-04-01

    Functional disorders of platelets can involve any aspect of platelet physiology, with many different effects or outcomes. These include platelet numbers (thrombocytosis or thrombocytopenia); changes in platelet production or destruction, or capture to the liver (Ashwell receptor); altered adhesion to vascular injury sites and/or influence on hemostasis and wound healing; and altered activation or receptor functions, shape change, spreading and release reactions, procoagulant and antifibrinolytic activity. Procoagulant membrane alterations, and generation of thrombin and fibrin, also affect platelet aggregation. The above parameters can all be studied, but standardization and quality control of assay methods have been limited despite several efforts. Only after a comprehensive clinical bleeding assessment, including family history, information on drug use affecting platelets, and exclusion of coagulation factor, and tissue deficits, should platelet function testing be undertaken to confirm an abnormality. Current diagnostic tools include blood cell counts, platelet characteristics according to the cell counter parameters, peripheral blood smear, exclusion of pseudothrombocytopenia, whole blood aggregometry (WBA) or light transmission aggregometry (LTA) in platelet-rich plasma, luminescence, platelet function analysis (PFA-100) for platelet adhesion and deposition to collagen cartridges under blood flow, and finally transmission electron microscopy to exclude rare structural defects leading to functional deficits. The most validated test panels are included in WBA, LTA, and PFA. Because platelets are isolated from their natural environment, many simplifications occur, as circulating blood and interaction with vascular wall are omitted in these assays. The target to reach a highly specific platelet disorder diagnosis in routine clinical management can be exhaustive, unless needed for genetic counseling. The elective overall assessment of platelet function disorder

  14. Validation of a P2Y12-receptor specific whole blood platelet aggregation assay.

    Science.gov (United States)

    Amann, Michael; Ferenc, Miroslaw; Valina, Christian M; Bömicke, Timo; Stratz, Christian; Leggewie, Stefan; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-11-01

    Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen's κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

  15. Platelet-derived growth factors and their receptors in normal and malignant hematopoiesis

    Science.gov (United States)

    Demoulin, Jean-Baptiste; Montano-Almendras, Carmen P.

    2012-01-01

    Platelet-derived growth factors (PDGF) bind to two closely related receptor tyrosine kinases, PDGF receptor α and β, which are encoded by the PDGFRA and PDGFRB genes. Aberrant activation of PDGF receptors occurs in myeloid malignancies associated with hypereosinophilia, due to chromosomal alterations that produce fusion genes, such as ETV6-PDGFRB or FIP1L1-PDGFRA. Most patients are males and respond to low dose imatinib, which is particularly effective against PDGF receptor kinase activity. Recently, activating point mutations in PDGFRA were also described in hypereosinophilia. In addition, autocrine loops have been identified in large granular lymphocyte leukemia and HTLV-transformed lymphocytes, suggesting new possible indications for tyrosine kinase inhibitor therapy. Although PDGF was initially purified from platelets more than 30 years ago, its physiological role in the hematopoietic system remains unclear. Hematopoietic defects in PDGF-deficient mice have been reported but appear to be secondary to cardiovascular and placental abnormalities. Nevertheless, PDGF acts directly on several hematopoietic cell types in vitro, such as megakaryocytes, platelets, activated macrophages and, possibly, certain lymphocyte subsets and eosinophils. The relevance of these observations for normal human hematopoiesis remains to be established. PMID:22432087

  16. Efficacy of the controlled release of concentrated platelet lysate from a collagen/gelatin scaffold for dermis-like tissue regeneration.

    OpenAIRE

    Ito, Ran; Morimoto, Naoki; Pham, Liem Hieu; Taira, Tsuguyoshi; Kawai, Katsuya; Suzuki, Shigehiko

    2013-01-01

    Introduction: A collagen/gelatin scaffold (CGS) can provide a sustained release of basic fibroblast growth factor (bFGF), which promotes wound healing. However, bFGF is approved for clinical use in Japan and China only. One potential alternative to bFGF is platelet lysate (PL), a safe and easily attainable source of a wide range of growth factors necessary for tissue repair. In the present study, we investigated the use of PL with CGS to repair wounds and identified the optimal concentration ...

  17. Effectiveness of Agents Like Platelet-rich Plasma, Oxidized Regenerated Cellulose and Microfibrillar Collagen in Hard-tissue Healing: Validation of t heir Comparative Effectiveness

    Directory of Open Access Journals (Sweden)

    Khalid Al Fouzan

    2015-07-01

    Full Text Available Autologous platelet-rich plasma (PRP can be a valid agent that is effective in inducing and accelerating bone healing for the treatment of periodontal defects and also to accelerate alveolar bone regeneration. Early healing appeared to be impaired by the presence of microfibrillar collagen (MFC and impeded by the presence of oxidized regenerated cellulose (ORC. In contrast, alkylene oxide copolymer (AOC did not inhibit bone healing and suggest that AOC may be a better bone hemo - static material for procedures where bony fusion is critical and immediate hemostasis required

  18. C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets.

    Science.gov (United States)

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol A; Kunapuli, Satya P

    2015-01-15

    C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

  19. Diversity and Impact of Rare Variants in Genes Encoding the Platelet G Protein-Coupled Receptors

    Science.gov (United States)

    Jones, Matthew L.; Norman, Jane E.; Morgan, Neil V.; Mundell, Stuart J.; Lordkipanidzé, Marie; Lowe, Gillian C.; Daly, Martina E.; Simpson, Michael A.; Drake, Sian; Watson, Steve P.

    2015-01-01

    Summary Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes. PMID:25567036

  20. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

    Science.gov (United States)

    Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

    2015-04-01

    Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

  1. Collagen advanced glycation inhibits its Discoidin Domain Receptor 2 (DDR2)-mediated induction of lysyl oxidase in osteoblasts.

    Science.gov (United States)

    Khosravi, Roozbeh; Sodek, Katharine L; Faibish, Michael; Trackman, Philip C

    2014-01-01

    Diabetes increases the risk of bone fracture. Organic and inorganic bone extracellular matrix components determine bone strength. Previous studies indicate that in diabetes, glycation of collagen causes abnormal arrangements of collagen molecules and fragile bones. Diabetic bone fragility is additionally attributed to reduced levels of lysyl oxidase enzyme-dependent collagen cross-links. The mechanism underlying the presence of lower enzymatic collagen cross-links in diabetic bone has not been directly investigated. Here we determine in primary osteoblast cultures the regulation of lysyl oxidase protein by type I collagen and collagen modified by carboxymethylation (CML-collagen), a form of advanced glycation endproducts. Data indicate that non-glycated collagen up-regulates lysyl oxidase levels both in primary non-differentiated and in differentiating mouse and rat osteoblast cultures, while CML-collagen fails to regulate lysyl oxidase in these cells. Collagen binding to Discoidin Domain Receptor-2 (DDR2) mediates lysyl oxidase increases, determined in DDR2 shRNA knockdown studies. DDR2 binding and activation were disrupted by collagen glycation, pointing to a mechanism for the diminished levels of lysyl oxidase and consequently low lysyl oxidase-derived cross-links in diabetic bone. Our studies indicate that collagen-integrin interactions may not play a major role in up-regulating lysyl oxidase. Furthermore, non-collagenous ligands for the receptor for advanced glycation end products (RAGE) failed to alter lysyl oxidase levels. Taken together with published studies a new understanding emerges in which diabetes- and age-dependent inhibition of normal collagen-stimulated DDR2- and integrin-signaling, and independent advanced glycation-stimulated RAGE-signaling, each contributes to different aspects of diabetic osteopenia.

  2. Inhibitory effects of peroxisome proliferator-activated receptor γ agonists on collagen IV production in podocytes.

    Science.gov (United States)

    Li, Yanjiao; Shen, Yachen; Li, Min; Su, Dongming; Xu, Weifeng; Liang, Xiubin; Li, Rongshan

    2015-07-01

    Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists have beneficial effects on the kidney diseases through preventing microalbuminuria and glomerulosclerosis. However, the mechanisms underlying these effects remain to be fully understood. In this study, we investigate the effects of PPAR-γ agonist, rosiglitazone (Rosi) and pioglitazone (Pio), on collagen IV production in mouse podocytes. The endogenous expression of PPAR-γ was found in the primary podocytes and can be upregulated by Rosi and Pio, respectively, detected by RT-PCR and Western blot. PPAR-γ agonist markedly blunted the increasing of collagen IV expression and extraction in podocytes induced by TGF-β. In contrast, adding PPAR-γ antagonist, GW9662, to podocytes largely prevented the inhibition of collagen IV expression from Pio treatment. Our data also showed that phosphorylation of Smad2/3 enhanced by TGF-β in a time-dependent manner was significantly attenuated by adding Pio. The promoter region of collagen IV gene contains one putative consensus sequence of Smad-binding element (SBE) by promoter analysis, Rosi and Pio significantly ameliorated TGF-β-induced SBE4-luciferase activity. In conclusion, PPAR-γ activation by its agonist, Rosi or Pio, in vitro directly inhibits collagen IV expression and synthesis in primary mouse podocytes. The suppression of collagen IV production was related to the inhibition of TGF-β-driven phosphorylation of Smad2/3 and decreased response activity of SBEs of collagen IV in PPAR-γ agonist-treated mouse podocytes. This represents a novel mechanistic support regarding PPAR-γ agonists as podocyte protective agents.

  3. Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

    Science.gov (United States)

    Ntokou, Aglaia; Klein, Friederike; Dontireddy, Daria; Becker, Sven; Bellusci, Saverio; Richardson, William D; Szibor, Marten; Braun, Thomas; Morty, Rory E; Seeger, Werner; Voswinckel, Robert; Ahlbrecht, Katrin

    2015-11-01

    A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

  4. Treatment of life-threatening wounds with a combination of allogenic platelet-rich plasma, fibrin glue and collagen matrix, and a literature review.

    Science.gov (United States)

    Asadi, Mehdi; Alamdari, Daryoush Hamidi; Rahimi, Hamid Reza; Aliakbarian, Mohsen; Jangjoo, Ali; Abdollahi, Abbas; Bahar, Mostafa Mehrabi; Azadmand, Ali; Forghani, Naser; Sadegh, Mohammad Nori; Khayamy, Mohammad Esmail; Seifalian, Alexander

    2014-08-01

    Currently there is no ideal procedure for the treatment of recalcitrant ulcers that are unresponsive to the majority of common treatments. However, several novel approaches have been proposed, including bone marrow stem cells, platelets, fibrin glue and collagen matrix. For the first approach treatment of a chronic wound, a non-invasive method is highly desirable. The present study was undertaken with the aim of evaluating the effect of a combination of platelets, fibrin glue and collagen matrix (PFC) in one treatment. A total of ten patients with aggressive, refractory, life-threatening wounds were recruited for the study and their treatment effects were evaluated. Initially, the ulcers were extensively debrided, measured and photographed at weekly intervals. The PFC combination was applied topically to the wound every two days. Following treatment, the wound was completely closed in nine patients and was markedly reduced in the other patient. The mean 100% healing time for the nine patients was 11.3±5.22 weeks. There was no evidence of local or systemic complications or any abnormal tissue formation, keloid or hypertrophic scarring. Therefore, the results of the present study indicate that in the first approach, the combination of PFC components may be used safely in order to synergize the effect of chronic wound healing.

  5. Preclinical Toxicology Studies of Recombinant Human Platelet-Derived Growth Factor-BB Either Alone or in Combination with Beta-Tricalcium Phosphate and Type I Collagen

    Directory of Open Access Journals (Sweden)

    Conan S. Young

    2010-01-01

    Full Text Available Human platelet-derived growth factor-BB (hPDGF-BB is a basic polypeptide growth factor released from platelets at the injury site. It is a multifunctional molecule that regulates DNA synthesis and cell division and induces biological effects that are implicated in tissue repair, atherosclerosis, inflammatory responses, and neoplastic diseases. This paper is an overview of the toxicology data generated from a broad testing platform to determine bone, soft tissue, and systemic responses following administration of rhPDGF-BB. Moreover, the systemic and local toxicity of recombinant human PDGF-BB (rhPDGF-BB in combination with either beta-tricalcium phosphate (β-TCP or collagen combined with β-TCP was studied to determine dermal sensitization, irritation, intramuscular tissue responses, pyrogenicity, genotoxicity, and hemolytic properties. All data strongly suggest that rhPDGF-BB either alone or in combination with β-TCP or collagen with β-TCP is biocompatible and has neither systemic nor local toxicity, supporting its safe use in enhancing wound healing in patients.

  6. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  7. A comparison of the fibrinogen receptor distribution on adherent platelets using both soluble fibrinogen and fibrinogen immobilized on gold beads.

    Science.gov (United States)

    Estry, D W; Mattson, J C; Mahoney, G J; Oesterle, J R

    1991-04-01

    The distribution of fibrinogen receptors was determined on the surface of adherent platelets using both direct labeling with the ligand fibrinogen which was immobilized on gold particles (Fg-Au) and indirect immunogold (Ig-Au) labeling of bound soluble fibrinogen identified with a rabbit polyclonal anti-fibrinogen antibody. Two distinctly different patterns of labeling were obtained and appeared to depend on whether solid phase fibrinogen (Fg-Au) or soluble phase released fibrinogen were bound to the membrane receptor. The membrane-bound Fg-Au reorganized in patterns that closely mimicked the organization of the underlying cytoskeleton. In approximately 18% of the adherent platelets, Fg-Au was seen in channels or vesicle-like structures lying deep to the platelet surface suggesting internalization into the open canalicular system and/or endocytosis. The labeling pattern obtained when identifying the location of membrane-bound soluble released fibrinogen by Ig-Au was diffuse and lacked the organizational patterns characteristic of Fg-Au. Unlike the Fg-Au probe, early dendritic platelets were heavily labeled by the soluble phase fibrinogen using the Ig-Au technique. Although the label covered the entire exposed platelet membrane in fully spread platelets, labeling over the peripheral web was more dense than that over the intermediate or granulomere zone. The diffuse organization and heavier peripheral distributional pattern of the glycoprotein IIb-IIIa (GP IIb-IIIa) receptor in fixed, adherent platelets, was also seen with the GP IIb-IIIa receptor-specific antibody AP-2. The binding of both the Fg-Au and Ig-Au were inhibited using the tetrapeptide Arg-Gly-Asp-Ser (RGDS) (93% and 98% inhibition, respectively), AP-2 (98% and 97%, respectively) and platelets from patients with Glanzmann's thrombasthenia (GT) (99% and 98%, respectively). The data presented provides the first report that receptor reorganization, following binding of fibrinogen, appears to be related to

  8. Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation.

    Science.gov (United States)

    Miyamoto, Mitsuko; Yamada, Naohiro; Ikezawa, Shiho; Ohno, Michihiro; Otake, Atsushi; Umemura, Kazuo; Matsushita, Teruo

    2003-11-01

    [4-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxy]-acetic acid N-Methyl-d-glucamine salt (TRA-418) has both thromboxane A2 (TP)-receptor antagonist and prostacyclin (IP)-receptor agonist properties. The present study examined the advantageous effects of TRA-418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA-418 and a glycoprotein alphaIIb/beta3 integrin (GPIIb/IIIa) inhibitor. TRA-418 inhibited platelet GPIIb/IIIa activation as well as P-selectin expression induced by adenosine 5'-diphosphate, thrombin receptor agonist peptide 1-6 (Ser-Phe-Leu-Leu-Arg-Asn-NH2), and U-46619 in the presence of epinephrine (U-46619+ epinephrine). TRA-418 also inhibited platelet aggregation induced by those platelet-stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK). The TP-receptor antagonist SQ-29548 inhibited only U-46619+epinephrine-induced GPIIb/IIIa activation, P-selectin expression, and platelet aggregation. The IP-receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK. The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P-selectin expression. TRA-418 is more advantageous as an antiplatelet agent than TP-receptor antagonists or IP-receptor agonists separately used. TRA-418 showed a different inhibitory profile from abciximab in the effects on P-selectin expression.

  9. Molecular interaction studies of hemostasis: fibrinogen ligand-human platelet receptor interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Imshik; Marchant, Roger E

    2003-10-15

    The interactions between fibrinogen ligands and platelet receptor {alpha}{sub IIb}{beta}{sub 3} were studied under physiological conditions by atomic force microscopy (AFM). Two linear peptide sequences in fibrinogen, RGD and HHLGGAKQAGDV, play central roles in the regulation of hemostasis and thrombosis by facilitating adhesion and aggregation of platelets. In order to measure the interactions (i.e., debonding force), oligopeptides, GSSSGaaa, where aaa is -RGDSPA or -HHLGGAKQAGDV, were synthesized and grafted on to the surface of AFM probe tips. The interaction forces between a peptide-modified AFM probe tip and platelet surface were determined from pN to nN levels using AFM force measurements. Our results show that the zero kinetic off-rate, K{sub off}(0), for RGDSPA is significantly smaller than that for HHLGGAKQAGDV, under the consideration of flexible receptor surfaces. From our analysis, the K{sub off}(0), the single molecular binding energy E{sub b}, and the transition state x{sub b}, were extracted from the data, and estimated to be 1.53 s{sup -1}, -2.64x10{sup -20} J and 1.03 A for the RGD-{alpha}{sub IIb}{beta}{sub 3} system, and 47.58 s{sup -1}, 2.67x10{sup -20}, 1.09 A for the HHLGGAKQAGDV-{alpha}{sub IIb}{beta}{sub 3} system, respectively.

  10. The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism.

    Science.gov (United States)

    Labrador, J P; Azcoitia, V; Tuckermann, J; Lin, C; Olaso, E; Mañes, S; Brückner, K; Goergen, J L; Lemke, G; Yancopoulos, G; Angel, P; Martínez, C; Klein, R

    2001-05-01

    The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2-deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2-/- mice exhibit a reduced proliferative response compared with wild-type littermates. In vitro, fibroblasts derived from DDR2-/- mutants proliferate more slowly than wild-type fibroblasts, a defect that is rescued by introduction of wild-type but not kinase-dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation.

  11. Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

    Science.gov (United States)

    Herfindal, Lars; Nygaard, Gyrid; Kopperud, Reidun; Krakstad, Camilla; Døskeland, Stein Ove; Selheim, Frode

    2013-08-09

    The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations.

  12. Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis.

    Directory of Open Access Journals (Sweden)

    Sebastian Hofmann

    Full Text Available BACKGROUND: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation--platelet adhesion and aggregation--have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. OBJECTIVE: The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. METHODS AND RESULTS: We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84(-/- platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84(-/- mice. In vivo, Cd84(-/- mice exhibited unaltered hemostatic function and arterial thrombus formation. CONCLUSION: These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.

  13. The Augmentation of a Collagen/Glycosaminoglycan Biphasic Osteochondral Scaffold with Platelet-Rich Plasma and Concentrated Bone Marrow Aspirate for Osteochondral Defect Repair in Sheep

    Science.gov (United States)

    Henson, Frances; Skelton, Carrie; Herrera, Emilio; Brooks, Roger; Fortier, Lisa A.; Rushton, Neil

    2012-01-01

    Objective: This study investigates the combination of platelet-rich plasma (PRP) or concentrated bone marrow aspirate (CBMA) with a biphasic collagen/glycosaminoglycan (GAG) osteochondral scaffold for the treatment of osteochondral defects in sheep. Design: Acute osteochondral defects were created in the medial femoral condyle (MFC) and the lateral trochlea sulcus (LTS) of 24 sheep (n = 6). Defects were left empty or filled with a 6 × 6-mm scaffold, either on its own or in combination with PRP or CBMA. Outcome measures at 6 months included mechanical testing, International Cartilage Repair Society (ICRS) repair score, modified O’Driscoll histology score, qualitative histology, and immunohistochemistry for type I, II, and VI collagen. Results: No differences in mechanical properties, ICRS repair score, or modified O’Driscoll score were detected between the 4 groups. However, qualitative assessments of the histological architecture, Safranin O content, and collagen immunohistochemistry indicated that in the PRP/scaffold groups, there was a more hyaline cartilage–like tissue repair. In addition, the addition of CBMA and PRP to the scaffold reduced cyst formation in the subchondral bone of healed lesions. Conclusion: There was more hyaline cartilage–like tissue formed in the PRP/scaffold group and less subchondral cystic lesion formation in the CBMA and PRP/scaffold groups, although there were no quantitative differences in the repair tissue formed. PMID:26069645

  14. Purification and reconstitution of the human platelet. cap alpha. /sub 2/-adrenergic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Regan, J.W.; Cerione, R.A.; Nakata, H.; Benovic, J.L.; DeMarinis, R.M.; Caron, M.G.; Lefkowitz, R.J.

    1986-05-01

    Human platelet ..cap alpha../sub 2/-adrenergic receptors have been purified approx.80,000 fold to apparent homogeneity by a five step chromatographic procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M/sub r/ 64,000. The competitive binding of ligands to the purified receptor protein shows the proper ..cap alpha../sub 2/-adrenergic specificity. The ..cap alpha../sub 2/-adrenergic receptor contains an essential sulfhydryl residues. Thus, exposure of the purified receptor to the sulfhydryl specific reagent, phenylmercuric chloride (PMC), resulted in a 80% loss of binding activity. This loss of binding activity was prevented when exposure to PMC was done in the presence of ..cap alpha../sub 2/-adrenergic ligands and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified ..cap alpha../sub 2/-adrenergic receptors was obtained with S. aureus V-8 protease, ..cap alpha..-chymotrypsin and papain. In a comparison with purified ..beta../sub 2/-adrenergic receptors no common partial proteolytic products were found. Partially purified preparations of the ..cap alpha../sub 2/-adrenergic receptor were successfully reconstituted into phospholipid vesicles with the inhibitory guanyl nucleotide-binding regulatory protein, N/sub i/. In these reconstituted preparations, epinephrine could stimulate, and phentolamine could block, the GTPase activity of N/sub i/.

  15. Increased expression of the collagen internalization receptor uPARAP/Endo180 in the stroma of head and neck cancer

    DEFF Research Database (Denmark)

    Sulek, Jay; Wagenaar-Miller, Rebecca A; Shireman, Jessica

    2007-01-01

    Local growth, invasion, and metastasis of malignancies of the head and neck involve extensive degradation and remodeling of the underlying, collagen-rich connective tissue. Urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 is an endocytic receptor recently shown to play...... a critical role in the uptake and intracellular degradation of collagen by mesenchymal cells. As a step toward determining the putative function of uPARAP/Endo180 in head and neck cancer progression, we used immunohistochemistry to determine the expression of this collagen internalization receptor in 112...... sections with two different monoclonal antibodies against two non-overlapping epitopes on uPARAP/Endo180 and by the use of isotype-matched non-immune antibodies. uPARAP/Endo180 expression was observed in stromal fibroblast-like, vimentin-positive cells. Furthermore, expression of the collagen...

  16. Platelet aggregation following trauma

    DEFF Research Database (Denmark)

    Windeløv, Nis A; Sørensen, Anne M; Perner, Anders

    2014-01-01

    We aimed to elucidate platelet function in trauma patients, as it is pivotal for hemostasis yet remains scarcely investigated in this population. We conducted a prospective observational study of platelet aggregation capacity in 213 adult trauma patients on admission to an emergency department (ED......). Inclusion criteria were trauma team activation and arterial cannula insertion on arrival. Blood samples were analyzed by multiple electrode aggregometry initiated by thrombin receptor agonist peptide 6 (TRAP) or collagen using a Multiplate device. Blood was sampled median 65 min after injury; median injury...... severity score (ISS) was 17; 14 (7%) patients received 10 or more units of red blood cells in the ED (massive transfusion); 24 (11%) patients died within 28 days of trauma: 17 due to cerebral injuries, four due to exsanguination, and three from other causes. No significant association was found between...

  17. The canonical transient receptor potential 6 (TRPC6) channel is sensitive to extracellular pH in mouse platelets.

    Science.gov (United States)

    Berna-Erro, Alejandro; Albarran, Letizia; Dionisio, Natalia; Redondo, Pedro C; Alonso, Nieves; Gomez, Luis J; Salido, Gines M; Rosado, Juan A

    2014-01-01

    The canonical transient receptor potential-6 (TRPC6) is a receptor-activated non-selective Ca(2+) channel regulated by a variety of modulators such as diacylglycerol, Ca(2+)/calmodulin or phosphorylation. The present study is aimed to investigate whether different situations, such as acidic pH, exposure to reactive oxygen species (ROS) or hypoxic-like conditions modulate TRPC6 channel function. Here we show normal aggregation and Ca(2+) mobilization stimulated by thrombin in TRPC6 KO platelets; however, OAG (1-oleoyl-2-acetyl-sn-glycerol)-evoked Ca(2+) entry was attenuated in the absence of TRPC6. Exposure of mouse platelets to acidic pH resulted in abolishment of thrombin-evoked aggregation and attenuated platelet aggregation induced by thapsigargin (TG) or OAG. Both OAG-induced Ca(2+) entry and platelet aggregation were greatly attenuated in cells expressing TRPC6 channels. Exposure of platelets to H2O2 or deferoxamine did not clearly alter thrombin, TG or OAG-induced platelet aggregation. Our results indicate that TRPC6 is sensitive to acidic pH but not to exposure to ROS or hypoxic-like conditions, which might be involved in the pathogenesis of the altered platelet responsiveness to DAG-generating agonists in disorders associated to acidic pH.

  18. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts.

    Science.gov (United States)

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi

    2017-03-07

    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  19. Characterization of canine platelet adhesion to extracellular matrix proteins.

    Science.gov (United States)

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  20. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    Institute of Scientific and Technical Information of China (English)

    Chong Chen; Shi-Hai Xia; Hong Chen; Xiao-Hong Li

    2008-01-01

    Acute pancreatitis (AP) causes release of plateletactivating factor (PAF),which induces systemic effects that contribute to circulatory disturbances and multiple organ failure.PAF is a cell surface secretion of bioactive lipid,which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R).Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activating factor receptor antagonists (PAF-RAs) could significantly reduce local and systemic events after AP.PAF has also been implicated as a key mediator in the progression of severe AP,which can lead to complications and unacceptably high mortality rates.Several classes of PAF-RA show PAFRAs significant local and systemic effects on reducing inflammatory changes.As a preventive treatment,PAF-RA could block a series of PAF-mediatedinflammatory injury and thus improve the prognosis of AR This review introduces the important role of PAF-RA in the treatment of AP.

  1. Signaling mechanisms mediated by G-protein coupled receptors in human platelets

    Institute of Scientific and Technical Information of China (English)

    Sheikh Arshad SAEED; Huma RASHEED; Faisal A Wahed FECTO; Mohammad Ilyas ACHAKZAI; Rahmat ALI; John Dennis CONNOR; Anwar-ul-Hassan GILANI

    2004-01-01

    AIM: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA). METHODS: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 ℃ using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time. RESULTS: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1μmol/L) and AA (0.2μmol/L) was inhibited by the α2-receptor antagonist (yohimbine, IC50=0.6 μmol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC50=0.25 μmol/L).In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by calcium channel blockers, verapamil (IC50=0.4 μtmol/L) and diltiazem (IC50=2.5 μmol/L), as well as by low concentrations of inhibitors of phospholipase C (PLC) (U73122; IC50=0.2 μmol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC50=3.8 μmol/L). Herbimycin A, a specific inhibitor of tyrosine light chain kinase (TLCK), showed inhibition at IC50 value of 15 μmol/L, whereas chelerythrine, a protein kinase C (PKC)inhibitor, had no effect up to 20 μmol/L. CONCLUSION: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC,COX, and MAP kinase-signaling pathways.

  2. Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Catalán, Mabel; Smolic, Christian [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Contreras, Ariel [Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Ayala, Pedro; Olmedo, Ivonne; Copaja, Miguel; Boza, Pía; Vivar, Raúl; Avalos, Yennifer [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Lavandero, Sergio [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile); Instituto Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile (Chile); Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX (United States); Velarde, Victoria [Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago (Chile); Díaz-Araya, Guillermo, E-mail: gadiaz@ciq.uchile.cl [Centro de estudios moleculares de la célula, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile (Chile)

    2012-06-15

    Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. Methods: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-β1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca{sup +2} levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. Results: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca{sup 2+} levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca{sup 2+} levels in CF; however, after preincubation for 1 h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca{sup 2+} levels. Finally, DAKD increased intracellular Ca{sup 2+} levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. Conclusion: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was

  3. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition......The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass...... spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high...

  4. Expression and functional characterization of platelet-derived growth factor receptor-like gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry in CRC and colorectal normal tissues.PDGFRL prokaryotic expression vector was carried out in Escherichia coli(E.coli),and purified by immobilized metal affinity chromatography.The effect of PDGFRL protein on CRC HCT-116 cells was det...

  5. Platelet-derived growth factor type BB and collagen matrix for soft tissue reconstruction after muco-epidermoid carcinoma removal: A possible therapeutic option

    Directory of Open Access Journals (Sweden)

    Marco Cicciù

    2015-01-01

    Full Text Available Muco-epidermoid carcinoma (MEC is a rare malignant tumor occurring in major and minor salivary glands. The described case shows a patient undergoing tumor resection without neck dissection. A quick diagnosis performed through clinical investigation and incisional biopsy revealed the nature of the tumor. A porcine collagen matrix was applied after the surgery in order to improve soft tissue healing. The matrix was saturated with platelet-derived growth factor type BB in order to favorite healing process and then fixed on the palate with a dental support device. Follow-up visit performed at first, second, and third weeks highlighted a quick healing of oral mucosa. Here reported is a case of a 34-year-old man who developed a muco-epidermoid oral carcinoma localized in the left upper jaw palatal side. The clinical, radiographic, and histopathologic findings, plus differential diagnoses of the case and reconstructive treatment options are also presented.

  6. CCK1 and CCK2 Receptors Are Expressed on Pancreatic Stellate Cells and Induce Collagen Production

    Science.gov (United States)

    Berna, Marc J.; Seiz, Oliver; Nast, Jan Friso; Benten, Daniel; Bläker, Michael; Koch, Johannes; Lohse, Ansgar W.; Pace, Andrea

    2010-01-01

    The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC. PMID:20843811

  7. Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

    Energy Technology Data Exchange (ETDEWEB)

    Bienz, D.; Clemetson, K.J.

    1989-01-05

    Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with /sup 125/I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/(/sup 3/H)NaBH/sub 4/. Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects.

  8. A dual laminin/collagen receptor acts in peripheral nerve regeneration.

    OpenAIRE

    Toyota, B; Carbonetto, S; David, S.

    1990-01-01

    A regeneration chamber was created in vivo by suturing a synthetic tube sealed at its distal end onto the proximal stump of a severed rat sciatic nerve. Nerves regenerated into tubes coated with laminin at a rate of 0.33 mm/day after a lag of about 2 days. At 25 days, regenerating nerves had extended 23% farther into laminin-coated tubes as compared with uncoated ones. Monoclonal antibody 3A3, which functionally interferes with a dual laminin/collagen receptor, inhibited nerve regeneration in...

  9. Platelet-derived growth factor receptor-β in myocyte was upregulated by angiotensin II

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To observe the regulation of platelet-derived growth factor (PDGF) receptor-βin myocyte stimulated by angiotensin II (AngII) at both integrated and cellular levels and reveal the signal transduction mechanism in cell, two kidneys, one clip (2K1C) renal hypertension were performed by placing a sliver clip around the left renal artery. Blood pressure and the ratio of left ventricular weight to body weight were measured at 4 and 8 weeks after operation. The content of AngII in heart was detected by radioimmunology assay; the protein level of PDGF receptor-βin heart was measured by Western blot analysis. The alteration of PDGF receptor-βstimulated by AngII and several inhibitors was observed on cultured neonatal rat ventricular myocyte (NRVM). The content of AngII in heart of 2K1C renal hypertensive rat at 4 and 8 weeks after operation was increased. Compared with sham group, 4 and 8 weeks after operation, PDGF receptor-βin heart of 2K1C group was upregulated by 100.3% and 127.1% (P < 0.05), respectively. This upregulation could be inhibited by captopril. For cultured myocyte, PDGF receptor-βwas increased by 47.1% after being stimulated by AngII and this upregulation could be inhibited by losartan which was an inhibitor of AT1 receptor. PLC inhibitor (U73122) and MEK inhibitor (PD98059) could partly inhibit PDGF receptor-βupregulation induced by AngII. These results suggested that AngII could upregulate PDGF receptor-βin myocyte by its AT1 receptor and this effect was at least partly dependent on PLC and extracellular signal-regulated kinase (ERK).

  10. Inhibitory effects of ginkgolide B on CD40 Ligand expression in collagen-induced platelet activation%银杏内酯B抑制血小板CD40Ligand表达的分子机制研究

    Institute of Scientific and Technical Information of China (English)

    闫琰; 赵革新; 陈北冬; 鲍利; 吴伟; 齐若梅

    2012-01-01

    目的 探讨银杏内酯B(ginkgolide B)对活化血小板CD40 Ligand(CD40L)的影响以及相关的分子机制.方法 取正常人血分离血小板,用不同浓度银杏内酯B孵育血小板5 min,然后用胶原(collagen)刺激血小板活化.用Western blot分析PI3K表达,Akt磷酸化,CD40L的变化.结果 ①用collagen刺激血小板聚集,银杏内酯B(0.2、0.4、0.6 g*L-1)预处理血小板5 min,血小板聚集率明显降低,聚集率分别为77%,60%和48%.②Western blot结果显示胶原刺激血小板活化后CD40L表达明显增加,银杏内酯B以剂量依赖方式抑制了CD40L表达.③银杏内酯B对胶原刺激的血小板PI3K表达无明显影响.④collagen刺激血小板活化后Akt的磷酸化增加,银杏内酯B抑制了Akt磷酸化.结论 银杏内酯B能够有效抑制collagen诱导的血小板聚集以及CD40L的表达,并明显抑制了Akt磷酸化,表明银杏内酯B能够通过PI3K/Akt信号传导通路抑制血小板活化.%Aim To investigate the effects of ginkgolide B on the CD40Ligand ( CD40L ) expression in collagen-induced platelet activation and the related mechanism. Methods Anti-coagulated blood was collected from health donor. Platelets were isolated through cen-trifugation. Platelets were pre-incubated by the various concentrations of ginkgolide B for 5 min, then platelets were stimulated by collagen. Protein expression was detected by Western blot. Results 1. 10 mg · L-1 of collagen induced platelet aggregation, and 0. 2 g · L-1,0. 4 g · L-1 and 0. 6 g · L-1 of ginkgolide B potently inhibited platelet aggregation in a dose-dependent manner. 2. The expression of CD40L was increased in collagen-induced platelet activation. Ginkgolide B significantly attenuated the increase of CD40L expression in activated platelets. 3. Ginkgolide B had no effect on PI3K expression in collagen-induced platelet activation. 4. Ginkgolide B obviously abolished Akt phospho-rylation in activated platelets. Conclusions Ginkgolide B can

  11. 2-Arachidonoylglycerol enhances platelet formation from human megakaryoblasts.

    Science.gov (United States)

    Gasperi, Valeria; Avigliano, Luciana; Evangelista, Daniela; Oddi, Sergio; Chiurchiù, Valerio; Lanuti, Mirko; Maccarrone, Mauro; Valeria Catani, Maria

    2014-01-01

    Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles. 2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB1 and CB2 receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation. In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases.

  12. Implication of platelet-derived growth factor receptor alpha in prostate cancer skeletal metastasis

    Institute of Scientific and Technical Information of China (English)

    Qingxin Liu; Danielle Jernigan; Yun Zhang; Alessandro Fatatis

    2011-01-01

    Metastasis represents by far the most feared complication of prostate carcinoma and is the main cause of death for patients.The skeleton is frequently targeted by disseminated cancer cells andrepresents the sole site of spread in more than 80% of prostate cancer cases.Compatibility between select malignant phenotypes and the microenvironment of colonized tissues is broadly recognized as the culprit for the organ-tropism of cancer cells.Here,we review our recent studies showing that the expression of platelet-derived growth factor receptor alpha (PDGFRα) supports the survival and growth of prostate cancer cells in the skeleton and that the soluble fraction of bone marrow activates PDGFRα in a ligand-independent fashion.Finally,we offer pre-clinical evidence that this receptor is a viable target for therapy.

  13. Single prostacyclin receptor of gel-filtered platelets provides a correlation with antiaggregatory potency of PGI2 mimics

    Energy Technology Data Exchange (ETDEWEB)

    Eggerman, T.L.; Hartzell, C.J.; Selfe, S.; Andersen, N.H.

    1987-03-01

    Gel-filtered human platelets (GFP) display only a single binding site for (/sup 3/H)-PGI2: KD = 61nM, 234 fmol/10(8) platelets (1410 sites/platelet). Platelet-rich plasma (PRP) displays the same receptor density but the KD value increases to 123 nM due to protein binding of PGI2 which lowers its effective concentration. The (/sup 3/H)-PGI2/GFP binding assay has been used to evaluate the molecular basis of aggregation inhibition for prostacyclin analogs and mimics, three PGE type structures, and PGD2. Antiaggregatory IC50s and radioligand binding IC50s correlate for PGE2, E1, and six PGI2 analogs. PGD2, and to a lesser extent 6-oxo-PGE1, display greater antiaggregatory potency than expected based on PGI2-binding site affinity data.

  14. Calpain Activity and Toll-Like Receptor 4 Expression in Platelet Regulate Haemostatic Situation in Patients Undergoing Cardiac Surgery and Coagulation in Mice

    Directory of Open Access Journals (Sweden)

    Jui-Chi Tsai

    2014-01-01

    Full Text Available Human platelets express Toll-like receptors (TLR 4. However, the mechanism by which TLR4 directly affects platelet aggregation and blood coagulation remains to be explored. Therefore, in this study, we evaluated the platelet TLR4 expression in patients who underwent CABG surgery; we explored the correlation between platelet TLR4 expression and the early outcomes in hospital of patients. Additionally, C57BL/6 and C57BL/6-TlrLPS−/− mice were used to explore the roles of platelet TLR4 in coagulation by platelet aggregometry and rotation thromboelastometry. In conclusion, our results highlight the important roles of TLR4 in blood coagulation and platelet function. Of clinical relevance, we also explored novel roles for platelet TLR4 that are associated with early outcomes in cardiac surgery.

  15. Aldosterone and mineralocorticoid receptor antagonists modulate elastin and collagen deposition in human skin.

    Science.gov (United States)

    Mitts, Thomas F; Bunda, Severa; Wang, Yanting; Hinek, Aleksander

    2010-10-01

    We have shown that the steroid hormone aldosterone, recognized for its action on the kidney and the cardiovascular system, also modulates deposition of extracellular matrix in human skin. We have shown that treatment of primary cultures of normal skin fibroblasts with aldosterone (10 n-1 μM), in addition to stimulation of collagen type I expression, induces elastin gene expression and elastic fiber deposition. We have further shown that the elastogenic effect of aldosterone, which can be enhanced in the presence of mineralocorticoid receptor (MR) antagonists spironolactone and eplerenone, is executed in a MR-independent manner via amplification of IGF-I receptor-mediated signaling. Because aldosterone applied alone stimulates both collagen and elastin deposition in cultures of fibroblasts and in cultures of skin explants derived from dermal stretch marks, we postulate that this steroid should be used in the treatment of damaged skin that loses its volume and elasticity. Moreover, aldosterone applied in conjunction with spironolactone or eplerenone induces matrix remodeling and exclusively enhances elastogenesis in cultures of fibroblasts and explants derived from dermal scars and keloids. We therefore propose that intra-lesional injection of these factors should be considered in therapy for disfiguring dermal lesions and especially in prevention of their recurrence after surgical excision.

  16. Central and peripheral benzodiazepine receptors in rat brain and platelets: effects of treatment with diazepam and clobazam.

    Science.gov (United States)

    Larkin, J G; Thompson, G G; Scobie, G; Brodie, M J

    1992-09-01

    Tolerance to the effects of benzodiazepines (BZ) may be mediated by changes in benzodiazepine receptors (BZRs). Peripheral BZRs (in brain and platelets) and central BZRs (in brain) were measured in rats following intraperitoneal administration of diazepam and clobazam each for 4 and 12 days. BZRs were measured by binding assays using [3H] PK 11195 (peripheral) and [3H] flunitrazepam (central) as radioligands. Diazepam, but not clobazam, increased peripheral BZR numbers in platelets (both P < 0.005), but not in brain, after 4 and 12 days' treatment compared with appropriate controls. Neither drug altered central BZR affinities or numbers in rat brain. BZ effects on peripheral BZRs in platelets cannot be extrapolated to predict changes in brain receptors, either peripheral or central.

  17. Identification of a functional genetic variant driving racially dimorphic platelet gene expression of the thrombin receptor regulator, PCTP.

    Science.gov (United States)

    Kong, Xianguo; Simon, Lukas M; Holinstat, Michael; Shaw, Chad A; Bray, Paul F; Edelstein, Leonard C

    2017-03-02

    Platelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression.

  18. Platelet-derived growth factor receptor/platelet-derived growth factor (PDGFR/PDGF) system is a prognostic and treatment response biomarker with multifarious therapeutic targets in cancers.

    Science.gov (United States)

    Appiah-Kubi, Kwaku; Wang, Ying; Qian, Hai; Wu, Min; Yao, Xiaoyuan; Wu, Yan; Chen, Yongchang

    2016-08-01

    Progress in cancer biology has led to an increasing discovery of oncogenic alterations of the platelet-derived growth factor receptors (PDGFRs) in cancers. In addition, their overexpression in numerous cancers invariably makes PDGFRs and platelet-derived growth factors (PDGFs) prognostic and treatment markers in some cancers. The oncologic alterations of the PDGFR/PDGF system affect the extracellular, transmembrane and tyrosine kinase domains as well as the juxtamembrane segment of the receptor. The receptor is also involved in fusions with intracellular proteins and receptor tyrosine kinase. These discoveries undoubtedly make the system an attractive oncologic therapeutic target. This review covers elementary biology of PDGFR/PDGF system and its role as a prognostic and treatment marker in cancers. In addition, the multifarious therapeutic targets of PDGFR/PDGF system are discussed. Great potential exists in the role of PDGFR/PDGF system as a prognostic and treatment marker and for further exploration of its multifarious therapeutic targets in safe and efficacious management of cancer treatments.

  19. Alpha/sub 2/-adrenergic receptors on a platelet precursor cell line, HEL

    Energy Technology Data Exchange (ETDEWEB)

    McKernan, R.M.; Motulsky, H.J.; Rozansky, D.; Insel, P.A.

    1986-03-01

    The authors have identified ..cap alpha../sub 2/-adrenergic receptors on human erythroleukemia HEL cells, a suspension-growing, bone-marrow-derived cell line related to human platelets. Intact HEL cells were studied using radioligand binding and cAMP accumulation assays. The authors identified saturable specific binding of the ..cap alpha../sub 2/-antagonist (/sup 3/H)yohimbine (yoh) in cells incubated at 37/sup 0/C for 1 hr (B/sub max/ 5900 +/- 2100 sites/cell, K/sub d/ 3.6 +/- 0.9 nM, n = 7). Competition for (/sup 3/H)yoh binding sites with antagonists confirmed that these sites were similar to human ..cap alpha../sub 2/-adrenoceptors from platelets and other resources, as typified by their high affinity for WY-26392, yohimbine and idazoxan, and very low affinity for prazosin. Studies at 37/sup 0/C revealed a low affinity of these sites for catecholamines (K/sub i/ for (-)-epinephrine, 21 ..mu..M; (-)-norepinephrine, 45 ..mu..M, (+)-epinephrine, 80 ..mu..M). When experiments were conducted at 4 /sup 0/C, (-)-epinephrine was able to compete for only 50-60% of the sites specifically labelled by (/sup 3/H)yoh at 37/sup 0/, but (-)-epinephrine had an approximately 10-fold greater affinity for these sites (K/sub i/ at 4 /sup 0/C = 2.4 ..mu..M). In addition, epinephrine inhibited cAMP accumulation stimulated by forskolin and PGE/sub 1/ in HEL cells; this response was inhibited by pertussis toxin. The authors conclude that HEL cells possess ..cap alpha../sub 2/-adrenergic receptors linked to G/sub i/ and thus should serve as a useful model to explore metabolism and regulation of these receptors in human cells.

  20. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    OpenAIRE

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma,Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.; Bray, Paul F.

    2014-01-01

    White individuals have a high frequency of the common PAR4 gene (F2RL3) variant Ala120; blacks have a high frequency of Thr120.PAR4 Thr120 induces greater signaling and is associated with greater platelet aggregation and reduced inhibition by a PAR4 antagonist.

  1. Fusaric acid, a mycotoxin, and its influence on blood coagulation and platelet function.

    Science.gov (United States)

    Devaraja, Sannaningaiah; Girish, Kesturu S; Santhosh, Martin S; Hemshekhar, Mahadevappa; Nayaka, Siddaiah C; Kemparaju, Kempaiah

    2013-06-01

    The current study intended to explore the effect of fusaric acid on blood coagulation including plasma coagulation and platelet aggregation. Fusaric acid exhibited biphasic effects on citrated human plasma recalcification time. At concentrations below 50 ng, fusaric acid decreased the clotting time of plasma dose-dependently from 130 ± 3s control value to 32 ± 3s; however, above 50 ng, fusaric acid increased the clotting time from 32 ± 3s and reached a maximum of 152 s at 100 ng and remained unaltered thereafter for the increased dose of fusaric acid. Fusaric acid without damaging red blood cells and platelets, inhibited agonists such as collagen, ADP, thrombin, and epinephrine-induced aggregation of both platelet-rich plasma (PRP) and washed platelets preparations of human. Interestingly, fusaric acid showed biphasic effects only in thrombin-induced platelet aggregation of washed platelets, and at lower concentration (below 900 ng) it activated platelet aggregation; however, in increased concentration (above 900 ng) it inhibited the platelet aggregation of washed platelets. In addition, fusaric acid also inhibited the agonist ADP-induced platelet aggregation of washed platelet suspension but did not show biphasic effect. Further, fusaric acid did not induce the platelets to generate reactive oxygen species (ROS) that clearly suggests that the induction of platelet function could be the result of the fusaric acid-mediated receptor interaction but not through the morphological shape change.

  2. The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.

    Science.gov (United States)

    Debili, N; Wendling, F; Cosman, D; Titeux, M; Florindo, C; Dusanter-Fourt, I; Schooley, K; Methia, N; Charon, M; Nador, R

    1995-01-15

    The Mpl receptor (Mpl-R) is a cytokine receptor belonging to the hematopoietin receptor superfamily for which a ligand has been recently characterized. To study the lineage distribution of Mpl-R in normal hematopoietic cells, we developed a monoclonal antibody (designated M1 MoAb) by immunizing mice with a soluble form of the human Mpl-R protein. With few exceptions, Mpl-R was detected by indirect immunofluorescent analysis on all human leukemic hematopoietic cell lines with pluripotential and megakaryocytic phenotypes, but not on other cell lines. By immunoprecipitation and immunoblotting, M1 MoAb recognized a band at 82 to 84 kD corresponding to the expected size of the glycosylated receptor. Among normal hematopoietic cells, M1 MoAb strongly stained megakaryocytes (MK) and Mpl-R was detected on platelets by indirect immunofluorescence staining or immunoblotting. On purified CD34+ cells, less than 2% of the population was stained, but the labeling was weak and just above the threshold of detection. However, dual-labeling with the M1 and antiplatelet glycoprotein MoAbs showed that most Mpl-R+/CD34+ cells coexpressed CD41a, CD61, or CD42a, suggesting that cell surface appearance of Mpl-R and platelet glycoproteins could be coordinated. M1-positive and M1-negative subsets were sorted from purified CD34+ cell populations. Colony assays showed that the absolute number of hematopoietic progenitors was extremely low and no primitive progenitors were present in the CD34+/Mpl-R+ fraction. However, this cell fraction was significantly enriched in low proliferative colony-forming units-MK. When the CD34+/Mpl-R+ fraction was grown in liquid culture containing human aplastic serum and a combination of growth factors, mature MK were seen as early as day 4, whereas the predominant cell population was erythroblasts on day 8. Similar data were also obtained with the CD34+/Mpl-R- fraction with, however, a delay in the time of appearance of both MK and erythroblasts. In conclusion

  3. Platelet-derived Toll-like receptor 4 (Tlr-4 is sufficient to promote microvascular thrombosis in endotoxemia.

    Directory of Open Access Journals (Sweden)

    Ryan J Stark

    Full Text Available Endotoxin (lipopolysaccharide, LPS produced by gram-negative bacteria initiates a host of pro-inflammatory effects through Toll-like receptor 4 (TLR-4. We reported previously that LPS enhances microvascular thrombosis in cremaster venules of wild-type mice, but had no effect in mice deficient in TLR-4. Since TLR-4 is expressed on various cell types, the cellular origin of TLR-4 responsible for the LPS-enhanced thrombosis remains undetermined. Platelets are known to express functional TLR-4. Platelet-derived TLR-4 has been suggested to mediate various inflammatory responses in endotoxemia, including production of tumor necrosis factor alpha (TNF-α and interleukin-1 beta (IL-1β, two cytokines reported to enhance microvascular thrombosis. We determined whether platelet-derived TLR-4 was sufficient to mediate the enhanced thrombosis induced by endotoxin and whether these responses were accompanied by systemic increases in TNF-α and IL-1β. We isolated platelets from wild-type mice and transfused them into either of two strains of TLR-4-deficient mice (C57BL/10ScN and B6.B10ScN-TLR-4(lps-del/Jth. The mice were then injected with LPS or saline, and the kinetics of thrombosis were studied 4 hours later. Transfusion of wild-type platelets restored responsiveness to LPS in TLR-4-deficient mice with regards to microvascular thrombosis but not to plasma levels of TNF-α or IL-1β. The accelerated rates of microvascular thrombosis induced by platelet transfusions were specific to TLR-4, since isolation and transfusion of platelets derived from TLR-4-deficient donors did not restore responsiveness to LPS. These studies demonstrate that platelet-derived TLR-4 is sufficient to promote microvascular thrombosis in endotoxemia, independent of systemic increases in TNF-α or IL-1β.

  4. Identification of a Platelet Membrane Glycoprotein as a Falciparum Malaria Sequestration Receptor

    Science.gov (United States)

    Ockenhouse, Christian F.; Tandon, Narendra N.; Magowan, Cathleen; Jamieson, G. A.; Chulay, Jeffrey D.

    1989-03-01

    Infections with the human malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected with mature forms of the parasite. Sequestration of infected erythrocytes appears to be critical for survival of the parasite and to mediate immunopathological abnormalities in severe malaria. A leukocyte differentiation antigen (CD36) was previously suggested to have a role in sequestration of malaria-infected erythrocytes. CD36 was purified from platelets, where it is known as GPIV, and was shown to be a receptor for binding of infected erythrocytes. Infected erythrocytes adhered to CD36 immobilized on plastic; purified CD36 exhibited saturable, specific binding to infected erythrocytes; and purified CD36 or antibodies to CD36 inhibited and reversed binding of infected erythrocytes to cultured endothelial cells and melanoma cells in vitro. The portion of the CD36 molecule that reverses cytoadherence may be useful therapeutically for rapid reversal of sequestration in cerebral malaria.

  5. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    Science.gov (United States)

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows

  6. Macrophage Receptor with Collagenous Structure (MARCO Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

    Directory of Open Access Journals (Sweden)

    Seishiro Hirano

    Full Text Available The Macrophage Receptor with COllagenous structure (MARCO protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

  7. Antiplatelet Agents Inhibit the Generation of Platelet-Derived Microparticles

    Science.gov (United States)

    Giacomazzi, Alice; Degan, Maurizio; Calabria, Stefano; Meneguzzi, Alessandra; Minuz, Pietro

    2016-01-01

    Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 μg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 μmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347–2946 vs. 3061, 446–4901 PMPs/μL; median ad range, n = 9, P PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified. PMID:27695417

  8. Pulp revascularization using platelet rich plasma autologous or in conjunction with a collagen matrix as a therapeutic possibility for teeth with an open apex necrotic pulp and / or periapical pathology.

    OpenAIRE

    Camargo Guevara, Paula Alejandra; Sossa Rojas, Henry

    2014-01-01

    Objective: Describe the use of the Autologous Platelet-Rich Plasma or in combination with a collagen matrix as a potential revascularization therapy in teeth with open apices, necrotic pulp and/or periapical lesion. Methods: A thematic literature searchwas conducted from 2007 to 2013 using electronic means,  databases and journals with high impact on endodontic. Results: Regenerative endodontic is a biological based procedure  designed to replace structures that have been damaged, diseased or...

  9. Effects of angiotensin Ⅱ receptor antagonist on expression of collagen Ⅲ, collagen Ⅴ, and transforming growth factor β1 in the airway walls of sensitized rats

    Institute of Scientific and Technical Information of China (English)

    杜永成; 许建英; 张韶君

    2004-01-01

    Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling, the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma, we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ, collagen Ⅴ, and transforming growth factor β1 (TGF-β1) mRNA and protein in the airway walls of sensitized rats.Methods Forty Wistar rats were randomly divided into 5 groups: control group, sensitized group, and valsartan groups 1, 2, and 3. The rats in the sensitized group and in valsartan groups 1, 2, and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1, 2, and 3 were drenched with valsartan (10 μg, 20 μg, or 30 μg, respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ, collagen Ⅴ, and TGF-β1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen Ⅴ was significantly higher in rats from the sensitized group (7.73±0.81, 1.34±0.28) and from valsartan groups 1, 2, and 3 (5.73±0.64, 1.13±0.15; 4.96±0.51, 0.98±0.08; 4.43±0.35, 0.93±0.06, respectively) than those in the control group (2.65±0.38, 0.67±0.08, P<0.05). In addition, collagen levels were significantly lower in valsartan groups 1, 2, and 3 than those from the sensitized group (P<0.05). The expression of TGF-β1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49%±3.46%, 29.73%±3.25%) and from valsartan groups 1, 2, and 3 (16.47%±1.94%, 19.41%±1.87%; 14

  10. Identification and characterization of a putative human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ receptor

    Energy Technology Data Exchange (ETDEWEB)

    Saussy, D.L. Jr.

    1986-01-01

    The thromboxane A/sub 2/ (TXA/sub 2/) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15..cap alpha beta..-omega-tetranor TXA/sub 2/ (I-PTA-OH) was characterized as a competitive antagonist of TXA/sub 2/ mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA/sub 2//PGH/sub 2/, and did not inhibit platelet TXA/sub 2/ synthesis. (/sup 125/I)-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k/sub 1/ of 1.35 x 10/sup 6/ M/sup -1/ x min/sup -1/ and a k..sqrt../sub 1/ of 0.032 min/sup -1/, K/sub d/ = k..sqrt../sub 1//k/sub 1/ = 24 nM. The subcellular localization of the putative TXA/sub 2//PGH/sub 2/ receptor was determined using (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding as a marker for the receptor. (/sup 125/I)-PTA-OH binding, was coenriched with markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules.

  11. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    Science.gov (United States)

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes.

  12. The research progress of collagen and its receptors%胶原及其受体的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵海亚; 张晓姝

    2010-01-01

    细胞外基质蛋白中的胶原在多细胞动物的进化中扮演重要的角色.在组织中,细胞锚定在含有胶原的结构上,通常细胞间通过基质蛋白连接的方式相互作用,但是细胞也表达受体,受体具有直接结合胶原中三股螺旋主链的能力.越来越多的证据表明,这些受体通过与胶原中特定基序结合发挥作用.%The collagen family of extracellular matrix proteins plays a fundamental role in the evolution of multicellular organism. In tissues, cells are anchored to collagenous structures. The interaction between cells and collagen can be indirect and mediated by matrix glycoproteins. Cells also express receptors, which directly bind to the triple helical domains of collagens. In addition, increasing evidence indicates that receptors bind to the specific motifs of collagens.

  13. Platelet lipidomic.

    Science.gov (United States)

    Dolegowska, B; Lubkowska, A; De Girolamo, L

    2012-01-01

    Lipids account for 16-19 percent dry platelet matter and includes 65 percent phospholipids, 25 percent neutral lipids and about 8 percent glycosphingolipids. The cell membrane that surrounds platelets is a bilayer that contains different types phospholipids symmetrically distributed in resting platelets, such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, and sphingomyelin. The collapse of lipid asymmetry is exposure of phosphatidylserine in the external leaflet of the plasma bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. During activation, this asymmetrical distribution becomes disrupted, and PS and PE become exposed on the cell surface. The transbilayer movement of phosphatidylserine is responsible for the platelet procoagulant activity. Exposure of phosphatidylserine is a flag for macrophage recognition and clearance from the circulation. Platelets, stored at room temperature for transfusion for more than 5 days, undergo changes collectively known as platelet storage lesions. Thus, the platelet lipid composition and its possible modifications over time are crucial for efficacy of platelet rich plasma therapy. Moreover, a number of substances derived from lipids are contained into platelets. Eicosanoids are lipid signaling mediators generated by the action of lipoxygenase and include prostaglandins, thromboxane A2, 12-hydroxyeicosatetraenoic acid. Isoprostanes have a chemical structure similar to this of prostanoids, but are differently produced into the particle, and are ligands for prostaglandins receptors, exhibiting biological activity like thromboxane A2. Endocannabinoids are derivatives from arachidonic acid which could reduce local pain. Phospholipids growth factors (sphingolipids, lysophosphatidic acid, platelet-activating factor) are involved in tissue

  14. Platelet-derived growth factor receptor beta is critical for zebrafish intersegmental vessel formation.

    Directory of Open Access Journals (Sweden)

    Katie M Wiens

    Full Text Available BACKGROUND: Platelet-derived growth factor receptor beta (PDGFRbeta is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRbeta functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the role of PDGFRbeta in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRbeta. We found that pdgfrbeta is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRbeta, and a dominant negative PDGFRbeta transgenic line, we found that PDGFRbeta is necessary for angiogenesis of the intersegmental vessels. SIGNIFICANCE/CONCLUSION: Our data provide the first evidence that PDGFRbeta signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRbeta signaling that regulates vascular angiogenesis in the absence of mural cells.

  15. GH receptor blocker administration and muscle-tendon collagen synthesis in humans

    DEFF Research Database (Denmark)

    Nielsen, Rie Harboe; Doessing, Simon; Goto, Kazushige;

    2011-01-01

    The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis stimulates collagen synthesis in tendon and skeletal muscle, but no studies have investigated the effect of reducing IGF-I on collagen synthesis in healthy humans.......The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis stimulates collagen synthesis in tendon and skeletal muscle, but no studies have investigated the effect of reducing IGF-I on collagen synthesis in healthy humans....

  16. Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

    Science.gov (United States)

    Ren, Xiaoyan; Bischoff, David; Weisgerber, Daniel W; Lewis, Michael S; Tu, Victor; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2015-05-01

    Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

  17. The role of collagen receptors Endo180 and DDR-2 in the foreign body reaction against non-crosslinked collagen and gelatin.

    Science.gov (United States)

    Ye, Qingsong; Harmsen, Martin C; Ren, Yijin; Bank, Ruud A

    2011-02-01

    Despite the use of collagen-derived scaffolds in regenerative medicine, little is known about the degradation mechanisms of these scaffolds in vivo. Non-crosslinked dermal sheep (NDSC) and gelatin disks were implanted subcutaneously in mice. NDSC disks showed a very low degradation rate, despite the presence of high numbers of macrophages and the influx of neutrophils. This was attributed to the presence of the matrix metalloproteinase inhibitor TIMP-1. The limited degradation occurred mainly in the later stages of the foreign body reaction, and could be attributed to (1) phagocytosis by macrophages due to a co-expression of Endo180 and MT1-MMP on these cells (intracellular degradation) and (2) the presence of MMP-13 due to an upregulation of the expression of the DDR-2 receptor (extracellular degradation). In contrast, gelatin disks degraded quickly, due to the efficient formation of large giant cells as well as the presence of MMP-13; the inhibitor TIMP-1 was absent. The DDR-2 receptor was not expressed in the gelatin disks. Endo180 and MT1-MMP were expressed, but at most times no co-expression was seen. We conclude that the physical state of collagen (native or denatured) had a dramatic outcome on the degradation rate and provoked a completely different foreign body reaction.

  18. Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Karl Egan

    Full Text Available Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

  19. Effectiveness of xenogenous-based bovine-derived platelet gel embedded within a three-dimensional collagen implant on the healing and regeneration of the Achilles tendon defect in rabbits

    Science.gov (United States)

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid; Koohi-Hosseinabadi, Omid

    2014-01-01

    Background and objective: Tissue engineering is an option in reconstructing large tendon defects and managing their healing and regeneration. We designed and produced a novel xenogeneic-based bovine platelet, embedded it within a tissue-engineered collagen implant (CI) and applied it in an experimentally induced large tendon defect model in rabbits to test whether bovine platelets could stimulate tendon healing and regeneration in vivo. Methods: One hundred twenty rabbits were randomly divided into two experimental and pilot groups. In all the animals, the left Achilles tendon was surgically excised and the tendon edges were aligned by Kessler suture. Each group was then divided into three groups of control (no implant), treated with CI and treated with collagen-platelet implant. The pilot groups were euthanized at 10, 15, 30 and 40 days post-injury (DPI), and their gross and histologic characteristics were evaluated to study host–graft interaction mechanism. To study the tendon healing and its outcome, the experimental animals were tested during the experiment using hematologic, ultrasonographic and various methods of clinical examinations and then euthanized at 60 DPI and their tendons were evaluated by gross pathologic, histopathologic, scanning electron microscopic, biophysical and biochemical methods. Results: Bovine platelets embedded within a CI increased inflammation at short term while it increased the rate of implant absorption and matrix replacement compared with the controls and CI alone. Treatment also significantly increased diameter, density, amount, alignment and differentiation of the collagen fibrils and fibers and approximated the water uptake and delivery behavior of the healing tendons to normal contralaterals (p tendons and reduced peritendinous adhesion, muscle fibrosis and atrophy, and therefore, it improved the clinical scores and physical activity related to the injured limb when compared with the controls (p tendon in rabbit. This

  20. Antiplatelet Agents Inhibit The Generation Of Platelet-Derived Microparticles

    Directory of Open Access Journals (Sweden)

    Alice Giacomazzi

    2016-09-01

    Full Text Available Platelet microparticles (PMPs contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMP shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid, U46619, collagen type-I (10 and 1.5 µg/mL, epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 µmol/L, SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMP shedding. The strongest response was to high concentration collagen. ADP-triggered PMP shedding was dose-independent. ASA reduced PMPs induced by arachidonic acid- (645, 347-2946 vs 3061, 446-4901 PMPs/µL; median ad range, n=9, P<0.001, collagen 10 µg/mL (5317, 2027-15935 vs 10252, 4187-46316 PMPs/µL; n=13, P<0.001, collagen 1.5 µg/mL (1078, 528-2820 vs 1465, 582-5948 PMPs/µL; n=21, P<0.001 and TRAP-6 (2008, 1621-2495 vs 2840, 2404-3031 PMPs/µL; n=3, P<0.01 but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395-2908 vs 3045, 1119-5494 PMPs/µL, n=6, P<0.05, collagen 1.5 µg/mL (1006, 780-1309 vs 2422, 1839-3494 PMPs/µL, n=3, P<0.01 and TRAP-6 (904, 761-1224 vs 2840, 2404-3031 PMPs/µL, n=3, P<0.01. The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMP release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

  1. Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA, an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f. var. glaucocalyx (Maxim. Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml significantly inhibited platelet aggregation induced by collagen (P<0.001 and CRP (P<0.01, a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

  2. Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

    Directory of Open Access Journals (Sweden)

    Szarka E

    2012-04-01

    Full Text Available Eszter Szarka1*, Zsuzsa Neer1*, Péter Balogh2, Monika Ádori1, Adrienn Angyal1, József Prechl3, Endre Kiss1,3, Dorottya Kövesdi1, Gabriella Sármay11Department of Immunology, Eötvös Loránd University, 1117 Budapest, 2Department of Immunology and Biotechnology, University of Pécs, Pécs, 3Immunology Research Group of the Hungarian Academy of Science at Eötvös Loránd University, 1117 Budapest, Hungary*These authors contributed equally to this workAbstract: Antibodies specific for bovine type II collagen (CII and Fcγ receptors play a major role in collagen-induced arthritis (CIA, a mouse model of rheumatoid arthritis (RA. Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2. These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro

  3. Transgenic expression of an altered angiotensin type I AT1 receptor resulting in marked modulation of vascular type I collagen.

    Science.gov (United States)

    Yu, Jun; Taylor, Linda; Rich, Celeste; Toselli, Paul; Stone, Philip; Green, Daniel; Warburton, Rod; Hill, Nicholas; Goldstein, Ronald; Polgar, Peter

    2012-05-01

    The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor β (TGFβ) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.

  4. Formyl-Peptide Receptor 2/3/Lipoxin A4 Receptor Regulates Neutrophil-Platelet Aggregation and Attenuates Cerebral Inflammation: Impact for Therapy in Cardiovascular Disease.

    Science.gov (United States)

    Vital, Shantel A; Becker, Felix; Holloway, Paul M; Russell, Janice; Perretti, Mauro; Granger, D Neil; Gavins, Felicity N E

    2016-05-31

    Platelet activation at sites of vascular injury is essential for hemostasis, but it is also a major pathomechanism underlying ischemic injury. Because anti-inflammatory therapies limit thrombosis and antithrombotic therapies reduce vascular inflammation, we tested the therapeutic potential of 2 proresolving endogenous mediators, annexin A1 N-terminal derived peptide (AnxA1Ac2-26) and aspirin-triggered lipoxin A4 (15-epi-lipoxin A4), on the cerebral microcirculation after ischemia/reperfusion injury. Furthermore, we tested whether the lipoxin A4 receptor formyl-peptide receptor 2/3 (Fpr2/3; ortholog to human FPR2/lipoxin A4 receptor) evoked neuroprotective functions after cerebral ischemia/reperfusion injury. Using intravital microscopy, we found that cerebral ischemia/reperfusion injury was accompanied by neutrophil and platelet activation and neutrophil-platelet aggregate formation within cerebral microvessels. Moreover, aspirin-triggered lipoxin A4 activation of neutrophil Fpr2/3 regulated neutrophil-platelet aggregate formation in the brain and inhibited the reactivity of the cerebral microvasculature. The same results were obtained with AnxA1Ac2-26 administration. Blocking Fpr2/lipoxin A4 receptor with the antagonist Boc2 reversed this effect, and treatments were ineffective in Fpr2/3 knockout mice, which displayed an exacerbated disease severity, evidenced by increased infarct area, blood-brain barrier dysfunction, increased neurological score, and elevated levels of cytokines. Furthermore, aspirin treatment significantly reduced cerebral leukocyte recruitment and increased endogenous levels of aspirin-triggered lipoxin A4, effects again mediated by Fpr2/3. Fpr2/lipoxin A4 receptor is a therapeutic target for initiating endogenous proresolving, anti-inflammatory pathways after cerebral ischemia/reperfusion injury. © 2016 American Heart Association, Inc.

  5. GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5 induced platelet activation and direct toward glycans as potential inhibitors.

    Directory of Open Access Journals (Sweden)

    Houyuan Hu

    Full Text Available BACKGROUND: Staphylococcus aureus (S. aureus is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5 has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. METHODOLOGY/PRINCIPAL FINDINGS: In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. CONCLUSIONS/SIGNIFICANCE: These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.

  6. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

    Science.gov (United States)

    Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T.; Mundell, Stuart J.; Coxon, Carmen H.

    2016-01-01

    Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

  7. Platelet-derived growth factor receptor alpha in glioma: a bad seed

    Institute of Scientific and Technical Information of China (English)

    Kun-Wei Liu; Bo Hu; Shi-Yuan Cheng

    2011-01-01

    Recent collaborative,large-scale genomic profiling of the most common and aggressive brain tumor glioblastoma multiforme(GBM) has significantly advanced our understanding of this disease.The gene encoding platelet-derived growth factor receptor alpha (PDGFRα) was identified as the third of the top 11 amplified genes in clinical GBM specimens.The important roles of PDGFRα signaling during normal brain development also implicate the possible pathologic consequences of PDGFRα over-activation in glioma.Although the initial clinical trials using PDGFR kinase inhibitors have been predominantly disappointing,diagnostic and treatment modalities involving genomic profiling and personalized medicine are expected to improve the therapy targeting PDGFRα signaling.In this review,we discuss the roles of PDGFRα signaling during development of the normal central nervous system (CNS) and in pathologic conditions such as malignant glioma.We further compare various animal models of PDGF-induced gliomagenesis and their potential as a novel platform of pre-clinical drug testing.We then summarize our recent publication and how these findings will likely impact treatments for gliomas driven by PDGFRα overexpression.A better understanding of PDGFRα signaling in glioma and their microenvironment,through the use of human or mouse models,is necessary to design a more effective therapeutic strategy against gliomas harboring the aberrant PDGFRα signaling.

  8. Peptide-based targeting of the platelet-derived growth factor receptor beta.

    Science.gov (United States)

    Askoxylakis, Vasileios; Marr, Annabell; Altmann, Annette; Markert, Annette; Mier, Walter; Debus, Jürgen; Huber, Peter E; Haberkorn, Uwe

    2013-04-01

    The aim of this work is to identify new ligands targeting the platelet-derived growth factor receptor beta (PDGFRβ). Biopanning was carried out with a 12-amino-acid phage display library against the recombinant extracellular domain of PDGFRβ. The identified peptide PDGFR-P1 was chemically synthesized and labeled with (125)I or (131)I. In vitro studies were performed on the PDGFRβ-expressing cell lines BxPC3 and MCF7 and on PDGFRβ-transfected HEK cells in comparison to negative control wtHEK293 and CaIX-transfected HEK cells. Biodistribution experiments were performed in Balb/c nude mice, carrying subcutaneously BxPC3 tumors. In vitro studies demonstrated a higher binding to BxPC3, MCF7, and PDGFRβ-tr-HEK cells in comparison to negative control cell lines. Binding was inhibited up to 90% by the unlabeled PDGFR-P1 peptide. Organ distribution studies revealed a higher accumulation in BxPC3 tumors than in most organs. PDGFR-P1 is a promising candidate for targeting human PDGFRβ.

  9. Purification and characterization of the human platelet. cap alpha. /sub 2/-adrenergic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Shreeve, S.M.; Kerlavage, A.R.; Fraser, C.M.; Mariani, A.P.; Venter, J.C.

    1986-05-01

    The ..cap alpha../sub 2/-receptor (..cap alpha../sub 2/-R) from human platelets has been purified to homogeneity using a four step process. An affinity column was prepared by coupling p-aminoclonidine to CH-Sepharose 4B via the p-NH/sub 2/ group. Digitonin solubilized ..cap alpha../sub 2/-R bound to the affinity matrix were eluted with 100 ..mu..M phentolamine and directly applied to a DEAE-Sepharose column. Bound receptors were eluted with a linear gradient of 0-500 mM NaCl, pooled and chromatographed on HPLC size exclusion columns. Three peaks of ..cap alpha../sub 2/-R binding were eluted from HPLC columns (t = 33, 42, 47 min). Radioiodination of HPLC eluates and analysis by SDS-PAGE indicated that ..cap alpha../sub 2/-R binding was associated with a 75-85 kDa protein. These data suggest that the ..cap alpha../sub 2/-R may exist in monomeric and oligomeric forms in the purified state and support previous target size data which indicate that the ..cap alpha../sub 2/-R exists as a dimer in the native membrane. The pure radioiodinated ..cap alpha../sub 2/-R (77-85 kDa) is a glycoprotein with terminal sialic acid or N-acetylglucosamine residues and has a pI of 4.1 on column isoelectric focusing. These data are consistent with those previously reported on the partially purified ..cap alpha../sub 2/-R. Electron micrographs confirm the oligomeric nature and size of the pure ..cap alpha../sub 2/-R.

  10. Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.

    Science.gov (United States)

    Schraw, Todd D; Rutledge, Tara W; Crawford, Garland L; Bernstein, Audrey M; Kalen, Amanda L; Pessin, Jeffery E; Whiteheart, Sidney W

    2003-09-01

    It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.

  11. Structures of a platelet-derived growth factor/propeptide complex and a platelet-derived growth factor/receptor complex

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Ann Hye-Ryong; Liu, Heli; Focia, Pamela J.; Chen, Xiaoyan; Lin, P. Charles; He, Xiaolin (Vanderbilt); (NWU)

    2010-07-13

    Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFR{beta}, showing that two PDGF-B protomers clamp PDGFR{beta} at their dimerization seam. The PDGF-B:PDGFR{beta} interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFR{beta}. Calorimetric data also shows that the membrane-proximal homotypic PDGFR{alpha} interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.

  12. Rac1 is essential for phospholipase C-gamma2 activation in platelets

    DEFF Research Database (Denmark)

    Pleines, Irina; Elvers, Margitta; Strehl, Amrei

    2008-01-01

    Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta...... isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC...... regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation...

  13. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen.

    Science.gov (United States)

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka

    2015-03-24

    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  14. Homoerydictyl-7-O-β-D-glycosidc—A Receptor Antagonist of Platelet-activating Factor (PAF)

    Institute of Scientific and Technical Information of China (English)

    GuanZengwei; WangYinye; YangXiuwei; CuiYuxin

    2001-01-01

    Homoeriodictyl-7-O-β-D-glycoside, a flavonoid compound isolated from the Chinese medic inalherb, viscum coloratura inhibited platelet aggregation induced by platelet-activating factor(PAF), but it had no inhibitory activity on adenosine diphosphate-induced platelet aggregation. In the present study, we intended to get an insight into the mechanism of its anti-PAF action. Using [3H]PAF receptor binding assay we found that the compound exhibited inhibitory activity. The inhibitory rate was 18.5%, 28.4%, 58.7%, 78% and 78%, respestively, at concentrations of 10-8, 10-7, 10-6, 10-5 and 10-4 mol.L-1, There was a visible dose-effect relationship as well as a correlation between different concentrations and their inhibitory rates (r=0.985, P<0.05) when the dose was equal to or less than 1×10-5 mol.L-1, and its IC50 was 8.0×10-7 mol.L-1. The inhibitory rate didn't increase with increasing concentration of the compound when it went beyond1×10-5 mol.L-1 indicating competitive inhibition of binding of [3H]PAF to PAF receptor reached saturation.

  15. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

    OpenAIRE

    Gronwald, R G; Grant, F J; Haldeman, B A; Hart, C E; O'Hara, P J; Hagen, F S; Ross, R.; Bowen-Pope, D F; Murray, M. J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately ...

  16. Influence of platelet-derived growth factor-AB on tissue development in autologous platelet-rich plasma gels.

    Science.gov (United States)

    Wirz, Simone; Dietrich, Maren; Flanagan, Thomas C; Bokermann, Gudrun; Wagner, Wolfgang; Schmitz-Rode, Thomas; Jockenhoevel, Stefan

    2011-07-01

    Fibrin-based scaffolds are widely used in tissue engineering. We postulated that the use of platelet-rich plasma (PRP) in contrast to platelet-poor plasma and pure fibrinogen as the basic material leads to an increased release of autologous platelet-derived growth factor (PDGF)-AB, which may have a consequent positive effect on tissue development. Therefore, we evaluated the release of PDGF-AB during the production process and the course of PDGF release during cultivation of plasma gels with and w/o platelets. The influence of PDGF-AB on the proliferation rate of human umbilical cord artery smooth muscle cells (HUASMCs) was studied using XTT assay. The synthesis of extracellular matrix by HUASMCs in plasma- and fibrin gels was measured using hydroxyproline assay. The use of PRP led to an increase in autologous PDGF-AB release. Further, the platelet-containing plasma gels showed a prolonged release of growth factor during cultivation. Both PRP and platelet-poor plasma gels had a positive effect on the production of collagen. However, PDGF-AB as a supplement in medium and in pure fibrin gel had neither an effect on cell proliferation nor on the collagen synthesis rate. This observation may be due to an absence of PDGF receptors in HUASMCs as determined by flow cytometry. In conclusion, although the prolonged autologous production of PDGF-AB in PRP gels is possible, the enhanced tissue development by HUASMCs within such gels is not PDGF related.

  17. Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) Ly-GDI in platelet function: a spatial systems approach.

    Science.gov (United States)

    Ngo, Anh T P; Thierheimer, Marisa L D; Babur, Özgün; Rocheleau, Anne D; Huang, Tao; Pang, Jiaqing; Rigg, Rachel A; Mitrugno, Annachiara; Theodorescu, Dan; Burchard, Julja; Nan, Xiaolin; Demir, Emek; McCarty, Owen J T; Aslan, Joseph E

    2017-02-01

    Upon activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. While RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

  18. Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

    Directory of Open Access Journals (Sweden)

    Daniel Fábio Kawano

    2011-12-01

    Full Text Available Chagas disease (CD causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs, which is the main characteristic of the G protein-coupled receptors (GPCRs, including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

  19. Restoration of responsiveness of phospholipase Cγ2-deficient platelets by enforced expression of phospholipase Cγ1.

    Directory of Open Access Journals (Sweden)

    Yongwei Zheng

    Full Text Available Receptor-mediated platelet activation requires phospholipase C (PLC activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2, which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT, PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbβ3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbβ3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistently, αIIbβ3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbβ3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbβ3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2.

  20. Discoidin domain receptors promote α1β1- and α2β1-integrin mediated cell adhesion to collagen by enhancing integrin activation.

    Directory of Open Access Journals (Sweden)

    Huifang Xu

    Full Text Available The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx'GEx". The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.

  1. The human megakaryocytic cell line UT-7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor.

    Science.gov (United States)

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Yokota, Hiroshi

    2010-09-01

    We have demonstrated that a unique megakaryocytic cell line UT-7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT-7/TPO was found to express GPVI and its associate signal molecule of FcRgamma (Fc receptor gamma chain). When UT-7/TPO was stimulated with the GPVI agonist convulxin, the [Ca2+]i (intracellular Ca2+) was elevated in a convulxin concentration-dependent manner, and [Ca2+]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT-7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)-1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca2+]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin-stimulated platelets failed to produce TX, it is suggested that UT-7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT-7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.

  2. The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

    Science.gov (United States)

    Bhadriraju, Kiran; Chung, Koo-Hyun; Spurlin, Tighe A; Haynes, Ross J; Elliott, John T; Plant, Anne L

    2009-12-01

    Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

  3. The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function.

    Directory of Open Access Journals (Sweden)

    Olivia A Lin

    Full Text Available There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and

  4. Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

    Science.gov (United States)

    Ezumi, Y; Nishida, E; Uchiyama, T; Takayama, H

    1999-07-22

    Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway. Copyright 1999 Academic Press.

  5. Flavanols and Platelet Reactivity

    Directory of Open Access Journals (Sweden)

    Debra A. Pearson

    2005-01-01

    Full Text Available Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

  6. Effect of ionizing radiation on platelet function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kalovidouris, A.E.; Papayannis, A.G. (Evangelismos Hospital, Athens (Greece))

    1981-01-01

    The effect of ionizing radiation on platelet function was investigated in vitro. Platelet-rich plasma (300x10/sup 9//l) was irradiated with doses of 1, 4, 10, 20 and 50 Gy. Platelet function tests were performed on both irradiated and control (non-irradiated) platelet samples. The platelet function tests were (1) platelet aggregation by ADP (1, 2, 4 ..mu..mol final concentration), adrenaline and collagen, (2) ADP-release from platelets, (3) clot retraction and (4) platelet factor-3 availability. It was found that roentgen irradiation of platelets in vitro did not affect these platelet function tests.

  7. The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis.

    Science.gov (United States)

    Senis, Yotis A; Tomlinson, Michael G; Ellison, Stuart; Mazharian, Alexandra; Lim, Jenson; Zhao, Yan; Kornerup, Kristin N; Auger, Jocelyn M; Thomas, Steve G; Dhanjal, Tarvinder; Kalia, Neena; Zhu, Jing W; Weiss, Arthur; Watson, Steve P

    2009-05-14

    Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

  8. Effect of fluvoxamine on platelet 5-HT2A receptors as studied by [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy volunteers.

    Science.gov (United States)

    Spigset, O; Mjörndal, T

    1997-09-01

    Alterations in platelet 5-HT2A receptor characteristics have been reported in major depression as well as in other psychiatric diseases, and some effort has been made to utilize platelet 5-HT2A receptor status as a biological correlate to antidepressant drug response. In order to investigate whether treatment with a selective serotonin reuptake inhibitor affects platelet 5-HT2A receptors, we have studied platelet [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy subjects treated with fluvoxamine in increasing dosage once weekly for 4 weeks. After 1 week of fluvoxamine treatment (25 mg/day), both Bmax and Kd were significantly lower than before the start of the treatment (19.9 versus 25.5 fmol/mg protein, P = 0.005 for Bmax; 0.45 versus 0.93 nM, P = 0.006 for Kd). Bmax returned to baseline during week 2, whereas Kd was lower than the baseline value throughout the treatment period. After discontinuation of fluvoxamine treatment, there was a significant increase in Kd (0.50 nM before discontinuation vs. 1.14 nM after discontinuation; P = 0.001), but not in Bmax. The study demonstrates that fluvoxamine affects platelet 5-HT2A receptor status irrespective of underlying psychiatric disease, and that this effect is evident already after 1 week at a subtherapeutic fluvoxamine dose.

  9. Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro.

    Science.gov (United States)

    Sokolova, Ekaterina V; Byankina, Anna O; Kalitnik, Alexandra A; Kim, Yong H; Bogdanovich, Larisa N; Solov'eva, Tamara F; Yermak, Irina M

    2014-05-01

    The influence of sulfated polysaccharides (λ-, κ-, and κ/β-carrageenan and porphyran) - on platelet activation was studied. Carrageenans were much weaker inhibitors of a coagulation process than heparin, while porphyran had not that effect. Results of the aPTT and PT assays suppose that carrageenans affected mostly intrinsic pathway of coagulation, while their effect on the extrinsic pathway is extremely low (λ and κ/β) or absent (κ, LMW derivative of κ-carrageenan). λ-Carrageenan was the most potent anticoagulant agent in TT, aPTT, PT, and anti-factor Xa activity. This sample was also the strongest inhibitor of collagen-induced platelet aggregation in PRP. Generally, the correlation of anticoagulant and antithrombotic action in PRP is preserved for carrageenans but not for heparin. Carrageenans and porphyran affected platelet adhesion to collagen by influencing glycoprotein VI. Low molecular weight κ-carrageenan had a similar effect on platelet adhesion mediated with both major collagen receptors: integrin α2 β1 and glycoprotein VI as native polysaccharide had. Carrageenans resulted in activation of platelets under platelet adhesion mediated by integrin αIIb β3 with less degree than heparin. The least sulfated κ/β-carrageenan that possessed an inhibiting effect on thrombin- and collagen-induced aggregation of washed platelets and on the PT test but it had no significant effect on TT was the weakest promoter of integrin αIIb β3 mediated platelet activation. In summary, our study showed that the polysaccharide action was complex, since it depended on its molecular mass, sulfation degree, and monosaccharide contents (3,6-anhydrogalactose).

  10. Airway epithelial platelet-activating factor receptor expression is markedly upregulated in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Shukla SD

    2014-08-01

    Full Text Available Shakti Dhar Shukla,1,* Sukhwinder Singh Sohal,1,* Malik Quasir Mahmood,1 David Reid,2 Hans Konrad Muller,1 Eugene Haydn Walters1 1NHMRC Centre for Research Excellence for Chronic Respiratory Disease and Lung Ageing, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia; 2Queensland Institute of Medical Research, Iron Metabolism Laboratory, Brisbane, Queensland, Australia *Shakti Dhar Shukla and Sukhwinder Singh Sohal are joint first authors Background: We recently published that platelet-activating factor receptor (PAFr is upregulated on the epithelium of the proximal airways of current smokers and also in bronchial epithelial cells exposed to cigarette smoke extract. These treated cells also showed upregulation of Streptococcus pneumoniae adhesion. Bacterial wall phosphorylcholine specifically binds to PAFr expressed on airway epithelium, thus facilitating adherence and tissue invasion, which may be relevant to chronic obstructive pulmonary disease (COPD. Moreover, the use of inhaled corticosteroids (ICS in COPD patients is associated with an increased risk of invasive respiratory pneumococcal infections. Objective: In this study, we have investigated whether PAFr expression is especially upregulated in airway epithelium in COPD patients and whether this expression may be modulated by ICS therapy. Methods: We cross-sectionally evaluated PAFr expression in bronchial biopsies from 15 COPD patients who were current smokers (COPD-smokers and 12 COPD-ex-smokers, and we compared these to biopsies from 16 smokers with normal lung function. We assessed immunostaining with anti-PAFr monoclonal antibody. We also used material from a previous double-blinded randomized placebo-controlled 6-month ICS intervention study in COPD patients to explore the effect of ICS on PAFr expression. We employed computer-aided image analysis to quantify the percentage of epithelium stained for PAFr. Results: Markedly enhanced expression of PAFr was found

  11. The nature of interactions between tissue-type plasminogen activator and platelets

    Energy Technology Data Exchange (ETDEWEB)

    Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E. (Washington Univ., St. Louis, MO (USA))

    1990-07-15

    To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.

  12. GH receptor blocker administration and muscle-tendon collagen synthesis in humans

    DEFF Research Database (Denmark)

    Nielsen, Rie Harboe; Doessing, Simon; Goto, Kazushige

    2011-01-01

    Collagen is the predominant structural protein in tendons and ligaments, and can be controlled by hormonal changes. In animals, injections of insulin-like growth factor I (IGF-I) has been shown to increase collagen synthesis in tendons and ligaments and to improve structural tissue healing......, but the effect of local IGF-I administration on tendon collagen synthesis in human has not been studied. The purpose of this study was to study whether local injections of IGF-I would have a stimulating effect on tendon collagen synthesis. Twelve healthy nonsmoking men [age 62 ± 1 years (mean ± SEM), BMI 27 ± 1......] participated. Two injections of either human recombinant IGF-I (0.1 mL Increlex©) or saline (control) into each patellar tendon were performed 24-h apart, respectively. Tendon collagen fractional synthesis rate (FSR) was measured by stable isotope technique in the hours after the second injection...

  13. Crosstalk between Protease-activated Receptor 1 and Platelet-activating Factor Receptor Regulates Melanoma Cell Adhesion Molecule (MCAM/MUC18) Expression and Melanoma Metastasis*

    Science.gov (United States)

    Melnikova, Vladislava O.; Balasubramanian, Krishnakumar; Villares, Gabriel J.; Dobroff, Andrey S.; Zigler, Maya; Wang, Hua; Petersson, Frederik; Price, Janet E.; Schroit, Alan; Prieto, Victor G.; Hung, Mien-Chie; Bar-Eli, Menashe

    2009-01-01

    The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis. PMID:19703903

  14. Platelet-derived growth factor and platelet-derived growth factor receptor-α expression in the normal human thymus and thymoma

    Science.gov (United States)

    Cimpean, Anca Maria; Ceauşu, Raluca; Encică, Svetlana; Gaje, Pusa Nela; Ribatti, Domenico; Raica, Marius

    2011-01-01

    Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) are strongly involved in the normal development of several organs, tumour angiogenesis and malignant progression and metastasis. Few studies concerning their expression, distribution and role in normal and pathological human thymus are available in the literature. The aim of this study has been to analyse the immunohistochemical expression of PDGF and PDGFR-α in prenatal and postnatal normal human thymus and thymomal biopsy specimens. The results demonstrated immunoreactivity to both PDGF and PDGFR-α in all specimens, but the intensity, distribution and number of positive cells were different in normal thymus and thymomas, and also among different tumour types. PDGF and PDGFR-α were weakly expressed in foetal and postnatal humans with a different distribution between cortex and medulla in both blood vessels and epithelial cells, whereas they were overexpressed in thymoma, especially in type B2 and B3, in the tumour epithelial cells. Overall, these data suggest that PDGF and PDGFR-α may be involved in the pathophysiology of the human thymus. PMID:21645144

  15. Platelet-derived growth factor and platelet-derived growth factor receptor-α expression in the normal human thymus and thymoma.

    Science.gov (United States)

    Cimpean, Anca Maria; Ceauşu, Raluca; Encică, Svetlana; Gaje, Pusa Nela; Ribatti, Domenico; Raica, Marius

    2011-10-01

    Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) are strongly involved in the normal development of several organs, tumour angiogenesis and malignant progression and metastasis. Few studies concerning their expression, distribution and role in normal and pathological human thymus are available in the literature. The aim of this study has been to analyse the immunohistochemical expression of PDGF and PDGFR-α in prenatal and postnatal normal human thymus and thymomal biopsy specimens. The results demonstrated immunoreactivity to both PDGF and PDGFR-α in all specimens, but the intensity, distribution and number of positive cells were different in normal thymus and thymomas, and also among different tumour types. PDGF and PDGFR-α were weakly expressed in foetal and postnatal humans with a different distribution between cortex and medulla in both blood vessels and epithelial cells, whereas they were overexpressed in thymoma, especially in type B2 and B3, in the tumour epithelial cells. Overall, these data suggest that PDGF and PDGFR-α may be involved in the pathophysiology of the human thymus.

  16. Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants.

    Science.gov (United States)

    Jantan, I; Rafi, I A A; Jalil, J

    2005-01-01

    Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).

  17. Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Yin-Ying Lu; Chun-Ping Wang; Lin Zhou; Yan Chen; Shu-Hui Su; Yong-Yi Feng; Yong-Ping Yang

    2008-01-01

    AIM:To determine the platelet-activating factor (PAF)synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induce dcirrhosis.METHODS:Kupffer cells,isolated from the livers of control and CCl4-induced cirrhotic rats,were placed in serum-free medium overnight.PAF saturation binding,ET-1 saturation and competition binding were assayed.ET-1 induced PAF synthesis,mRNA expression of PAF,preproendothelin-1,endothelin A (ETA) and endothelin B (ETB) receptors were also determined.RESULTS:A two-fold increase of PAF synthesis (1.42±0.14 vs 0.66±0.04 pg/μg DNA) and a 1.48-fold increase of membrane-bound PAF (1.02±0.06 vs 0.69±0.07 Pg/μg DNA) were observed in activated Kupffer cells of cirrhotic rats.The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor,but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells.In activated Kupffer cells,PAF receptor expression and PAF binding capacity were markedly enhanced.Activated Kupffer cells raised the [125I]-ET-1 binding capacity,but changed neither the affinity of the receptors,nor the expression of ETA receptor.CONCLUSION:Kupffer cells in the course of CCl4-induced cirrhosis are the main source of increased PAF.ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine.ETA receptor did not appear in activated Kupffer cells,which may exacerbate the hepatic and extrahepatic complications of cirrhosis.

  18. RESIDUAL PLATELET REACTIVITY DURING THERAPY WITH INHIBITORS OF CYCLOOXIGENASE OR ADENOSINE DIPHOSPHATE RECEPTORS

    Directory of Open Access Journals (Sweden)

    A. A. Lomonosova

    2012-01-01

    Full Text Available Aim. To compare effects of acetylsalicylic acid (ASA and two clopidogrel drugs on residual platelet aggregative reactivity (RPAR. Material and methods. Patients (n=40 with ischemic heart disease aged under 70 years were involved into the crossover study. Clinical examination included questionnaire survey , blood pressure (BP measurement, ECG registration, 24-hour ECG and BP monitoring, determination of blood levels of total cholesterol, high density lipoproteins, triglycerides, transaminases, and creatinine, complete blood cell count, including platelets number and hemoglobin level. Besides evaluation of the platelet aggregation by optical aggregometry was performed initially , after one week ASA treatment and after every next 3 week clopidogrel treatment period.  Results. RPAR during ASA monotherapy was 56.4±0.3%. There were no significant differences in effects of original and generic clopidogrel on RPAR. Сlopidogrel therapy reduced RPAR more significantly (42.2±0.2% than ASA monotherapy did (p=0.0003. Authors proposed definition for high level of RPAR during therapy - it is platelet aggregation more than 46%. Data analysis taking into account this criterion showed that a number of patients with high RPAR was 70 and 30% among patients treated with enterosoluble ASA and clopidogrel, respectively. Conclusion. Study results show that a significant number of patients receiving antiplatelet monotherapy does not achieve the target level of RPAR(<46%. These results may be a rationale for combined therapy in patients of this type.

  19. Platelet function in dogs

    DEFF Research Database (Denmark)

    Nielsen, Line A.; Zois, Nora Elisabeth; Pedersen, Henrik D.

    2007-01-01

    Cairn Terriers, 10 Boxers, and 11 Labrador Retrievers) were included in the study. Platelet function was assessed by whole-blood aggregation with ADP (1, 5, 10, and 20 µM) as agonist and by PFA-100 using collagen and epinephrine (Col + Epi) and Cpæ + ADP as agonists. Plasma thromboxane B2 concentration......Background: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. Objectives: The objective of this study was to investigate platelet function in clinically healthy dogs of 4...... different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Methods: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12...

  20. Case-control study of platelet glycoprotein receptor Ib and IIb/IIIa expression in patients with acute and chronic cerebrovascular disease.

    Directory of Open Access Journals (Sweden)

    Peter Kraft

    Full Text Available Animal models have been instrumental in defining thrombus formation, including the role of platelet surface glycoprotein (GP receptors, in acute ischemic stroke (AIS. However, the involvement of GP receptors in human ischemic stroke pathophysiology and their utility as biomarkers for ischemic stroke risk and severity requires elucidation.To determine whether platelet GPIb and GPIIb/IIIa receptors are differentially expressed in patients with AIS and chronic cerebrovascular disease (CCD compared with healthy volunteers (HV and to identify predictors of GPIb and GPIIb/IIIa expression.This was a case-control study of 116 patients with AIS or transient ischemic attack (TIA, 117 patients with CCD, and 104 HV who were enrolled at our University hospital from 2010 to 2013. Blood sampling was performed once in the CCD and HV groups, and at several time points in patients with AIS or TIA. Linear regression and analysis of variance were used to analyze correlations between platelet GPIb and GPIIb/IIIa receptor numbers and demographic and clinical parameters.GPIb and GPIIb/IIIa receptor numbers did not significantly differ between the AIS, CCD, and HV groups. GPIb receptor expression level correlated significantly with the magnitude of GPIIb/IIIa receptor expression and the neutrophil count. In contrast, GPIIb/IIIa receptor numbers were not associated with peripheral immune-cell sub-population counts. C-reactive protein was an independent predictor of GPIIb/IIIa (not GPIb receptor numbers.Platelet GPIb and GPIIb/IIIa receptor numbers did not distinguish between patient or control groups in this study, negating their potential use as a biomarker for predicting stroke risk.

  1. SU-F-SPS-08: Measuring the Interaction Of DDR Cell Receptors and Extracellular Matrix Collagen in Prostate Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, J; Sarkar, A; Hoffmann, P [Wayne State University, Detroit, MI (United States); Suhail, A; Fridman, R [Wayne State University School of Medicine, Detroit, MI (United States)

    2016-06-15

    Purpose: Discoidin domain receptors (DDR) have recently been recognized as important players in cancer progression. DDRs are cell receptors that interact with collagen, an extracellular matrix (ECM) protein. However the detailed mechanism of their interaction is unclear. Here we attempted to examine their interaction in terms of structural (surface topography), mechanical (rupture force), and kinetic (binding probability) information on the single molecular scale with the use of atomic force microscopy (AFM). Methods: The Quantitative Nano-mechanical property Mapping (QNM) mode of AFM allowed to assess the cells in liquid growth media at their optimal physiological while being viable. Human benign prostate hyperplasia (BPH-1) cell line was genetically regulated to suppress DDR expression (DDR- cells) and was compared with naturally DDR expressing cells (DDR+). Results: Binding force measurements (n = 1000) were obtained before and after the two groups were treated with fibronectin (FN), an integrin-inhibiting antibody to block the binding of integrin. The quantification indicates that cells containing DDR bind with collagen at a most probable force of 80.3–83.0 ±7.6 pN. The probability of them binding is 0.167 when other interactions (mainly due to integrin-collagen binding) are minimized. Conclusion: Together with further force measurements at different pulling speeds will determine dissociation rate, binding distance and activation barrier. These parameters in benign cells provides some groundwork in understanding DDR’s behavior in various cell microenvironments such as in malignant tumor cells. Funding supported by Richard Barber Interdisciplinary Research Program of Wayne State University.

  2. Platelets and hemostasis

    Directory of Open Access Journals (Sweden)

    M. A. Panteleev

    2014-09-01

    Full Text Available Platelets are anuclear cell fragments playing important role in hemostasis, termination of bleeding after damage, as well as in pathological thrombus formation. The main action of platelets is the formation of aggregates, overlapping the injury. They obtained the ability to aggregate by the transition process called activation. Despite the relatively simple and definite function platelet structure is very difficult: they have almost a full set of organelles, including the endoplasmic reticulum, mitochondria and other entities. When activated platelets secrete various granules interact with plasma proteins and red blood cells and other tissues. Their activation is controlled by multiple receptors and complex signaling cascades. In this review platelet structure, mechanisms of its functioning in health and disease, diagnostic methods of platelet function and approaches to their correction were considered. Particular attention will be given to those areas of the science of platelets, which still lay hidden mysteries.

  3. Effects of hormones on platelet aggregation.

    Science.gov (United States)

    Farré, Antonio López; Modrego, Javier; Zamorano-León, José J

    2014-04-01

    Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

  4. Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling.

    Science.gov (United States)

    Lamy, Sylvie; Beaulieu, Edith; Labbé, David; Bédard, Valérie; Moghrabi, Albert; Barrette, Stéphane; Gingras, Denis; Béliveau, Richard

    2008-05-01

    Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet-derived growth factor (PDGF)-BB receptor-beta [platelet-derived growth factor receptor-beta (PDGFR-beta)] in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-beta was very rapid and led to the inhibition of PDGF-BB-induced activation of extracellular signal-regulated kinase (ERK)-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a three-dimensional coculture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies.

  5. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay.

    Science.gov (United States)

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm(2))/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s(-1)), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s(-1)) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s(-1) revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s(-1) or 1000 s(-1), and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s(-1) (compared to fibrin formed at 100 s(-1)) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway

    DEFF Research Database (Denmark)

    Liu, Daoyan; Maier, Alexandra; Scholze, Alexandra;

    2008-01-01

    Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels....

  7. The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue

    NARCIS (Netherlands)

    Beljaars, L.; Weert, B; Geerts, Albert; Meijer, D.K F; Poelstra, Klaas

    2003-01-01

    Platelet derived growth factor (PDGF) is a key factor in the induction and progression of fibrotic diseases with the activated fibroblast as its target cell. Drug targeting to the PDGF-receptor is explored as a new approach to treat this disease. Therefore, we constructed a macromolecule with affini

  8. In vivo and protease-activated receptor-1-mediated platelet activation but not response to antiplatelet therapy predict two-year outcomes after peripheral angioplasty with stent implantation.

    Science.gov (United States)

    Gremmel, T; Steiner, S; Seidinger, D; Koppensteiner, R; Panzer, S; Kopp, C W

    2014-03-01

    Data linking the response to antiplatelet therapy with clinical outcomes after angioplasty and stenting for lower extremity artery disease (LEAD) are scarce. Moreover, associations of in vivo and thrombin-inducible platelet activation with the occurrence of adverse events have not been investigated in these patients, so far. We therefore assessed clinical outcomes and on-treatment platelet reactivity by four test systems in 108 patients receiving dual antiplatelet therapy after infrainguinal angioplasty and stenting for LEAD. Further, in vivo and thrombin receptor-activating peptide (TRAP)-6-inducible glycoprotein (GP) IIb/IIIa activation and P-selectin expression were measured as sensitive parameters of platelet activation. The primary endpoint was defined as the composite of atherothrombotic events and target vessel restenosis or reocclusion. Residual platelet reactivity to adenosine diphosphate and arachidonic acid was similar between patients without and with adverse outcomes within two-year follow-up (all p>0.05). Further, the occurrence of clinical endpoints did not differ significantly between patients without and with high on-treatment residual platelet reactivity by all test systems (all p>0.05). In contrast, in vivo and TRAP-6-inducible platelet activation were significantly more pronounced in patients with subsequent adverse events (all pangioplasty and stenting for LEAD.

  9. Lack of the specific influence of histamine and histamine H1, H2 and H3 receptor ligands on the serotonin uptake and release in rat blood platelets.

    Science.gov (United States)

    Pawlak, D; Malinowska, B; Wollny, T; Godlewski, G; Buczko, W

    1996-01-01

    This work was designed to investigate the influence of histamine, and H1 receptor agonist 2-(2-thiazolyl)ethylamine, H2 receptor agonist dimaprit and H3 receptor agonist R-(-)-alpha-methylhistamine on the serotonin uptake and release in rat blood platelets. Histamine and R-(-)-alpha-methylhistamine (up to 1 mmol/l), 2-(2-thiazolyl)ethylamine (up to 10 mumol/l) and dimaprit (up to 1 mumol/l) failed to affect the serotonin uptake. The concentration-dependent inhibitory effects of higher concentrations of 2-(2-thiazolyl)ethylamine and dimaprit (up to 1 mmol/l) were not diminished by the H1 receptor antagonist dimetindene and the H2 receptor antagonist ranitidine (1 and 100 mumol/l each), respectively. Histamine, 2-(2-thiazolyl)ethylamine, dimaprit and R-(-)-alpha-methylhistamine (up to 10 mumol/l) did not change the serotonin release from rat blood platelets. Our results demonstrate that histamine and histamine H1, H2 and H3 receptor agonists do not affect in a specific manner the serotonin uptake and release in rat blood platelets.

  10. Peroxisome proliferator-activated receptor-gamma abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator.

    Science.gov (United States)

    Ghosh, Asish K; Bhattacharyya, Swati; Wei, Jun; Kim, Suyeon; Barak, Yaacov; Mori, Yasuji; Varga, John

    2009-09-01

    Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

  11. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  12. Effect of platelet-rich plasma on the healing of intra-bony defects treated with a natural bone mineral and a collagen membrane.

    NARCIS (Netherlands)

    Dori, F.; Huszar, T.; Nikolidakis, D.; Arweiler, N.B.; Gera, I.; Sculean, A.

    2007-01-01

    BACKGROUND: Regenerative periodontal therapy with a combination of platelet-rich plasma (PRP)+a natural bone mineral (NBM)+guided tissue regeneration (GTR) has been shown to result in significantly higher probing depth reductions and clinical attachment-level gains compared with treatment with open

  13. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  14. Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

    Directory of Open Access Journals (Sweden)

    Stefanie Krajewski

    Full Text Available Extracorporeal circulation (ECC and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2Y(12 and P(2Y(1 blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2Y(12 antagonist 2-MeSAMP, the P(2Y(1 antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls. Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2Y blockers (p<0.05, while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05. P(2Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05. Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2Y and PI3K blockade (p<0.05. Combined blockade of P

  15. The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers

    DEFF Research Database (Denmark)

    Nielsen, Christoffer Fagernæs; van Putten, Sander Maarten; Lund, Ida Katrine

    2017-01-01

    A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelia...

  16. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin a1 and discoidin domain receptor 2: a protein kinase C-dependent pathway

    NARCIS (Netherlands)

    Vonk, L.A.; Doulabi, B.Z.; Huang, C.L.; Helder, M.N.; Everts, V.; Bank, R.A.

    2011-01-01

    Objectives. To investigate whether maintaining the chondrocyte’s native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and/or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role

  17. Differential actions of the endocytic collagen receptor uPARAP/Endo180 and the collagenase MMP-2 in bone homeostasis

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Jürgensen, Henrik J; Ingvarsen, Signe;

    2013-01-01

    the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone...... quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone...... structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency...

  18. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    Science.gov (United States)

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders.

  19. Endocytic collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe;

    2012-01-01

    it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked......Fibrosis of the liver and its end-stage, cirrhosis, represent major health problems worldwide. In these fibrotic conditions, activated fibroblasts and hepatic stellate cells display a net deposition of collagen. This collagen deposition is a major factor leading to liver dysfunction, thus making...... up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional...

  20. β1 integrin-mediated signals are required for platelet granule secretion and hemostasis in mouse.

    Science.gov (United States)

    Petzold, Tobias; Ruppert, Raphael; Pandey, Dharmendra; Barocke, Verena; Meyer, Hannelore; Lorenz, Michael; Zhang, Lin; Siess, Wolfgang; Massberg, Steffen; Moser, Markus

    2013-10-10

    Integrins are critical for platelet adhesion and aggregation during arterial thrombosis and hemostasis. Although the platelet-specific αIIbβ3 integrin is known to be crucial for these processes, the in vivo role of β1 integrins is a matter of debate. Here we demonstrate that mice expressing reduced levels of β1 integrins or an activation-deficient β1 integrin show strongly reduced platelet adhesion to collagen in vitro and in a carotis ligation model in vivo. Interestingly, hypomorphic mice expressing only 3% of β1 integrins on platelets show normal bleeding times despite reduced platelet adhesion. The residual 3% of β1 integrins are able to trigger intracellular signals driving Rac-1-dependent granule release required for platelet aggregation and hemostasis. Our findings support a model, in which platelet β1 integrins serve as an important signaling receptor rather than an adhesion receptor in vivo and therefore promote β1 integrins as a promising and so far clinically unemployed antithrombotic target.

  1. Anti-thrombotic effects of selective estrogen receptor modulator tamoxifen.

    Science.gov (United States)

    Nayak, Manasa K; Singh, Sunil K; Roy, Arnab; Prakash, Vivek; Kumar, Anand; Dash, Debabrata

    2011-10-01

    Tamoxifen is a known anti-cancer drug and established estrogen receptor modulator. Few clinical studies have earlier implicated the drug in thrombotic complications attributable to lower anti-thrombin and protein S levels in plasma. However, action of tamoxifen on platelet signalling machinery has not been elucidated in detail. In the present report we show that tamoxifen is endowed with significant inhibitory property against human platelet aggregation. From a series of in vivo and in vitro studies tamoxifen was found to inhibit almost all platelet functions, prolong tail bleeding time in mouse and profoundly prevent thrombus formation at injured arterial wall in mice, as well as on collagen matrix perfused with platelet-rich plasma under arterial shear against the vehicle dimethylsulfoxide (DMSO). These findings strongly suggest that tamoxifen significantly downregulates platelet responses and holds potential as a promising anti-platelet/anti-thrombotic agent.

  2. Stage III-IV Uterine Prolapse Risk Factors: Sacrouterine Ligaments High Estrogen Receptor Alpha and Collagen III Expression and Low Elastin Expression

    Directory of Open Access Journals (Sweden)

    I Wayan Megadhana

    2016-08-01

    Full Text Available Background: Uterine prolapse is common, non-life-threatening, but has a negative impact on women psychosocial and economic life. Damage to levator ani muscle is the early onset of uterine prolapse, while the damage of sacrouterine ligaments aggravates the stage. The strength of sacrouterine ligament depends on tissue cellularity, the formation of collagen I/III ratio, and the decreased expression of elastin. The lower the ratio of collagen I/III, the higher the risk of stage III-IV uterine prolapse. The ratio of collagen I/III formation is allegedly influencing through the expression of estrogen receptor alpha, by increasing collagen III synthesis and decreasing the degradation. Objective: We aimed to investigate whether high estrogen receptor alpha and collagen III expression, and the low elastin expression in the sacrouterine ligaments were stage III-IV uterine prolapse risk factors. Method: In March to August 2014, a non-matching case control study was conducted in 3 hospitals in Denpasar, and the materials were processed in the Faculty of Veterinary Medicine Laboratory of Udayana University. The case was uterine prolapse stage III-IV, the control was the non-uterine prolapse. We collected 1.5 cm residual sacrouterine ligaments from the edge of the cervix fixed with 10% buffered formalin from patients who underwent a total hysterectomy. They were examined immunohistochemically to identify estrogen receptor alpha expression, collagen III, and elastin. Results: Our sample was 44, divided equally between the case and control group. Compared to the control, in the case group, the proportion was significantly higher for the high estrogen receptor alpha expression (OR=5.71, 95%CI 1.56-20.93, p=0.007, high collagen III (OR=6.50, 95% CI 1.64- 25.76, p=0.005, and low elastin (OR=5.40, 95%CI 1.37-21.26, p=0.012. Conclusion: the high expression of estrogen receptor alpha and collagen III and low expressions of elastin in sacrouterine ligaments served as

  3. Signal Transduction of Platelet-Induced Liver Regeneration and Decrease of Liver Fibrosis

    Directory of Open Access Journals (Sweden)

    Soichiro Murata

    2014-03-01

    Full Text Available Platelets contain three types of granules: alpha granules, dense granules, and lysosomal granules. Each granule contains various growth factors, cytokines, and other physiological substances. Platelets trigger many kinds of biological responses, such as hemostasis, wound healing, and tissue regeneration. This review presents experimental evidence of platelets in accelerating liver regeneration and improving liver fibrosis. The regenerative effect of liver by platelets consists of three mechanisms; i.e., the direct effect on hepatocytes, the cooperative effect with liver sinusoidal endothelial cells, and the collaborative effect with Kupffer cells. Many signal transduction pathways are involved in hepatocyte proliferation. One is activation of Akt and extracellular signal-regulated kinase (ERK1/2, which are derived from direct stimulation from growth factors in platelets. The other is signal transducer and activator of transcription-3 (STAT3 activation by interleukin (IL-6 derived from liver sinusoidal endothelial cells and Kupffer cells, which are stimulated by contact with platelets during liver regeneration. Platelets also improve liver fibrosis in rodent models by inactivating hepatic stellate cells to decrease collagen production. The level of intracellular cyclic adenosine monophosphate (cyclic AMP is increased by adenosine through its receptors on hepatic stellate cells, resulting in inactivation of these cells. Adenosine is produced by the degradation of adenine nucleotides such as adenosine diphosphate (ADP and adenosine tri-phosphate (ATP, which are stored in abundance within the dense granules of platelets.

  4. Morphological adaptation of muscle collagen and receptor of advanced glycation end product (RAGE) in osteoarthritis patients with 12 weeks of resistance training

    DEFF Research Database (Denmark)

    Mattiello-Sverzut, Ana Claudia; Petersen, Susanne G; Kjaer, Michael

    2013-01-01

    The aim of this study was to investigate the effect of 12-week resistance training on morphological presence of collagen and RAGE (receptor for advanced glycation end products) in skeletal muscle of patients with knee osteoarthritis (OA). Little is known about the influence of exercise on the ske......The aim of this study was to investigate the effect of 12-week resistance training on morphological presence of collagen and RAGE (receptor for advanced glycation end products) in skeletal muscle of patients with knee osteoarthritis (OA). Little is known about the influence of exercise....... The patients (age 55-69 years) were divided into three groups, treated with NSAID, glucosamine or placebo. In addition, the muscle samples were analysed by immunohistochemistry for collagen types, RAGE and capillaries ratio. An increment in immunoreactivity for type IV collagen after the training period...... was observed in 72 % of all biopsies when compared with their respective baseline samples. Reduced immunoreactivity of collagen type I was observed in all patients treated with glucosamine. A significant increase with training in the amount of RAGE was detected in the placebo group only (p ...

  5. Neuropilin 1 binds platelet-derived growth factor (PDGF)-D and is a co-receptor in PDGF-D/PDGF receptor β signaling.

    Science.gov (United States)

    Muhl, Lars; Folestad, Erika Bergsten; Gladh, Hanna; Wang, Yixin; Moessinger, Christine; Jakobsson, Lars; Eriksson, Ulf

    2017-03-02

    Platelet-derived growth factor (PDGF)-D is a PDGF receptor β (PDGFRβ) specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological function remains incompletely understood.In this study, we demonstrate that PDGF-D binds directly to NRP1, with the requirement of the C-terminal Arg residue of PDGF-D. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRβ/NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell-cell junctions in endothelial cells, independent of PDGFRβ, altering the availability of NRP1 for VEGF-A/VEGF receptor 2 signaling. PDGF-D showed differential effects on pericyte behavior in ex vivo sprouting assays, compared to PDGF-B. Furthermore, PDGF-D induced PDGFRβ/NRP1 interaction in the trans-configuration between endothelial cells and pericytes.In summary, we show that NRP1 can act as a co-receptor for PDGF-D in PDGFRβ signaling, possibly implicated in intercellular communication in the vascular wall.

  6. Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis.

    Science.gov (United States)

    Gaudreault, Eric; Stankova, Jana; Rola-Pleszczynski, Marek

    2005-01-01

    Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.

  7. Clopidogrel discontinuation and platelet reactivity following coronary stenting

    LENUS (Irish Health Repository)

    2011-01-01

    Summary. Aims: Antiplatelet therapy with aspirin and clopidogrel is recommended for 1 year after drug-eluting stent (DES) implantation or myocardial infarction. However, the discontinuation of antiplatelet therapy has become an important issue as recent studies have suggested a clustering of ischemic events within 90 days of clopidogrel withdrawal. The objective of this investigation was to explore the hypothesis that there is a transient ‘rebound’ increase in platelet reactivity within 3 months of clopidogrel discontinuation. Methods and Results: In this prospective study, platelet function was assessed in patients taking aspirin and clopidogrel for at least 1 year following DES implantation. Platelet aggregation was measured using a modification of light transmission aggregometry in response to multiple concentrations of adenosine diphosphate (ADP), epinephrine, arachidonic acid, thrombin receptor activating peptide and collagen. Clopidogrel was stopped and platelet function was reassessed 1 week, 1 month and 3 months later. Thirty-two patients on dual antiplatelet therapy were recruited. Discontinuation of clopidogrel increased platelet aggregation to all agonists, except arachidonic acid. Platelet aggregation in response to ADP (2.5, 5, 10, and 20 μm) and epinephrine (5 and 20 μm) was significantly increased at 1 month compared with 3 months following clopidogrel withdrawal. Thus, a transient period of increased platelet reactivity to both ADP and epinephrine was observed 1 month after clopidogrel discontinuation. Conclusions: This study demonstrates a transient increase in platelet reactivity 1 month after clopidogrel withdrawal. This phenomenon may, in part, explain the known clustering of thrombotic events observed after clopidogrel discontinuation. This observation requires confirmation in larger populations.

  8. The role of collagen receptors Endo180 and DDR-2 in the foreign body reaction against non-crosslinked collagen and gelatin

    NARCIS (Netherlands)

    Ye, Qingsong; Harmsen, Martin C.; Ren, Yijin; Bank, Ruud A.

    2011-01-01

    Despite the use of collagen-derived scaffolds in regenerative medicine, little is known about the degradation mechanisms of these scaffolds in vivo. Non-crosslinked dermal sheep (NDSC) and gelatin disks were implanted subcutaneously in mice. NDSC disks showed a very low degradation rate, despite the

  9. Cognitive and socio-emotional deficits in platelet-derived growth factor receptor-β gene knockout mice.

    Directory of Open Access Journals (Sweden)

    Phuong Thi Hong Nguyen

    Full Text Available Platelet-derived growth factor (PDGF is a potent mitogen. Extensive in vivo studies of PDGF and its receptor (PDGFR genes have reported that PDGF plays an important role in embryogenesis and development of the central nervous system (CNS. Furthermore, PDGF and the β subunit of the PDGF receptor (PDGFR-β have been reported to be associated with schizophrenia and autism. However, no study has reported on the effects of PDGF deletion on mice behavior. Here we generated novel mutant mice (PDGFR-β KO in which PDGFR-β was conditionally deleted in CNS neurons using the Cre/loxP system. Mice without the Cre transgene but with floxed PDGFR-β were used as controls. Both groups of mice reached adulthood without any apparent anatomical defects. These mice were further examined by conducting several behavioral tests for spatial memory, social interaction, conditioning, prepulse inhibition, and forced swimming. The test results indicated that the PDGFR-β KO mice show deficits in all of these areas. Furthermore, an immunohistochemical study of the PDGFR-β KO mice brain indicated that the number of parvalbumin (calcium-binding protein-positive (i.e., putatively γ-aminobutyric acid-ergic neurons was low in the amygdala, hippocampus, and medial prefrontal cortex. Neurophysiological studies indicated that sensory-evoked gamma oscillation was low in the PDGFR-β KO mice, consistent with the observed reduction in the number of parvalbumin-positive neurons. These results suggest that PDGFR-β plays an important role in cognitive and socioemotional functions, and that deficits in this receptor may partly underlie the cognitive and socioemotional deficits observed in schizophrenic and autistic patients.

  10. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  11. Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI.

    Science.gov (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Yanagi, S; Yamamura, H; Okuma, M

    1995-11-24

    Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')2-anti-p62 in a way insensitive to elevation of cAMP. In contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive activation of c-Src and Syk was also observed in collagen but not thrombin-stimulated platelets. Moreover, either F(ab')2-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.

  12. Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

    Directory of Open Access Journals (Sweden)

    Maria José Dantas Coêlho

    2011-01-01

    Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

  13. Effect of angiotensin Ⅱ and angiotensin Ⅱ type 1 receptor antagonist on the proliferation,contraction and collagen synthesis in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; GONG Hao; ZHANG Zhong-tao; WANG Yu

    2008-01-01

    Background Angiotensin Ⅱ(Ang Ⅱ)is a very important vasoactive peptide that acts upon hepatic stellate cells(HSCs),which are major effector cells in hepatic cirrhosis and portal hypertension.The present study was aimed to investigate the effects of Ang Ⅱ and angiotensin Ⅱ type 1 receptor antagonist(AT1RA)on the proliferation,contraction and collagen synthesis in HSCs.Methods HSC-T6 rat hepatic stellate cell Iine was studied.The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by3 H-thymidine incorporation.The effects of angiotensin Ⅱ and AT1 RA on HSCs contraction were studied by analVSIs of the contraction of the collagen Iattice.CelI culture media were analyzed by RT-PCR to detect secretion of collagen Ⅰ(Col Ⅰ),collagen Ⅲ(Col Ⅲ)and transforming growth factor β1 (TGF-β1)by enzyme Iinked Immunosorbent assay.HSC was harvested to measure collagen Ⅰ,collagen Ⅲ and tissue inhibitor of metalloproteinase-1(TIMP-1)mRNA expression.Results Ang Ⅱ((1 x10-10-1×10-4)mol/L)stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells.AT1 RA inhibited angiotensin Ⅱ induced proliferation of HSCs.A Iinear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin Ⅱ(1×10-9-1×10-5mol/L)and with time over 48 hours.ATlRA blocks angiotensin Ⅱ induced contraction of collagen Iattice.Coll,Col Ⅲ and TGF-β1 levels of the Ang Ⅱ group were higher than those of control group and this increase was downregulated by AT1RA.The mRNA expressions of ColⅠ,CoI Ⅲ and TIMP-1 were higher in HSCs from the Ang Ⅱ group than the control group and downregulated by AT1RA.Conclusions Angiotensin Ⅱ increased DNA synthesis and proliferation of HSCs in a dose-dependent manner,stimulated the contraction of HSCs dose-and time-dependently.Angiotensin also promoted excretion of Col Ⅰ,ColⅢand TGF-β1 Ievels and stimulated Col Ⅰ,Col Ⅲ and

  14. Influence of caffeine on blood pressure and platelet aggregation

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    José Wilson S. Cavalcante

    2000-08-01

    Full Text Available OBJECTIVE: Studies have demonstrated that methylxanthines, such as caffeine, are A1 and A2 adenosine receptor antagonists found in the brain, heart, lungs, peripheral vessels, and platelets. Considering the high consumption of products with caffeine in their composition, in Brazil and throughout the rest of the world, the authors proposed to observe the effects of this substance on blood pressure and platelet aggregation. METHODS: Thirteen young adults, ranging from 21 to 27 years of age, participated in this study. Each individual took 750mg/day of caffeine (250mg tid, over a period of seven days. The effects on blood pressure were analyzed through the pressor test with handgrip, and platelet aggregation was analyzed using adenosine diphosphate, collagen, and adrenaline. RESULTS: Diastolic pressure showed a significant increase 24 hours after the first intake (p<0.05. This effect, however, disappeared in the subsequent days. The platelet aggregation tests did not reveal statistically significant alterations, at any time during the study. CONCLUSION: The data suggest that caffeine increases diastolic blood pressure at the beginning of caffeine intake. This hypertensive effect disappears with chronic use. The absence of alterations in platelet aggregation indicates the need for larger randomized studies.

  15. A point mutation in the EGF-4 domain of β(3) integrin is responsible for the formation of the Sec(a) platelet alloantigen and affects receptor function.

    Science.gov (United States)

    Sachs, Ulrich J; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2012-01-01

    Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbβ3. The location of Sec(a) on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the β3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbβ3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbβ3 receptor function.

  16. Activation of platelet-activating factor receptor in SZ95 sebocytes results in inflammatory cytokine and prostaglandin E2 production.

    Science.gov (United States)

    Zhang, Qiwei; Seltmann, Holger; Zouboulis, Christos C; Travers, Jeffrey B

    2006-10-01

    Platelet-activating factor (PAF) is a group of phosphocholines with various biological effects mediated by the PAF receptor (PAF-R). Activation of the epidermal PAF-R induces the expression of inflammatory mediators, including cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). The upregulation of COX-2 expression has been shown to be involved in sebocyte proliferation, sebaceous gland inflammation and carcinogenesis. The present study was designed to investigate whether PAF-R activation could induce the expression of COX-2 and production of PGE(2), as well as secretion of the inflammatory cytokine, interleukin-8 (IL-8), in the immortalized sebaceous gland cell line SZ95. Using calcium mobilization studies, we first confirmed that PAF can signal through PAF-R in SZ95 sebocytes. We then found that the production of IL-8 was induced following treatment with PAF-R agonist, however blocked by a specific PAF-R antagonist. Induction of COX-2 expression and increased PGE(2) production were observed in SZ95 sebocytes after PAF-R activation. Finally, it was demonstrated that the production of PGE(2), induced by PAF-R activation and mediated by COX-2 expression, was blocked following PAF-R antagonism in SZ95 sebocytes. These studies suggest that SZ95 sebocytes express functional PAF-Rs and PAF-Rs are involved in regulating the expression of inflammatory mediators, including COX-2, PGE(2) and IL-8.

  17. Lack of Platelet-Activating Factor Receptor Attenuates Experimental Food Allergy but Not Its Metabolic Alterations regarding Adipokine Levels

    Directory of Open Access Journals (Sweden)

    Nathália Vieira Batista

    2016-01-01

    Full Text Available Platelet-activating factor (PAF is known to be an important mediator of anaphylaxis. However, there is a lack of information in the literature about the role of PAF in food allergy. The aim of this work was to elucidate the participation of PAF during food allergy development and the consequent adipose tissue inflammation along with its alterations. Our data demonstrated that, both before oral challenge and after 7 days receiving ovalbumin (OVA diet, OVA-sensitized mice lacking the PAF receptor (PAFR showed a decreased level of anti-OVA IgE associated with attenuated allergic markers in comparison to wild type (WT mice. Moreover, there was less body weight and adipose tissue loss in PAFR-deficient mice. However, some features of inflamed adipose tissue presented by sensitized PAFR-deficient and WT mice after oral challenge were similar, such as a higher rate of rolling leukocytes in this tissue and lower circulating levels of adipokines (resistin and adiponectin in comparison to nonsensitized mice. Therefore, PAF signaling through PAFR is important for the allergic response to OVA but not for the adipokine alterations caused by this inflammatory process. Our work clarifies some effects of PAF during food allergy along with its role on the metabolic consequences of this inflammatory process.

  18. Lack of Platelet-Activating Factor Receptor Attenuates Experimental Food Allergy but Not Its Metabolic Alterations regarding Adipokine Levels

    Science.gov (United States)

    Batista, Nathália Vieira; Fonseca, Roberta Cristelli; Perez, Denise; Pereira, Rafaela Vaz Sousa; de Lima Alves, Juliana; Pinho, Vanessa; Faria, Ana Maria Caetano; Cara, Denise Carmona

    2016-01-01

    Platelet-activating factor (PAF) is known to be an important mediator of anaphylaxis. However, there is a lack of information in the literature about the role of PAF in food allergy. The aim of this work was to elucidate the participation of PAF during food allergy development and the consequent adipose tissue inflammation along with its alterations. Our data demonstrated that, both before oral challenge and after 7 days receiving ovalbumin (OVA) diet, OVA-sensitized mice lacking the PAF receptor (PAFR) showed a decreased level of anti-OVA IgE associated with attenuated allergic markers in comparison to wild type (WT) mice. Moreover, there was less body weight and adipose tissue loss in PAFR-deficient mice. However, some features of inflamed adipose tissue presented by sensitized PAFR-deficient and WT mice after oral challenge were similar, such as a higher rate of rolling leukocytes in this tissue and lower circulating levels of adipokines (resistin and adiponectin) in comparison to nonsensitized mice. Therefore, PAF signaling through PAFR is important for the allergic response to OVA but not for the adipokine alterations caused by this inflammatory process. Our work clarifies some effects of PAF during food allergy along with its role on the metabolic consequences of this inflammatory process. PMID:27314042

  19. Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses

    NARCIS (Netherlands)

    Tunjungputri, R.N.; Ven, A.J.A.M. van der; Riksen, N.P.; Rongen, G.A.P.J.M.; Tacke, S.; Berg, T.N.A. van den; Fijnheer, R.; Gomes, M.E.; Dinarello, C.A.; Veerdonk, F.L. van de; Gasem, M.H.; Netea, M.G.; Joosten, L.A.B.; Groot, P.G. de; Mast, Q. de

    2015-01-01

    Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist

  20. Effects of Buyang Huanwu decoction and Astragalus mongholicus on platelet activating factor receptor activity in rabbits in vitro

    Institute of Scientific and Technical Information of China (English)

    Hui Yao; Jiping Zhang; Zhixi Chen; Yongjie Wu; Zhiqiang Li

    2006-01-01

    BACKGROUND:The pharmacological action of traditional Chinese medicine compound is the comprehensive effect of the various ingredients,and the interactions of various ingredients are closely correlated with the final effect.In order to reveal the compatibility mechanism of BHD's prescription in treating and preventing ischemic cerebrovascular disease,we needed explore the effect and relation of ingredients in the prescription.OBJECTIVE:To observe the effect of Buyang Huanwu decoction(BHD)and Astragalus mongholicus on the activity of platelet activating factor receptor(PAFR)in the platelet of rabbits in vitro,and investigate the mechanism of Astragalus mongholicus.DESIGN:A decomposed recipes study.SETTING:Guangzhou University of Traditional Chinese Medicine.MATERIALS:Five New Zealand rabbits,weighing 2-3 kg,both sexes,were used.BHD was composed of Sheng Huang Qi 120 g,Dang Gui Wei 6 g,Chi Shao 4.5 g,Chuan Xiong 3 g,Di Long 3 g,Tao Ren 3 g,Hong Hua 3 g.The prescription for activating blood circulation consisted of Dang Gui Wei 6 g,Chi Shao 4.5 g,Chuan Xiong 3 g,Di Long 3 g,Tao Ren 3 g and Hong Hua 3 g.The prescription for invigorating qi consisted of 120 g Sheng Huang Qi.The prepared herbal pieces were purchased from the traditional Chinese medicine Dispensary of Foshan Second People's Hospital,and appraised by Professor Xu from Science of Chinese Materia Medica College,Guangzhou University of Traditional Chinese Medicine.3H-PAF was supplied by Amersham Co.,Ltd.(specific activity:6.475 TBq/mmol;batch number:200402);PAF standard by Biomol Co.,Ltd.(batch number:P1318V).METHODS:The experiments were carried out in the Laboratory of Nuclear Medicine,Guangzhou University of Traditional Chinese Medicine from September to December 2004.①Injections of BHD,prescriptions for activating blood circulation and invigorating qi were prepared by the decoction and alcohol sedimentation technique.Rabbit common carotid artery blood(40 mL)was drawn via intubation to prepare platelet

  1. RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets

    Science.gov (United States)

    Akbar, Huzoor; Duan, Xin; Saleem, Saima; Davis, Ashley K.; Zheng, Yi

    2016-01-01

    Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively. PMID:27681226

  2. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    Science.gov (United States)

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  3. Platelet surface glutathione reductase-like activity.

    Science.gov (United States)

    Essex, David W; Li, Mengru; Feinman, Richard D; Miller, Anna

    2004-09-01

    We previously found that reduced glutathione (GSH) or a mixture of GSH/glutathione disulfide (GSSG) potentiated platelet aggregation. We here report that GSSG, when added to platelets alone, also potentiates platelet aggregation. Most of the GSSG was converted to GSH by a flavoprotein-dependent platelet surface mechanism. This provided an appropriate redox potential for platelet activation. The addition of GSSG to platelets generated sulfhydryls in the beta subunit of the alpha(IIb)beta(3) fibrinogen receptor, suggesting a mechanism for facilitation of agonist-induced platelet activation.

  4. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    NARCIS (Netherlands)

    O.A. Hamad; P.H. Nilsson; D. Wouters; J.D. Lambris; K.N. Ekdahl; B. Nilsson

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfac

  5. Discoidin domain receptor 2-microRNA 196a-mediated negative feedback against excess type I collagen expression is impaired in scleroderma dermal fibroblasts.

    Science.gov (United States)

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Hirano, Ayaka; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Fukushima, Satoshi; Inoue, Yuji; Ihn, Hironobu

    2013-01-01

    Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

  6. Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets.

    Science.gov (United States)

    Park, Won-Hwan; Kim, Han-Kyu; Nam, Kyung-Soo; Shon, Yun-Hee; Jeon, Byung Hun; Moon, Sung-Kwon; Kim, Min-Gon; Kim, Cheorl-Ho

    2004-11-05

    Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.

  7. Platelet Activation and Thrombus Formation over IgG Immune Complexes Requires Integrin αIIbβ3 and Lyn Kinase.

    Science.gov (United States)

    Zhi, Huiying; Dai, Jing; Liu, Junling; Zhu, Jieqing; Newman, Debra K; Gao, Cunji; Newman, Peter J

    2015-01-01

    IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.

  8. Platelet Activation and Thrombus Formation over IgG Immune Complexes Requires Integrin αIIbβ3 and Lyn Kinase.

    Directory of Open Access Journals (Sweden)

    Huiying Zhi

    Full Text Available IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.

  9. The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet alpha(IIbbeta(3 function.

    Directory of Open Access Journals (Sweden)

    Francisca C Gushiken

    Full Text Available Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin alpha(IIbbeta(3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c interacts with alpha(IIbbeta(3, the role of PP1c in platelet reactivity is unclear.Using gamma isoform of PP1c deficient mice (PP1cgamma(-/-, we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4-activating peptide but not to adenosine diphosphate (ADP, collagen or collagen-related peptide (CRP. Thrombin-stimulated PP1cgamma(-/- platelets showed decreased alpha(IIbbeta(3 activation despite comparable levels of alpha(IIbbeta(3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin alpha(IIbbeta(3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cgamma(-/- platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cgamma(-/- mice. Phosphorylation of glycogen synthase kinase (GSK3beta-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cgamma(-/- platelets by an AKT independent mechanism. Inhibition of GSK3beta partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cgamma(-/- platelets.These studies illustrate a role for PP1cgamma in maintaining GSK3beta-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

  10. Collagen I induces discoidin domain receptor (DDR) 1 expression through DDR2 and a JAK2-ERK1/2-mediated mechanism in primary human lung fibroblasts.

    Science.gov (United States)

    Ruiz, Pedro A; Jarai, Gabor

    2011-04-15

    Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1β identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.

  11. BAT3 interacts with transforming growth factor-beta (TGF-beta) receptors and enhances TGF-beta1-induced type I collagen expression in mesangial cells.

    Science.gov (United States)

    Kwak, Joon Hyeok; Kim, Sung Il; Kim, Jin Kuk; Choi, Mary E

    2008-07-11

    Transforming growth factor-beta1 (TGF-beta1) plays essential roles in a wide array of cellular processes, such as in development and the pathogenesis of tissue fibrosis, including that associated with progressive kidney diseases. Tight regulation of its signaling pathways is critical, and proteins that associate with the TGF-beta receptors may exert positive or negative regulatory effects on TGF-beta signaling. In the present study we employed a yeast-based two-hybrid screening system to identify BAT3 (HLA-B-associated transcript 3) as a TGF-beta receptor-interacting protein. Analysis of endogenously expressed BAT3 in various tissues including the kidney reveals the existence of approximately 140-kDa full-length protein as well as truncated forms of BAT3 whose expression is developmentally regulated. Endogenous BAT3 protein interacts with TGF-beta receptors type I and type II in renal mesangial cells. Functional assays show that expression of full-length BAT3 results in enhancement of TGF-beta1-stimulated transcriptional activation of p3TP-Lux reporter, and these effects require the presence of functional TGF-beta signaling receptors as demonstrated in R-1B and DR-26 mutant cells. Moreover, expression of full-length BAT3, but not C-terminal truncated mutant of BAT3, enhanced TGF-beta1-induced type I collagen expression in mesangial cells, whereas knock down of BAT3 protein expression by small interfering RNA suppressed the expression of type I collagen induced by TGF-beta1. Our findings suggest that BAT3, a TGF-beta receptor-interacting protein, is capable of modulating TGF-beta signaling and acts as a positive regulator of TGF-beta1 stimulation of type I collagen expression in mesangial cells.

  12. Platelet-Derived Growth Factor-Receptor α Strongly Inhibits Melanoma Growth In Vitro and In Vivo1

    Science.gov (United States)

    Faraone, Debora; Aguzzi, Maria Simona; Toietta, Gabriele; Facchiano, Angelo M; Facchiano, Francesco; Magenta, Alessandra; Martelli, Fabio; Truffa, Silvia; Cesareo, Eleonora; Ribatti, Domenico; Capogrossi, Maurizio C; Facchiano, Antonio

    2009-01-01

    Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Rα may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Rα respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Rα. Proliferation was rescued by PDGF-Rα inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Rα mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Rα was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Rα show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Bα and a marked increase of p38γ, mitogen-activated protein kinase kinase 3, and signal regulatory protein α1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Rα reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Rα strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control. PMID:19649203

  13. Platelet-Derived Growth Factor-Receptor α Strongly Inhibits Melanoma Growth In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Debora Faraone

    2009-08-01

    Full Text Available Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Rα may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Rα respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Rα. Proliferation was rescued by PDGF-Rα inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Rα mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Rα was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Rα show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Bα and a marked increase of p38γ, mitogen-activated protein kinase kinase 3, and signal regulatory protein α1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Rα reached a significant 70% inhibition of primary melanoma growth (P < .001 and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Rα strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

  14. Platelet-Derived Growth Factor (PDGF/PDGF Receptors (PDGFR Axis as Target for Antitumor and Antiangiogenic Therapy

    Directory of Open Access Journals (Sweden)

    Anca Maria Cimpean

    2010-03-01

    Full Text Available Angiogenesis in normal and pathological conditions is a multi-step process governed by positive and negative endogenous regulators. Many growth factors are involved in different steps of angiogenesis, like vascular endothelial growth factors (VEGF, fibroblast growth factor (FGF-2 or platelet-derived growth factors (PDGF. From these, VEGF and FGF-2 were extensively investigated and it was shown that they significantly contribute to the induction and progression of angiogenesis. A lot of evidence has been accumulated in last 10 years that supports the contribution of PDGF/PDGFR axis in developing angiogenesis in both normal and tumoral conditions. The crucial role of PDGF-B and PDGFR-β in angiogenesis has been demonstrated by gene targeting experiments, and their expression correlates with increased vascularity and maturation of the vascular wall. PDGF and their receptors were identified in a large variety of human tumor cells. In experimental models it was shown that inhibition of PDGF reduces interstitial fluid pressure in tumors and enhances the effect of chemotherapy. PDGFR have been involved in the cardiovascular development and their loss leads to a disruption in yolk sac blood vessels development. PDGFRβ expression by pericytes is necessary for their recruitment and integration in the wall of tumor vessels. Endothelial cells of tumor-associated blood vessels can express PDGFR. Based on these data, it was suggested the potential benefit of targeting PDGFR in the treatment of solid tumors. The molecular mechanisms of PDGF/PDGFR-mediated angiogenesis are not fully understood, but it was shown that tyrosine kinase inhibitors reduce tumor growth and angiogenesis in experimental xenograft models, and recent data demonstrated their efficacy in chemoresistant tumors. The in vivo effects of PDGFR inhibitors are more complex, based on the cross-talk with other angiogenic factors. In this review, we summarize data regarding the mechanisms and

  15. Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.

    Science.gov (United States)

    Greiling, D; Clark, R A

    1997-04-01

    After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

  16. Saccharomyces boulardii improves intestinal cell restitution through activation of the α2β1 integrin collagen receptor.

    Directory of Open Access Journals (Sweden)

    Alexandra Canonici

    Full Text Available Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

  17. Crystal structure of the MrkD1P receptor binding domain of Klebsiella pneumoniae and identification of the human collagen V binding interface.

    Science.gov (United States)

    Rêgo, Ana Toste; Johnson, Jeremiah G; Gelbel, Sebastian; Enguita, Francisco J; Clegg, Steven; Waksman, Gabriel

    2012-11-01

    Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD1P receptor domain (MrkDrd). The structure reveals a jelly-roll β-barrel fold comprising 17 β-strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n-acetyl-d-glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD1P mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD1P recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.

  18. Histological investigation of the effect of soybean (Glycine max extracts on the collagen layer and estrogen receptors in the skin of female rats

    Directory of Open Access Journals (Sweden)

    Belkiz Uyar

    2014-01-01

    Full Text Available OBJECTIVES: The purpose of this study was to analyze the effects of soybean extracts obtained using different extraction methods on the skin of female rats. METHOD: A total of 64 female Sprague-Dawley rats were divided into 8 equal groups. Various extracts were administered to the female rats by oral gavage for one month. The groups comprised carboxymethyl cellulose-free control, carboxymethyl cellulose-plus control, 100-mg/kg n-hexane extract, 200-mg/kg n-hexane extract, 100-mg/kg ethyl acetate extract, 200-mg/kg ethyl acetate extract, 100-mg/kg ethanol extract and 200-mg/kg ethanol extract groups. The thickness of the collagen layer and the number of estrogen receptor-positive cells were evaluated. RESULTS: All the extract-treated groups showed a statistically significant decrease in the number of estrogen receptor-positive cells compared with the control groups. Regarding the thickness of the collagen layer, only the 200-mg/kg ethyl acetate extract-treated group showed a significant increase compared with the control groups (p<0.05. CONCLUSIONS: Our data suggest that oral intake of three different total soybean extracts might have positive estrogenic effects on the skin and that only a high-dose ethyl acetate extract can increase the expression of collagen, which may prove to be beneficial for postmenopausal facial skin.

  19. The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers

    DEFF Research Database (Denmark)

    Nielsen, Christoffer Fagernæs; van Putten, Sander Maarten; Lund, Ida Katrine

    2017-01-01

    A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelia......A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non......-epithelial cancers, notably including sarcomas, glioblastomas and subsets of acute myeloid leukemia. In contrast, in healthy adult individuals, expression is restricted to minor subsets of mesenchymal cells. Functionally, uPARAP/Endo180 is a rapidly recycling endocytic receptor that delivers its cargo directly...... into the endosomal-lysosomal system, thus opening a potential route of entry into receptor-positive cells. This combination of specific expression and endocytic function appears well suited for targeting of uPARAP/Endo180-positive cancers by antibody-drug conjugate (ADC) mediated drug delivery. Therefore, we...

  20. Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis.

    Science.gov (United States)

    Medina, Carlos; Santos-Martinez, Maria Jose; Santana, Alfredo; Paz-Cabrera, Maria Cristina; Johnston, Michael J; Mourelle, Marisabel; Salas, Antonio; Guarner, Francisco

    2011-08-01

    Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.

  1. 5-HT receptor probe (/sup 3/H)8-OH-DPAT labels the 5-HT transporter in human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Ieni, J.R.; Meyerson, L.R.

    1988-01-01

    The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using (/sup 3/H)8-OH-DPAT as the radioligand. (/sup 3/H)8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average K/sub D/ of 43 nM and B/sub max/ of 1078 fmol/mg protein. Determinations of IC/sub 50/ values for various serotonergic characterizing agents in platelets for displacement of (/sup 3/H)8-OH-DPAT were performed. The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit (/sup 3/H) imipramine binding, however, it does inhibit (/sup 3/H)5-HT uptake in human platelets near 5-HT's K/sub m/ value (IC/sub 50/ = 2-4 ..mu..M). These results suggest that the human platelet site labelled by (/sub 3/H)8-OH-DPAT is pharmocologically different from the neuronal site and probably is a component of the 5-HT transporter. 32 references, 1 figure, 4 tables.

  2. Rho GTPases in platelet function.

    Science.gov (United States)

    Aslan, J E; McCarty, O J T

    2013-01-01

    The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low-molecular-weight or 'small' GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non-transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho-driven cellular processes. © 2012 International Society on Thrombosis and Haemostasis.

  3. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hua-Yu; Li, Chao; Zheng, Zhao; Zhou, Qin; Guan, Hao; Su, Lin-Lin; Han, Jun-Tao; Zhu, Xiong-Xiang; Wang, Shu-yue; Li, Jun, E-mail: lijunfmmu@163.com; Hu, Da-Hai, E-mail: hudahaifmmu@aliyun.com

    2015-03-27

    The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.

  4. Platelet mimicry

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities...

  5. Platelet Count

    Science.gov (United States)

    ... their spleen removed surgically Use of birth control pills (oral contraceptives) Some conditions may cause a temporary (transitory) increased ... increased platelet counts include estrogen and birth control pills (oral contraceptives). Mildly decreased platelet counts may be seen in ...

  6. Glycoxidized HDL, HDL enriched with oxidized phospholipids and HDL from diabetic patients inhibit platelet function.

    Science.gov (United States)

    Lê, Quang Huy; El Alaoui, Meddy; Véricel, Evelyne; Ségrestin, Bérénice; Soulère, Laurent; Guichardant, Michel; Lagarde, Michel; Moulin, Philippe; Calzada, Catherine

    2015-05-01

    High-density lipoproteins (HDL) possess atheroprotective properties including anti-thrombotic and antioxidant effects. Very few studies relate to the functional effects of oxidized HDL on platelets in type 2 diabetes (T2D). The objective of our study was to investigate the effects of in vitro glycoxidized HDL and HDL from patients with T2D on platelet aggregation and arachidonic acid signaling cascade. At the same time, the contents of hydroxylated fatty acids were assessed in HDL. Compared with control HDL, in vitro glycoxidized HDL had decreased proportions of linoleic (LA) and arachidonic (AA) acids in phospholipids and cholesteryl esters, and increased concentrations of hydroxy-octadecadienoic acids (9-HODE and 13-HODE) and 15-hydroxy-eicosatetraenoic acid (15-HETE), derived from LA and AA respectively, especially hydroxy derivatives esterified in phospholipids. Glycoxidized HDL dose-dependently decreased collagen-induced platelet aggregation by binding to scavenger receptor BI (SR-BI). Glycoxidized HDL prevented collagen-induced increased phosphorylation of platelet p38 MAPK and cytosolic phospholipase A2, as well as intracellular calcium mobilization. HDL enriched with oxidized phosphatidylcholine (PC), namely PC(16:0/13-HODE) dose-dependently inhibited platelet aggregation. Increased concentrations of 9-HODE, 13-HODE, and 15-HETE in phospholipids (2.1-, 2.1-, and 2.4-fold increase, respectively) were found in HDL from patients with T2D, and these HDL also inhibited platelet aggregation via SR-BI. Our results suggest that in vitro glycoxidized HDL as well as HDL from patients with T2D inhibit platelet aggregation, and suggest that oxidized LA-containing phospholipids may contribute to the anti-aggregatory effects of glycoxidized HDL and HDL from patients with T2D.

  7. Effect of Bexarotene on Platelet Activation and Apoptosis

    Directory of Open Access Journals (Sweden)

    Hang Cao

    2017-06-01

    Full Text Available Background/Aims: The retinoid X receptor (RXRs stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthylethynyl] benzoic acid is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i, which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP. The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml without or with an additional treatment with thrombin (0.01 U/ml or CRP (2 µg/ml or 5 µg/ml. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, and relative platelet volume from forward scatter. Results: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin

  8. Immunohistochemical examination of effects of kefir, koumiss and commercial probiotic capsules on platelet derived growth factor-c and platelet derived growth factor receptor-alpha expression in mouse liver and kidney.

    Science.gov (United States)

    Bakir, B; Sari, E K; Aydin, B D; Yildiz, S E

    2015-04-01

    We investigated using immunohistochemistry the effects of kefir, koumiss and commercial probiotic capsules on the expression of platelet derived growth factor-c (PDGF-C) and platelet derived growth factor receptor-alpha (PDGFR-α) in mouse liver and kidney. Mice were assigned to four groups: group 1 was given commercial probiotic capsules, group 2 was given kefir, group 3 was given koumiss and group 4 was untreated. After oral administration for 15 days, body weights were recorded and liver and kidney tissue samples were obtained. Hematoxylin and eosin staining was used to examine histology. PDGF-C and PDGFR-α in liver and kidney were localized using the streptavidin-biotin peroxidase complex method (ABC). We found that the weights of the mice in the kefir, koumiss and commercial probiotic capsules groups increased compared to the control group. No differences in liver and kidney histology were observed in any of the experimental groups. Kefir, koumiss and the commercial probiotic preparation increased PDGF-C and PDGFR-α expression.

  9. Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepal-6 cells and its relationship with collagen type Ⅳ

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type Ⅳ (collagen Ⅳ) in mouse hepatoma cell line Hepal-6 cells.METHODS: The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor (ActRII)and collagen Ⅳ expression were evaluated.RESULTS: The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). TheARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% ± 5.7% vS 48.1% ± 3.6%, P < 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells.CONCLUSION: These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

  10. Collagenous gastroduodenitis.

    Science.gov (United States)

    Rustagi, Tarun; Rai, Mridula; Scholes, John V

    2011-10-01

    Collagenous gastroduodenitis is a rare histopathologic entity characterized by marked subepithelial collagen deposition with associated mucosal inflammatory infiltrate. Only 4 cases have been reported, of which 3 had associated collagenous colitis. Collagenous gastroduodenitis without colonic involvement is exceptionally rare with only 1 case reported so far in the literature. We present a case of a 68-year-old woman with dyspepsia and mild anemia, who was found to have nodular gastric and duodenal mucosa on endoscopic examination. Histopathology showed collagenous gastroduodenitis. To the best of our knowledge, this is the second (and first in English literature) reported case of isolated collagenous gastroduodenitis.

  11. Glycoprotein IIb/IIIa and P2Y12 Induction by Oligochitosan Accelerates Platelet Aggregation

    Science.gov (United States)

    Halim, Ahmad Sukari; Hussein, Abdul Rahim; Ujang, Zanariah

    2014-01-01

    Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro. PMID:25247182

  12. Novel aspects of platelet aggregation

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    Roka-Moya Y. M.

    2014-01-01

    Full Text Available The platelet aggregation is an important process, which is critical for the hemostatic plug formation and thrombosis. Recent studies have shown that the platelet aggregation is more complex and dynamic than it was previously thought. There are several mechanisms that can initiate the platelet aggregation and each of them operates under specific conditions in vivo. At the same time, the influence of certain plasma proteins on this process should be considered. This review intends to summarize the recent data concerning the adhesive molecules and their receptors, which provide the platelet aggregation under different conditions.

  13. Exacerbation of Glycoprotein VI-Dependent Platelet Responses in a Rhesus Monkey Model of Type 1 Diabetes

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    J. F. Arthur

    2013-01-01

    Full Text Available Thrombosis is a life-threatening complication of diabetes. Platelet reactivity is crucial to thrombus formation, particularly in arterial vessels and in thrombotic complications causing myocardial infarction or ischaemic stroke, but diabetic patients often respond poorly to current antiplatelet medication. In this study, we used a nonhuman primate model of Type 1 diabetes to measure early downstream signalling events following engagement of the major platelet collagen receptor, glycoprotein (GPVI. Diabetic monkeys were given enough insulin to maintain their blood glucose levels either at ~8 mM (well-controlled diabetes or ~15 mM (poorly controlled diabetes. Flow cytometric analysis was used to measure platelet reactive oxygen species (ROS generation, calcium mobilisation, receptor surface expression, and immature platelet fraction. We observed exacerbated intracellular ROS and calcium flux associated with engagement of GPVI in monkeys with poorly controlled diabetes. GPVI surface levels did not differ between healthy monkeys or the two diabetic groups. Treatment of platelets with the specific Syk inhibitor BAY61-3606 inhibited GPVI-dependent ROS and, importantly, reduced ROS generation in the poorly controlled diabetes group to that observed in healthy monkeys. These data indicate that glycaemic control is important in reducing GPVI-dependent platelet hyperreactivity and point to a potential antithrombotic therapeutic benefit of Syk inhibition in hyperglycaemic diabetes.

  14. Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in rat hepatic stellate cells.

    Science.gov (United States)

    Koide, Shigeki; Kobayashi, Yoshimasa; Oki, Yutaka; Nakamura, Hirotoshi

    2004-09-01

    Prostaglandin (PG) E2 inhibits hepatic stellate cell (HSC) mitogenesis. PGE-specific receptors are divided into four subtypes that are coupled either to Ca2+ mobilization (EP1 and EP3) or to the stimulation of adenyl cyclase (EP2 and EP4). The aims of the current study were to identify PGE receptor subtypes in cultured rat HSC and to examine which PGE receptor subtype(s) mediates the inhibitory effect of PGE2 on platelet-derived growth factor (PDGF)-stimulated proliferation. Reverse transcription-polymerase chain reaction analysis was performed to detect PGE receptor subtype mRNA expression. Cell proliferation was determined by measuring [3H]thymidine incorporation, and intracellular cyclic AMP was measured by radioimmunoassay. Cultured rat HSC expressed mRNAs for all four subtypes of PGE receptor. PGE2- and EP2-selective agonist produced dose-dependent inhibitory effects on PDGF-stimulated proliferation. Neither EP1-, EP3-, nor EP4-selective agonists showed any inhibitory effect. An adenylate cyclase inhibitor strongly blunted the inhibition of DNA synthesis elicited by PGE2 and the EP2 agonist. The EP2 agonist generated higher and more prolonged increases in intracellular cyclic AMP than the EP4 agonist. Activation of the PGE EP2 receptor has an antiproliferative effect in HSC that may be mediated by cyclic AMP-related signal transduction pathways.

  15. Up and Down Expression of Androgen Receptor,Estrogen Receptor beta and Platelet Derived Growth Factor beta by Testosterone in Aortic Vascular Smooth Muscle Tissues

    Institute of Scientific and Technical Information of China (English)

    Wu Saizhu; Lv Hongsong; Zhou Kexiang; Sun Fei; Ma Rui; Zheng Hua; Wei Heming; Rong Zhiyi

    2004-01-01

    Objectives To investigate the effects of testosterone enanthate(TE) on serum lipids and lipoproteins metabolism and the expression of androgen receptor ( AR), estrogen receptor beta ( ER -β) and platelet derived growth factor beta (PDGFR-β ) in aortic vascular smooth muscle tissues(VSMTs). Methods Forty aged male rats were randomly divided into 4 groups, group A (placebo group),group B (2.5 mg/kg intramuscular injection of TE once a week ), group C (5.0 mg/kg intramuscular injection of TE once a week ), group D ( 10.0 mg,/kg intramuscular injection of TE once a week). All animals were fed freely during 16 - week treatment periods. The expression of AR , ER - βand PDGFR - β were studied by Western bolt. Results Average serum LDL - C was lower in group D than that in group A ( p < 0.01 ).Compared with the other groups, average serum TC was also lower in group D ( p < 0.05). AR expression in aortic vascular smooth muscle tissues could be regulated by TE: 99.50 ± 21.74, 125.38 ± 28.68 and 101.98 ±15.42 for TE concentrations at 2.5 mg/kg, 5.0 mg/kgand 10.0 mg/kg, respectively , the expression of ER -β could be regulated by TE: 92.34 ± 18.68, 47.72 ±18.12, 82.13 ±23.50, and the expression of PDGFR -β could be regulated as well by TE: 219.70 ± 45.59,50.16 ± 9.72, 125.36 ± 15.74 ( Data for AR , ER - βand PDGFR - β protein band intensity were expressed with x ± s, with control group taken as 100).Conclusions This study indicates that androgens have significant effects on serum lipids and lipoprotein metabolism. Testosterone enanthate at 5.0 mg/kg can stimulate the expression of AR, but inhibite the expression of PDGFR. Testosterone enanthate at the concentrations of 5.0 mg/kg and 10.0 mg/kg can inhibite the expression of ER - β.

  16. Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

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    Dalal, K.B.; Ebbe, S.; Mazoyer, E.; Carpenter, D.; Yee, T. (Lawrence Berkeley Laboratory, CA (USA))

    1990-02-01

    This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700-1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55 +/- 0.011 to 0.89 +/- 0.016 (P less than 0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of (14C)serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of (14C)serotonin in HC and normal platelets ranged from 78-94%. The percent of (14C)serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 microM acetylsalicylic acid into the aggregating medium suppressed the release of (14C)serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.

  17. Collagenous sprue

    DEFF Research Database (Denmark)

    Soendergaard, Christoffer; Riis, Lene Buhl; Nielsen, Ole Haagen

    2014-01-01

    Collagenous sprue is a rare clinicopathological condition of the small bowel. It is characterised by abnormal subepithelial collagen deposition and is typically associated with malabsorption, diarrhoea and weight loss. The clinical features of collagenous sprue often resemble those of coeliac dis...... disease and together with frequent histological findings like mucosal thinning and intraepithelial lymphocytosis the diagnosis may be hard to reach without awareness of this condition. While coeliac disease is treated using gluten restriction, collagenous sprue is, however, not improved...

  18. Platelet-activating factor receptor (PAF-R-dependent pathways control tumour growth and tumour response to chemotherapy

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    Rohde Ciro BS

    2010-05-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170. These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2, caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF and prostaglandin E2 (PGE2 were determined by ELISA, and levels of nitric oxide (NO by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained

  19. Estrogen, inflammation, and platelet phenotype.

    Science.gov (United States)

    Miller, Virginia M; Jayachandran, Muthuvel; Hashimoto, Kazumori; Heit, John A; Owen, Whyte G

    2008-01-01

    Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

  20. Decreased TGF-β1 and VEGF Release in Cystic Fibrosis Platelets: Further Evidence for Platelet Defects in Cystic Fibrosis

    Science.gov (United States)

    Maloney, James P.; Narasimhan, Jayashree; Biller, Julie

    2016-01-01

    Purpose Cystic fibrosis (CF) patients suffer from chronic lung inflammation. This inflammation may activate platelets. There are limited data on the role of platelet-secreted cytokines in CF. Platelet cytokines with inflammatory effects include vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1). As levels of these cytokines are tenfold greater in serum than plasma due to platelet release, serum levels may be one index of platelet content; but a more specific index is release during the aggregation of isolated platelets. We postulated that altered release of these platelet cytokines occurs in CF. Methods We obtained sera and plasma from CF outpatients (n=21) and from healthy controls (n=20), measured VEGF and TGF-β1, assessed for correlations with platelet number, analyzed cytokine release during platelet aggregation to collagen, and analyzed differences in maximal platelet aggregation. Results Platelet number and maximal aggregation levels were higher in CF. Plasma and serum levels of TGF-β1 and VEGF were higher in CF, but these levels were similar after adjusting for platelet number (serum cytokines correlated with platelet count). The release of VEGF and TGF-β1 during aggregation was decreased in CF platelets (by 52% and 29%, respectively). Conclusion Platelet release is not a source of the elevated blood proinflammatory cytokines TGF-β1 and VEGF in CF, as platelets from CF patients actually release less of these cytokines. These data provide further evidence for platelet defects in CF. PMID:27423781

  1. Inhibition of Wnt/β-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8.

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    Ismaïl Hendaoui

    Full Text Available The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs, which have a cysteine-rich domain (CRD structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18 inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.

  2. Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

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    Jacqueline Stockley

    Full Text Available The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12 could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =, both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =. Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

  3. Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

    DEFF Research Database (Denmark)

    Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

    2010-01-01

    Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation of h......-life of osteoblastic PDGFR-alpha mRNA, but did not decrease its promoter activity. In summary, our data show that PDGFR-alpha is downregulated in hOBs by co-cultivation with human primary endothelial cells through a p38 MAPK-dependent post-transcriptional mechanism.......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co-cultivation...... of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

  4. Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor.

    Science.gov (United States)

    Holdfeldt, André; Dahlstrand Rudin, Agnes; Gabl, Michael; Rajabkhani, Zahra; König, Gabriele M; Kostenis, Evi; Dahlgren, Claes; Forsman, Huamei

    2017-09-01

    Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2(-)) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2(-), producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2(-) through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off. © Society for Leukocyte Biology.

  5. Dopamine D1-like receptor stimulation inhibits hypertrophy induced by platelet-derived growth factor in cultured rat renal vascular smooth muscle cells.

    Science.gov (United States)

    Yasunari, K; Kohno, M; Kano, H; Yokokawa, K; Minami, M; Yoshikawa, J

    1997-01-01

    Vascular smooth muscle cell (VSMC) hypertrophy is believed to play some roles in atherosclerosis. To elucidate the role of vascular D1-like receptors in VSMC hypertrophy, the effects of dopamine and specific D1-like receptor agonists SKF 38393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC hypertrophy was studied. We observed that cells stimulated by PDGF-BB 5 ng/mL showed increased VSMC hypertrophy. These effects were prevented by coincubation with dopamine, SKF 38393, and YM 435 1-10 mumol/L, and this prevention was reversed by Sch 23390 1 to 10 mumol/L, a specific D1-like receptor antagonist. These actions are mimicked by forskolin 1 to 10 mumol/L, a direct activator of adenylate cyclase and 8-bromo-cAMP 0.1 to 1 mmol/L, and are blocked by a specific protein kinase A (PKA) inhibitor N-[2-(P-bromcoinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H89) but not blocked by its negative control. PDGF-BB (5 ng/mL)-mediated mitogen-activated protein kinase (MAPK) activity was significantly suppressed by coincubation with D1-like receptor agonists, which were reversed by PKA inhibitor H 89. These results suggest that vascular D1-like receptor agonists inhibit hypertrophy of VSMC, possibly through PKA activation and suppression of activated MAPK activity.

  6. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

    Science.gov (United States)

    Salles, Isabelle I; Thijs, Tim; Brunaud, Christine; De Meyer, Simon F; Thys, Johan; Vanhoorelbeke, Karen; Deckmyn, Hans

    2009-12-01

    Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

  7. Aromatic Interactions Promote Self-association of Collagen Triple-helical Peptides to Higher Order Structures

    Science.gov (United States)

    Kar, Karunakar; Ibrar, Sajjad; Nanda, Vikas; Getz, Todd M.; Kunapuli, Satya P.; Brodsky, Barbara

    2009-01-01

    Aromatic residues are relatively rare within the collagen triple-helix, but they appear to play a specialized role in higher order structure and function. The role of aromatic amino acids in the self-assembly of triple-helical peptides was investigated in terms of the kinetics of self-association, the nature of aggregated species formed, and the ability of these species to activate platelet aggregation. The presence of aromatic residues on both ends of a type IV collagen model peptide is observed to greatly accelerate the kinetics of self-association, decreasing the lag time and leading to insoluble, well defined linear fibrils as well as small soluble aggregates. Both macroscopic visible aggregates and small multi-molecular complexes in solution are capable of inducing platelet aggregation through the glycoprotein VI receptor on platelets. Proline-aromatic CH⋯π interactions are often observed within globular proteins and in protein complexes, and examination of molecular packing in the crystal structure of the integrin binding collagen peptide shows Phe interacts with Pro/Hyp in a neighboring triple-helical molecule. An intermolecular interaction between aromatic amino acids and imino acids within the triple-helix is also supported by the observed inhibitory effect of isolated Phe amino acids on the self-association of (Pro-Hyp-Gly)10. Given the high fraction of Pro and Hyp residues on the surface of collagen molecules, it is likely that imino acid-aromatic CH⋯π interactions are important in formation of higher order structure. It is suggested that the catalysis of type I collagen fibrillogenesis by non-helical telopeptides is due to specific intermolecular CH⋯π interactions between aromatic residues in the telopeptides and Pro/Hyp residues within the triple-helix. PMID:19610672

  8. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia

    Science.gov (United States)

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP. PMID:27321376

  9. Cyclosporine A enhances platelet aggregation.

    Science.gov (United States)

    Grace, A A; Barradas, M A; Mikhailidis, D P; Jeremy, J Y; Moorhead, J F; Sweny, P; Dandona, P

    1987-12-01

    In view of the reported increase in thromboembolic episodes following cyclosporine A (CyA) therapy, the effect of this drug on platelet aggregation and thromboxane A2 release was investigated. The addition of CyA, at therapeutic concentrations to platelet rich plasma from normal subjects in vitro was found to increase aggregation in response to adrenaline, collagen and ADP. Ingestion of CyA by healthy volunteers was also associated with enhanced platelet aggregation. The CyA-mediated enhancement of aggregation was further enhanced by the addition in vitro of therapeutic concentrations of heparin. Platelets from renal allograft recipients treated with CyA also showed hyperaggregability and increased thromboxane A2 release, which were most marked at "peak" plasma CyA concentration and less so at "trough" concentrations. Platelet hyperaggregability in renal allograft patients on long-term CyA therapy tended to revert towards normal following the replacement of CyA with azathioprine. Hypertensive patients with renal allografts on nifedipine therapy had normal platelet function and thromboxane release in spite of CyA therapy. These observations suggest that CyA-mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug. The increased release of thromboxane A2 (a vasoconstrictor) may also play a role in mediating CyA-related nephrotoxicity.

  10. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    Energy Technology Data Exchange (ETDEWEB)

    Kalayarasan, Srinivasan, E-mail: kalaivasanbio@gmail.com; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam, E-mail: sudhandiran@yahoo.com

    2013-09-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.

  11. [Mechanism of cooked blanched garlic leaves against platelet aggregation].

    Science.gov (United States)

    Wang, Xin-Hua; Di, Yan-Hui

    2014-06-01

    This study was purposed to explore the mechanism of cooked blanched garlic leave juice against platelet aggregation. The juice of blanched garlic leaves was mixed with platelet rich plasma (PRP), the human platelet aggregation, the activation of human platelets induced by adenosine diphosphate (ADP) and collagen were observed; the expression levels of the activated platelets (Fib-R) and P-selectin (CD62P), and the amount of platelet fibrinogen binding were detected by flow cytometry; 10 rabbits were randomly divided into two groups, in addition to the normal diet, they were fed with physiologic saline and cooked blanched garlic leave juice respectively. After 1, 3, 5 , 8 weeks, the maximum ratio of rabbit platelet aggregation induced by ADP and collagen were observed . The results showed that the cooked blanched garlic leave juice could significantly inhibit human platelet aggregation induced by ADP and collagen (P 0.05), but was able to inhibit platelet fibrinogen binding capacity (P garlic leave juice was significantly lower than that in control group (P garlic leave juice can inhibit platelet aggregation in vitro and in vivo, the inhibition of aggregation pathway mainly is blocking the combination of fibrinogen with Fib-R, which finally results in the inhibition of platelet aggregation. Therefore, regular consumption of cooked blanched garlic leaves may prevent cardiovascular thrombotic diseases.

  12. Extracts from Trifolium pallidum and Trifolium scabrum aerial parts as modulators of blood platelet adhesion and aggregation.

    Science.gov (United States)

    Kolodziejczyk-Czepas, Joanna; Olas, Beata; Malinowska, Joanna; Wachowicz, Barbara; Szajwaj, Barbara; Kowalska, Iwona; Oleszek, Wieslaw; Stochmal, Anna

    2013-01-01

    A growing number of reports indicate that some species of clover (Trifolium) may have remarkable medical importance; however, the effects of these plants on blood platelets and hemostasis are inadequately recognized. This work was designed to study the effects of Trifolium pallidum and Trifolium scabrum extracts on the functions of human blood platelets in vitro. Platelet suspensions were preincubated with extracts from aerial parts of T. pallidum (phenolic fraction and clovamide fraction) and T. scabrum (phenolic fraction) at the final concentrations of 12.5, 25, and 50 µg/ml. Then, for platelet activation thrombin (0.1 U/ml), thrombin receptor activating peptide (TRAP; 20 µM), or adenosine diphosphate (ADP; 1 µM) were used. The effects of Trifolium extracts on adhesion of blood platelets to fibrinogen and collagen were determined by enzyme-linked immunosorbent assay (ELISA) method. Platelet aggregation was monitored on a dual-channel Chronolog aggregometer. In these studies, we also compared the action of tested plant extracts with the effects of another antiplatelet plant-derived compound - resveratrol (3,4',5-trihydroxystilbene). The performed assays demonstrated that the tested extracts might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of all tested extracts on adhesion of thrombin-stimulated platelets to collagen was found. Both extracts from T. pallidum and from T. scabrum reduced the thrombin-induced platelet adhesion to fibrinogen. Furthermore, in the presence of all three extracts, the platelet aggregation induced by thrombin was slightly inhibited. Our results also indicate that the tested plant extracts (at the highest concentrations used of 50 µg/ml), similar to purified resveratrol, inhibit selected steps of platelet activation stimulated by both proteolytic (thrombin) and nonproteolytic agonists (TRAP or ADP). In the comparative studies, T. pallidum and T. scabrum extracts was not found

  13. Foreign-body reaction to dermal sheep collagen in interferon-gamma-receptor knock-out mice

    NARCIS (Netherlands)

    Khouw, IMSL; van Wachem, PB; Plantinga, JA; Haagmans, BL; de Leij, LFMH; van Luyn, MJA

    2000-01-01

    This study was performed to gain more insight into the role of interferon-gamma (IFN-gamma), a potent macrophage activator, in the foreign-body reaction to hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC). Because the results of earlier studies aimed at modulating the foreign-body

  14. Platelet proteomics.

    Science.gov (United States)

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  15. Association of Oxidative Stress and Platelet Receptor Glycoprotein GPIbα and GPVI Shedding During Nonsurgical Bleeding in Heart Failure Patients With Continuous-Flow Left Ventricular Assist Device Support.

    Science.gov (United States)

    Mondal, Nandan K; Chen, Zengsheng; Trivedi, Jaimin R; Sorensen, Erik N; Pham, Si M; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

    2017-09-26

    Nonsurgical bleeding (NSB) in heart failure (HF) patients with continuous-flow left ventricular assist device (CF-LVAD) support is the most common clinical complication. The aim of this study was to investigate the association between oxidative stress and platelet glycoproteins GPIbα and GPVI shedding on the incidence of NSB in CF-LVAD patients. Fifty-one HF patients undergoing CF-LVAD implantation and 11 healthy volunteers were recruited. Fourteen patients developed NSB (bleeder group) during 1 month follow-up duration, while others were considered nonbleeder group (n = 37). Several biomarkers of oxidative stress were quantified at baseline and weekly intervals in all patients. Surface expression and plasma elements of platelet receptor glycoproteins GPIbα and GPVI were measured. Oxidative stress biomarkers and platelet GPIbα and GPVI receptor-shedding (decreased surface expression and higher plasma levels) were found to be preexisting conditions in baseline samples of both groups of HF patients when compared with healthy volunteers. Significantly elevated oxidative stress biomarkers and platelet glycoprotein receptor shedding were observed in postimplant bleeder group temporarily when compared with nonbleeder group. Strong significant associations between biomarkers of oxidative stress and platelet glycoprotein receptor shedding were observed, suggesting a possible role of oxidative stress in platelet integrin shedding leading to NSB in CF-LVAD patients. Receiver operating characteristic analyses of GPIbα and GPVI indicated that the likelihood of NSB had a predictive power of bleeding complication in CF-LVAD patients. In conclusion, elevated oxidative stress may play a role in GPIbα and GPVI shedding in the event of NSB. Thus, oxidative stress and GPIbα and GPVI shedding may be used as potential biomarkers for bleeding risk stratification in those patients.

  16. Effects of the platelet-activating factor receptor antagonist BN 52021 on hematologic variables and blood loss during and after cardiopulmonary bypass.

    Science.gov (United States)

    Nathan, N; Mercury, P; Denizot, Y; Cornu, E; Laskar, M; Arnoux, B; Feiss, P

    1994-08-01

    Cardiopulmonary bypass (CPB)-induced thrombocytopenia and leukopenia is augmented after heparin reversal of protamine. Platelet-activating factor (PAF) might be implicated in these disorders. To evaluate the effects of PAF on the hematologic disorders and blood loss during and after CPB, patients were pretreated with BN 52021, a PAF receptor antagonist, or a placebo. BN 52021 (120 mg) (n = 13) or placebo (n = 15) were infused intravenously before vascular cannulation and before cross-clamp release. Platelet and leukocyte counts were assessed in venous blood before and after the first dose of BN 52021 or placebo, 2 min after the beginning of CPB (at the entry of the oxygenator), at the end of CPB, 1, 15, and 30 min after protamine infusion, and 6 and 24 h after CPB. The decrease in platelet and leukocyte counts were the same between groups during and after CPB and after protamine infusion. Bleeding times were not modified by the pretreatment of patients with BN 52021. During surgery, blood loss reached 1660 +/- 297 mL in the BN 52021 group and 1599 +/- 283 mL in the placebo group (P > 0.05). Forty-eight hours postoperatively, the chest tube outputs were not different between groups (1460 +/- 418 mL vs 1640 +/- 362 mL in the BN 52021 and placebo groups, respectively). This study shows that BN 52021 infusion did not change the hematologic variables studied. Moreover, a PAF antagonist pretreatment did not protect the patients against CPB- or protamine-induced hematologic changes.

  17. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel.

    Science.gov (United States)

    Dawood, Ban B; Lowe, Gillian C; Lordkipanidzé, Marie; Bem, Danai; Daly, Martina E; Makris, Mike; Mumford, Andrew; Wilde, Jonathan T; Watson, Steve P

    2012-12-13

    Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

  18. Involvement of P2X7 receptor signaling on regulating the differentiation of Th17 cells and type II collagen-induced arthritis in mice

    Science.gov (United States)

    Fan, Zhi-Dan; Zhang, Ya-Yuan; Guo, Yi-Hong; Huang, Na; Ma, Hui-Hui; Huang, Hui; Yu, Hai-Guo

    2016-01-01

    Interleukin (IL)-17 producing T helper (Th17) cells are major effector cells in the pathogenesis of rheumatoid arthritis (RA). The P2X7 receptor (P2X7R) has emerged as a potential site in the regulation of inflammation in RA but little is known of its functional role on the differentiation of Th17 cells. This study investigates the in vitro and in vivo effects of P2X7R on Th17 cell differentiation during type II collagen (CII) induced experimental arthritis model. In CII-treated dendritic cells (DCs) and DC/CD4+ T coculture system, pretreatment with pharmacological antagonists of P2X7R (Suramin and A-438079) caused strong inhibition of production of Th17-promoting cytokines (IL-1β, TGF-β1, IL-23p19 and IL-6). Exposure to CII induced the elevation of mRNAs encoding retinoic acid receptor-related orphan receptor α and γt, which were abolished by pretreatment with P2X7R antagonists. Furthermore, blocking P2X7R signaling abolished the CII-mediated increase in IL-17A. Blockade of P2X7R remarkably inhibited hind paw swelling and ameliorated pathological changes in ankle joint of the collagen-induced arthritis mice. Thus, we demonstrated a novel function for P2X7R signaling in regulating CII-induced differentiation of Th17 cells. P2X7R signaling facilitates the development of the sophisticated network of DC-derived cytokines that favors a Th17 phenotype. PMID:27775097

  19. Increase in phosphotidylinositide-3 kinase activity by nitrotyrosylation of lysates of platelets from patients with systemic sclerosis.

    Science.gov (United States)

    Chiang, Thomas M; Postlethwaite, Arnold E

    2006-01-01

    We have observed that the platelet non-integrin collagen receptor (65 kDa) and another protein (M(r) 185 kDa) are altered in the posttranslational modification by nitrotyrosylation in platelets from patients with systemic sclerosis (SSc). We reported the identification of nitrotyrosylated 65-kDa proteins in a previous study. In the present investigation, using Western blots, one- and two-dimensional gel electrophoreses and matrix assisted ionization/desorption-time of flight (MALDI-TOF) we have identified the 185-kDa protein as phosphoinositide kinase C2beta (PI 3-K). There is a positive correlation between the nitrotyrosylation of PI 3-K and activity of the enzyme, i.e., the nitrotyrosylation of PI 3-K increases its enzymatic activity. In addition, the activity of PI 3-K increases in nitrotyrosylated platelet lystaes from patients with SSc compared to normal volunteer controls, suggesting that this is an alteration in the posttranslational modification of PI 3-K in platelets from patients with SSc. The increased nitrotyrosylation of PI 3-K may contribute to the impairment of platelet function in patients with SSc by increasing platelet reactivity to matrix components within the vascular walls of patients with this disease.

  20. Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

    Science.gov (United States)

    Kahner, Bryan N; Dorsam, Robert T; Mada, Sripal R; Kim, Soochong; Stalker, Timothy J; Brass, Lawrence F; Daniel, James L; Kitamura, Daisuke; Kunapuli, Satya P

    2007-10-01

    Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

  1. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B;

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  2. Platelets and cardiac arrhythmia

    Directory of Open Access Journals (Sweden)

    Jonas S De Jong

    2010-12-01

    Full Text Available Sudden cardiac death remains one of the most prevalent modes of death in industrialized countries, and myocardial ischemia due to thrombotic coronary occlusion is its primary cause. The role of platelets in the occurrence of SCD extends beyond coronary flow impairment by clot formation. Here we review the substances released by platelets during clot formation and their arrhythmic properties. Platelet products are released from three types of platelet granules: dense core granules, alpha-granules, and platelet lysosomes. The physiologic properties of dense granule products are of special interest as a potential source of arrhythmic substances. They are released readily upon activation and contain high concentrations of serotonin, histamine, purines, pyrimidines, and ions such as calcium and magnesium. Potential arrhythmic mechanisms of these substances, e.g. serotonin and high energy phosphates, include induction of coronary constriction, calcium overloading, and induction of delayed after-depolarizations. Alpha-granules produce thromboxanes and other arachidonic acid products with many potential arrhythmic effects mediated by interference with cardiac sodium, calcium and potassium channels. Alpha-granules also contain hundreds of proteins that could potentially serve as ligands to receptors on cardiomyocytes. Lysosomal products probably do not have an important arrhythmic effect. Platelet products and ischemia can induce coronary permeability, thereby enhancing interaction with surrounding cardiomyocytes. Antiplatelet therapy is known to improve survival after myocardial infarction. Although an important part of this effect results from prevention of coronary clot formation, there is evidence to suggest that antiplatelet therapy also induces anti-arrhythmic effects during ischemia by preventing the release of platelet activation products.

  3. Platelets: crossroads of immunity and hemostasis.

    Science.gov (United States)

    Jenne, Craig N

    2014-07-31

    In this issue of Blood, Koupenova and colleagues report that platelets express functional TOLL-like receptor 7 (TLR7) and contribute to host survival during viral infection. Through a series of experiments utilizing mice deficient for TLR7 together with adoptive transfer of wild-type platelets, Koupenova et al demonstrate that platelets specifically respond to viral analogs and intact virus, leading to platelet activation and binding to various leukocyte subsets. Perhaps most importantly, this platelet activation appears absolutely essential for host survival during infection with some viral pathogens such as encephalomyocarditis virus (EMCV).

  4. Development of hygromas or severe edema during treatment with the tyrosine kinase inhibitor STI571 is not associated with platelet-derived growth factor receptor (PDGFR) gene polymorphisms.

    Science.gov (United States)

    Brück, Patrick; Wassmann, Barbara; Lopez, Elizabeth Ramos; Hoelzer, Dieter; Ottmann, Oliver G

    2004-11-01

    STI571 is active against Bcr/Abl-, c-kit- and platelet-derived growth factor receptor (PDGFR)-driven malignancies. Mild to moderate edema is common, whereas severe edema, body cavity effusions and subdural hygromas are rarely observed. These effects have been suggested to involve inhibition of PDGFR signaling, but predisposing factors are unknown. We examined SNPs in the PDGFR alpha and beta gene regions in STI571-treated patients with and without life-threatening edema or cerebral hygromas, and in healthy volunteers. By RFLP analysis of 15 SNPs, the frequencies of genotypes did not differ between the three groups. SNPs of PDGFR genes do not appear to play a role in patient's susceptibility to clinically severe edema formation during treatment with STI571.

  5. Bioengineered collagens

    Science.gov (United States)

    Ramshaw, John AM; Werkmeister, Jerome A; Dumsday, Geoff J

    2014-01-01

    Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications. PMID:24717980

  6. Rationale for the development of IMC-3G3, a fully human immunoglobulin G subclass 1 monoclonal antibody targeting the platelet-derived growth factor receptor alpha.

    Science.gov (United States)

    Shah, Gaurav D; Loizos, Nick; Youssoufian, Hagop; Schwartz, Jonathan D; Rowinsky, Eric K

    2010-02-15

    A large body of evidence suggests that the platelet-derived growth factor (PDGF) family and associated receptors are potential targets in oncology therapeutic development because of their critical roles in the proliferation and survival of various cancers and in the regulation and growth of the tumor stroma and blood vessels. Several small molecules that nonspecifically target the PDGF signaling axis are in current use or development as anticancer therapies. However, for the majority of these agents, PDGF and its receptors are neither the primary targets nor the principal mediators of anticancer activity. IMC-3G3, a fully human monoclonal antibody of the immunoglobulin G subclass 1, specifically binds to the human PDGF receptor alpha (PDGFRalpha) with high affinity and blocks PDGF ligand binding and PDGFRalpha activation. The results of preclinical studies and the frequent expression of PDGFRalpha in many types of cancer and in cancer-associated stroma support a rationale for the clinical development of IMC-3G3. Currently, IMC-3G3 is being evaluated in early clinical development for patients with several types of solid malignancies.

  7. Acceleration of Lung Regeneration by Platelet-Rich Plasma Extract through the Low-Density Lipoprotein Receptor-Related Protein 5-Tie2 Pathway.

    Science.gov (United States)

    Mammoto, Tadanori; Chen, Zhao; Jiang, Amanda; Jiang, Elisabeth; Ingber, Donald E; Mammoto, Akiko

    2016-01-01

    Angiogenesis, the growth of new blood vessels, plays a key role in organ development, homeostasis, and regeneration. The cooperation of multiple angiogenic factors, rather than a single factor, is required for physiological angiogenesis. Recently, we have reported that soluble platelet-rich plasma (PRP) extract, which contains abundant angiopoietin-1 and multiple other angiogenic factors, stimulates angiogenesis and maintains vascular integrity in vitro and in vivo. In this report, we have demonstrated that mouse PRP extract increases phosphorylation levels of the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) and thereby activates angiogenic factor receptor Tie2 in endothelial cells (ECs) and accelerates EC sprouting and lung epithelial cell budding in vitro. PRP extract also increases phosphorylation levels of Tie2 in the mouse lungs and accelerates compensatory lung growth and recovery of exercise capacity after unilateral pneumonectomy in mice, whereas soluble Tie2 receptor or Lrp5 knockdown attenuates the effects of PRP extract. Because human PRP extract is generated from autologous peripheral blood and can be stored at -80°C, our findings may lead to the development of novel therapeutic interventions for various angiogenesis-related lung diseases and to the improvement of strategies for lung regeneration.

  8. Hereditary sideroblastic anemia with associated platelet abnormalities.

    Science.gov (United States)

    Soslau, G; Brodsky, I

    1989-12-01

    A 62 year old male (R.H.) presented with a mild anemia (Hb 11-12 gm%) and a history of multiple hemorrhagic episodes. The marrow had 40-50% sideroblasts. Marrow chromosomes were normal. His wife was hematologically normal, while one daughter, age 30 years, had a sideroblastic anemia (Hb 11-12 gm%) with 40-50% sideroblasts in the marrow. Her anemia was first noted at age 15 years. Administration of vitamin B6 did not correct the anemia in either the father or daughter. Platelet abnormalities inherited jointly with this disorder are described for the first time. Both R.H. and his daughter had prolonged bleeding times, with normal PTT, PT times, fVIII:C, fVIII:Ag levels, and vWF multimers, which may rule out a von Willebrand's disease. They have normal platelet numbers but abnormally low platelet adhesiveness and greatly depressed ADP, collagen, and epinephrine responsiveness. Response to ristocetin was in the low normal range, and aggregation with thrombin was normal. While desmopressin completely normalized R.H.'s bleeding time, none of these platelet parameters were improved. No differences in the SDS PAGE protein patterns of RH platelets could be detected in comparison to normal samples. His platelets took up and released serotonin (5HT) normally, and electron micrographs defined no morphological abnormalities. However, no ATP was released from platelets activated with collagen, and when followed by thrombin about fourfold greater ATP was released by control platelets as compared to RH platelets. The dense granule fraction derived from RH platelets contained about 20% the level of ATP, 40% the level of ADP, and 50% the level of 5HT detected in a normal sample. The results indicate that the bleeding disorder is related to a non-classical heritable storage pool defect. The connection between the inherited sideroblastic anemia and platelet defects is obscure.

  9. Adenosine A2A Receptors Mediate Anti-Inflammatory Effects of Electroacupuncture on Synovitis in Mice with Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Qi-hui Li

    2015-01-01

    Full Text Available To study the role of adenosine A2A receptor (A2AR in mediating the anti-inflammatory effect of electroacupuncture (EA on synovitis in collagen-induced arthritis (CIA, C57BL/6 mice were divided into five treatment groups: Sham-control, CIA-control, CIA-EA, CIA-SCH58261 (A2AR antagonist, and CIA-EA-SCH58261. All mice except those in the Sham-control group were immunized with collagen II for arthritis induction. EA treatment was administered using the stomach 36 and spleen 6 points, and stimulated with a continuous rectangular wave for 30 min daily. EA treatment and SCH58261 were administered daily from days 35 to 49 (n=10. After treatment, X-ray radiography of joint bone morphology was established at day 60 and mouse blood was collected for ELISA determination of tumor necrosis factor alpha (TNF-α levels. Mice were sacrificed and processed for histological examination of pathological changes of joint tissue, including hematoxylin-eosin staining and immunohistochemistry of A2AR expression. EA treatment resulted in significantly reduced pathological scores, TNF-α concentrations, and bone damage X-ray scores. Importantly, the anti-inflammatory and tissue-protective effect of EA treatment was reversed by coadministration of SCH58261. Thus, EA treatment exerts an anti-inflammatory effect resulting in significant protection of cartilage by activation of A2AR in the synovial tissue of CIA.

  10. Inhibitory effect of soluble platelet-derived growth factor receptor β on intraosseous growth of breast cancer cells in nude mice.

    Science.gov (United States)

    Shan, Hongchao; Takahashi, Tetsuyuki; Bando, Yoshimi; Izumi, Keisuke; Uehara, Hisanori

    2011-10-01

    Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor β (PDGFRβ) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRβ signaling using soluble PDGFRβ (sPDGFRβ) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ. A stable transfectant (TNBCT/Bo-sPDGFRβ) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRβ cells. Intratibial injection of TNBCT/Bo-sPDGFRβ cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRβ is useful for the treatment of breast cancer bone metastasis.

  11. Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation.

    Science.gov (United States)

    Fanger, G R; Vaillancourt, R R; Heasley, L E; Montmayeur, J P; Johnson, G L; Maue, R A

    1997-01-01

    The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.

  12. Platelet Content of Nitric Oxide Synthase 3 Phosphorylated At Serine1177 Is Associated with the Functional Response of Platelets to Aspirin

    Science.gov (United States)

    Modrego, Javier; Azcona, Luis; Martín-Palacios, Naiara; Zamorano-León, José J.; Segura, Antonio; Rodríguez, Pablo; Guerra, Reddy; Tamargo, Juan; Macaya, Carlos; López-Farré, Antonio J.

    2013-01-01

    Objective To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO). Patients/Methods Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100). Results ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position −786 (T−786→C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. Conclusions Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser1177. PMID:24376548

  13. Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

    Directory of Open Access Journals (Sweden)

    Javier Modrego

    Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

  14. [Murine models of platelet diseases].

    Science.gov (United States)

    Lanza, F

    2007-05-01

    Platelet-related diseases correspond to functional defects or abnormal production (thrombopoiesis) of hereditary and immunological origins. Recent progress in the manipulation of the mouse genome (transgenesis, gene inactivation or insertion) has resulted in the generation of numerous strains exhibiting defective platelet function or production. Some strains reproduce known hereditary diseases affecting haemostasis (Glanzmann thrombasthenia, Bernard-Soulier syndrome (BSS) or thrombopoiesis (Wiscott-Aldrich or May-Hegglin syndrome). More often the mutated strains have no human equivalent and represent useful models to study: (i) the role of adhesive or signalling receptors or of signalling proteins in platelet-dependent haemostasis and thrombosis or; (ii) to study the poorly characterized mechanisms of thrombopoiesis, which implicate transcription factors (GATA, Fli1), growth factors and receptors (TPO, cMPL), and cytoskeletal or contractile proteins (tubulin, myosin). Additional mouse strains result from the selection of spontaneous mutants many of which affect intracellular platelet granules, representing models of storage pool diseases (SPD) such as the Gray platelet syndrome (alphaSPD) or Hermansky-Pudlack syndrome (deltaSPD). More recently, a systematic chemical mutagenesis approach has also identified genes involved in thrombopoiesis and platelet survival. Finally, mouse models of auto- or allo-immune thrombocytopenia have been developed to study the mechanisms of platelet destruction or removal.

  15. LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

    Science.gov (United States)

    Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

    2014-11-01

    Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Transcriptome analysis reveals an unexpected role of a collagen tyrosine kinase receptor gene, Ddr2, as a regulator of ovarian function.

    Science.gov (United States)

    Matsumura, Hirokazu; Kano, Kiyoshi; Marín de Evsikova, Caralina; Young, James A; Nishina, Patsy M; Naggert, Jürgen K; Naito, Kunihiko

    2009-10-07

    Mice homozygous for the smallie (slie) mutation lack a collagen receptor, discoidin domain receptor 2 (DDR2), and are dwarfed and infertile due to peripheral dysregulation of the endocrine system of unknown etiology. We used a systems biology approach to identify biological networks affected by Ddr2(slie/slie) mutation in ovaries using microarray analysis and validate findings using molecular, cellular, and functional biological assays. Transcriptome analysis indicated several altered gene categories in Ddr2(slie/slie) mutants, including gonadal development, ovulation, antiapoptosis, and steroid hormones. Subsequent biological experiments confirmed the transcriptome analysis predictions. For instance, a significant increase of TUNEL-positive follicles was found in Ddr2(slie/slie) mutants vs. wild type, which confirm the transcriptome prediction for decreased chromatin maintenance and antiapoptosis. Decreases in gene expression were confirmed by RT-PCR and/or qPCR; luteinizing hormone receptor and prostaglandin type E and F receptors in Ddr2(slie/slie) mutants, compared with wild type, confirm hormonal signaling pathways involved in ovulation. Furthermore, deficiencies in immunohistochemistry for DDR2 and luteinizing hormone receptor in the somatic cells, but not the oocytes, of Ddr2(slie/slie) mutant ovaries suggest against an intrinsic defect in germ cells. Indeed, Ddr2(slie/slie) mutants ovulated significantly fewer oocytes; their oocytes were competent to complete meiosis and fertilization in vitro. Taken together, our convergent data signify DDR2 as a novel critical player in ovarian function, which acts upon classical endocrine pathways in somatic, rather than germline, cells.

  17. Evaluation of platelet function using multiple electrode platelet aggregometry in dogs with septic peritonitis.

    Science.gov (United States)

    Li, Ronald H L; Chan, Daniel L

    2016-09-01

    To assess platelet function via multiple electrode platelet aggregometry (MEPA) in dogs with septic peritonitis and in healthy dogs. The secondary aim was to determine if there is prognostic significance to changes in platelet function observed in septic dogs. Prospective, observational cohort study conducted from January 2012 to March 2014. University teaching hospital. Twenty dogs with septic peritonitis and 23 healthy dogs. None. MEPA using arachidonic acid, adenosine diphosphate, and collagen (COL) as agonists was measured within 24 hours of diagnosis of sepsis. Compared to healthy dogs, platelet aggregation was reduced in dogs with septic peritonitis for all agonists (P peritonitis. Circulating platelets from dogs with septic peritonitis have diminished aggregation in response to multiple platelet agonists. MEPA may serve as an assessment tool for illness severity in this patient population. © Veterinary Emergency and Critical Care Society 2016.

  18. Serotonin 5-HT2A receptor binding in platelets from healthy subjects as studied by [3H]-lysergic acid diethylamide ([3H]-LSD): intra- and interindividual variability.

    Science.gov (United States)

    Spigset, O; Mjörndal, T

    1997-04-01

    In studies on platelet 5-HT2A receptor binding in patients with neuropsychiatric disorders, there has been a marked variability and a considerable overlap of values between patients and controls. The causes of the large variability in 5-HT2A receptor parameters is still unsettled. In the present study, we have quantified the intra- and interindividual variability of platelet 5-HT2A receptor binding in 112 healthy subjects and explored factors that may influence 5-HT2A receptor binding, using [3H]-lysergic acid diethylamide as radioligand. Age, gender, blood pressure, and metabolic capacity of the liver enzymes CYP2D6 and CYP2C19 did not influence Bmax and Kd values. Body weight and body mass index (BMI) showed a negative correlation with Kd (p = .04 and .03, respectively), but not with Bmax. Bmax was significantly lower in the light half of the year than in the dark half of the year (p = .001), and Kd was significantly lower in the fall than in the summer and winter (p < .001). In females, there was a significant increase in Bmax from week 1 to week 2 of the menstrual cycle (p = .03). Females taking contraceptive pills had significantly higher Kd than drug-free females in weeks 1 and 4 of the menstrual cycle (p = .04). This study shows that a number of factors should be taken into account when using platelet 5-HT2A receptor binding in studies of neuropsychiatric disorders.

  19. Involvement of nuclear factor {kappa}B in platelet CD40 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Hachem, Ahmed [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Yacoub, Daniel [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Zaid, Younes [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Mourad, Walid [Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Merhi, Yahye, E-mail: yahye.merhi@icm-mhi.org [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

  20. Acquired platelet function defect

    Science.gov (United States)

    Acquired qualitative platelet disorders; Acquired disorders of platelet function ... blood clotting. Disorders that can cause problems in platelet function include: Idiopathic thrombocytopenic purpura Chronic myelogenous leukemia Multiple ...

  1. Platelet Donation

    Science.gov (United States)

    ... of gratitude that washed over me when I saw those platelets going into my husband’s body. I ... Needles LGBTQ+ Donors Blood Donor Community SleevesUp Games Facebook Avatars and Badges Banners eCards Red Cross Information ...

  2. Modulatory effect of coffee on platelet function.

    Science.gov (United States)

    Bhaskar, Shobha; Rauf, Arun A

    2010-01-01

    Blood platelets play a major role in cardiovascular disease (CVD) and thrombosis. Conflicting information exists regarding the effect of coffee consumption on the cardiovascular system. We have investigated whether the consumption of moderate amount of coffee affect platelet functions and primary hemostasis in vivo in normal and high fat diet fed rats. Coffee fed group showed significant (P production from membrane arachidonic acid and it was decreased in coffee treated group. Platelet aggregation studies with ADP, collagen, arachidonic acid and epinephrine showed significant (P coffee fed group. Scanning electron microscopic studies revealed that platelet aggregation tendency increased in HFD group and was reduced in coffee fed group. These results indicate that coffee is active in inhibiting platelet aggregation, a critical step involved in thrombosis.

  3. Aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures.

    Science.gov (United States)

    Kar, Karunakar; Ibrar, Sajjad; Nanda, Vikas; Getz, Todd M; Kunapuli, Satya P; Brodsky, Barbara

    2009-08-25

    Aromatic residues are relatively rare within the collagen triple helix, but they appear to play a specialized role in higher-order structure and function. The role of aromatic amino acids in the self-assembly of triple-helical peptides was investigated in terms of the kinetics of self-association, the nature of aggregated species formed, and the ability of these species to activate platelet aggregation. The presence of aromatic residues on both ends of a type IV collagen model peptide is observed to greatly accelerate the kinetics of self-association, decreasing the lag time and leading to insoluble, well-defined linear fibrils as well as small soluble aggregates. Both macroscopic visible aggregates and small multimolecular complexes in solution are capable of inducing platelet aggregation through the glycoprotein VI receptor on platelets. Proline-aromatic CH...pi interactions are often observed within globular proteins and in protein complexes, and examination of molecular packing in the crystal structure of the integrin binding collagen peptide shows Phe interacts with Pro/Hyp in a neighboring triple-helical molecule. An intermolecular interaction between aromatic amino acids and imino acids within the triple helix is also supported by the observed inhibitory effect of isolated Phe amino acids on the self-association of (Pro-Hyp-Gly)(10). Given the high fraction of Pro and Hyp residues on the surface of collagen molecules, it is likely that imino acid-aromatic CH...pi interactions are important in formation of higher-order structure. We suggest that the catalysis of type I collagen fibrillogenesis by nonhelical telopeptides is due to specific intermolecular CH...pi interactions between aromatic residues in the telopeptides and Pro/Hyp residues within the triple helix.

  4. Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy

    Science.gov (United States)

    Enko, Dietmar; Mangge, Harald; Münch, Andreas; Niedrist, Tobias; Mahla, Elisabeth; Metzler, Helfried; Prüller, Florian

    2017-01-01

    Introduction The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%. Conclusions Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced. PMID:28392742

  5. Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa.

    Science.gov (United States)

    Al-Tamimi, Mohammad; Grigoriadis, George; Tran, Huy; Paul, Eldho; Servadei, Patricia; Berndt, Michael C; Gardiner, Elizabeth E; Andrews, Robert K

    2011-04-01

    This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

  6. Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex

    NARCIS (Netherlands)

    Brondijk, T.H.C.; Bihan, D.; Farndale, R.W.; Huizinga, E.G.

    2012-01-01

    Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High a

  7. Icariin attenuates high glucose-induced type IV collagen and fibronectin accumulation in glomerular mesangial cells by inhibiting transforming growth factor-β production and signalling through G protein-coupled oestrogen receptor 1.

    Science.gov (United States)

    Li, Yi-Chen; Ding, Xuan-Sheng; Li, Hui-Mei; Zhang, Cheng

    2013-09-01

    Icariin has been shown to attenuate diabetic nephropathy in rats by decreasing transforming growth factor-β (TGF-β) and type IV collagen expression, but its mode of action in glomerular mesangial cells is uncertain. The present study aimed to investigate the effect of icariin on excess mesangial type IV collagen and fibronectin accumulation induced by high glucose, and to determine the mechanism underlying its protective effects. Under high-glucose conditions, icariin diminished type IV collagen and fibronectin accumulation, as well as TGF-β production in human and rat mesangial cells. Mesangial cells treated with icariin after TGF-β1 exposure expressed less type IV collagen and fibronectin than those without icariin treatment, suggesting inhibition by icariin of TGF-β1 downstream pathways. On TGF-β1 stimulation, icariin inhibited TGF-β canonical Smad signalling and extracellular signal-regulated kinase (ERK)1/2 signalling by decreasing Smad2/3 and ERK1/2 phosphorylation in a dose-dependent manner. U0126, which blocked the ERK1/2 pathway, exerted an additive effect on the icariin suppression of type IV collagen and fibronectin expression, enhancing the beneficial effects of icariin. The G protein-coupled oestrogen receptor 1 (GPER) antagonist, G-15, abolished the icariin-induced inhibition of type IV collagen, and fibronectin overproduction and TGF-β signalling. Treatment of cells with fulvestrant, a downregulator of the oestrogen receptor, enhanced the action of icariin. In conclusion, icariin decreased type IV collagen and fibronectin accumulation induced by high glucose in mesangial cells by inhibiting TGF-β production, as well as Smad and ERK signalling in a GPER-dependent manner.

  8. [Collagenous colitis].

    Science.gov (United States)

    Lindström, C G

    1991-05-01

    Collagenous colitis is now regarded by an overwhelming majority of authors as a clinicopathological entity and has been taken up as a such in many text-books and diagnostic atlases (Morson & Dawson, 1990, Fenoglio-Preiser et al., 1989, Whitehead 1985, Whitehead 1989). A good, detailed review of cases of collagenous colitis published up to 1988 was performed by Perri et al. Collagenous colitis was also presented to a wider medical public through a clinicopathological conference case at Massachusetts General Hospital (Case 29-1988). Finally it may be added that collagenous colitis has been included in the new fourth edition of Robbins Pathologic Basis of Disease (Cotran, Kumar, Robbins, 1989), where the possibility of an autoimmune disease is stressed.

  9. Vascular endothelial (VEGF) and epithelial growth factor (EGF) as well as platelet-activating factor (PAF) and receptors are expressed in the early pregnant canine uterus.

    Science.gov (United States)

    Schäfer-Somi, S; Sabitzer, S; Klein, D; Reinbacher, E; Kanca, H; Beceriklisoy, H B; Aksoy, O A; Kucukaslan, I; Macun, H C; Aslan, S

    2013-02-01

    The aim of this study was to investigate the course of expression of platelet-activating factor (PAF), PAF-receptor (PAF-R), epidermal growth factor (EGF), EGF-R, vascular endothelial growth factor (VEGF), VEGF-R1 and VEGF-R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10-12 (n = 10), 18-25 (n = 5) and 28-45 (n = 5) days after mating, respectively. The pre-implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non-pregnant, bitches in diestrus (day 10-12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap-frozen in liquid nitrogen after embedding in Tissue Tec(®). Extraction of mRNA for RT-PCR was performed with Tri-Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre-implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy.

  10. Inhibition of Notch Signaling Ameliorates Acute Kidney Failure and Downregulates Platelet-Derived Growth Factor Receptor β in the Mouse Model.

    Science.gov (United States)

    Kramer, Jan; Schwanbeck, Ralf; Pagel, Horst; Cakiroglu, Figen; Rohwedel, Jürgen; Just, Ursula

    2016-01-01

    Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor β (Pdgfrb). Treatment of ischemic mice with the x03B3;-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis.

  11. BN 52021 (a platelet activating factor-receptor antagonist decreases alveolar macrophage-mediated lung injury in experimental extrinsic allergic alveolitis

    Directory of Open Access Journals (Sweden)

    J-L. Pérez-Arellano

    1998-01-01

    Full Text Available Several lines of research indirectly suggest that platelet activating factor (PAF may intervene in the pathogenesis of extrinsic allergic alveolitis (EAA. The specific aim of our study was to evaluate the participation of PAF on macrophage activation during the acute phase of EAA in an experimental model of this disease developed in guinea pigs. Initially we measured the concentration of PAF in bronchoalvedar lavage fluid, blood and lung tissue. In a second phase we evaluate the participation of PAF on alveolar macrophage activation and parenchymal lung injury. The effect of PAF on parenchymal lung injury was evaluated by m easuring several lung parenchymatous lesion indices (lung index, bronchoalvedar lavage fluid (BALF lactic hydrogenase activity and BALF alkaline phosphatase activity and parameters of systemic response to the challenge (acute phase reagents. We observed that induction of the experimental EAA gave rise to an increase in the concentration of PAF in blood and in lung tissue. The use of the PAF-receptor antagonist BN52021 decreases the release of lysosomal enzymes (β-glucuronidase and tartrate-sensitive acid phosphatase to the extracellular environment both in vivo and in vitro. Furthermore, antagonism of the PAF receptors notably decreases pulmonary parenchymatous lesion. These data suggest that lung lesions from acute EAA are partly mediated by local production of PAF.

  12. New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity

    Directory of Open Access Journals (Sweden)

    Hirz Taghreed

    2012-12-01

    Full Text Available Abstract Background Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. Results and discussion We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.. All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. Conclusions In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities

  13. Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase*

    Science.gov (United States)

    Ohkawa, Yuki; Momota, Hiroyuki; Kato, Akira; Hashimoto, Noboru; Tsuda, Yusuke; Kotani, Norihiro; Honke, Koichi; Suzumura, Akio; Furukawa, Keiko; Ohmi, Yuhsuke; Natsume, Atsushi; Wakabayashi, Toshihiko; Furukawa, Koichi

    2015-01-01

    There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas. PMID:25940087

  14. Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase.

    Science.gov (United States)

    Ohkawa, Yuki; Momota, Hiroyuki; Kato, Akira; Hashimoto, Noboru; Tsuda, Yusuke; Kotani, Norihiro; Honke, Koichi; Suzumura, Akio; Furukawa, Keiko; Ohmi, Yuhsuke; Natsume, Atsushi; Wakabayashi, Toshihiko; Furukawa, Koichi

    2015-06-26

    There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.

  15. 血小板衍化生长因子对血管平滑肌细胞增殖 及胶原蛋白合成的影响%Effects of platelet-derived growth factor on proliferation and collagen protein synthesis of vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    任雨笙; 崔芳; 贾国良; 俞世强; 汤朝武; 吴宗贵

    2001-01-01

    AIM:To investigate the effects of platelet-derived growth factor(PDGF) on proliferation and collagen protein synthesis of vascular smooth muscle cells (VSMC). METHODS:In the present experiment,the rabbit VSMC were cultured and effects of PDGF-BB on DNA synthesis and collagen protein synthesis of rabbit VSMC were observed by using 3H-TdR incorporation and 3H-proline incorporation in vitro. RESULTS:PDGF-BB could significantly promote DNA synthesis and collagen protein synthesis of quiescent rabbit VSMC in a dose-dependent manner,with a maximal response at a concentration of 40 μg/L at 36 h and 48 h respectively. CONCLUSION:PDGF-BB could promote proliferation and collagen protein synthesis of cultured rabbit VSMC.%目的:观察血小板衍化生长因子(PDGF)对培养的血管平滑肌细胞(VSMC)增殖及胶原蛋白合成的影响。方法:采用培养的兔动脉VSMC,应用3H-TdR的3H-脯氨酸掺入方法,观察PDGF-BB对兔VSMC DNA合成以及胶原蛋白合成的影响。结果:PDGF-BB可促进处于静止状态的兔VSMC DNA及胶原蛋白的合成,并呈现明显的浓度依赖关系,在40 μg/L的浓度时DNA及胶原蛋白的合成达到高峰,DNA及胶原蛋白分别处于36 h和48 h合成最为显著。结论:PDGF-BB可明显促进培养的VSMC增殖及胶原蛋白的合成。

  16. Evaluation of platelet aggregation in platelet concentrates: storage implications

    Directory of Open Access Journals (Sweden)

    Neiva Teresinha J.C.

    2003-01-01

    Full Text Available The use of hemo-derivatives is nowadays a fundamentally important therapeutic modality in the exercise of medicine. Among the various hemo-components employed, we have the platelet concentrate (PC, indicated in cases of hemorrhagic disturbances. We previously showed that platelet function in blood donors is reduced in their screening phase and after the separation process of PCs. Currently, we are providing evidence for the existence of biochemical and functional changes in PC preparations stored for three days at temperatures of 20 ± 2 ºC. Platelet concentrates from 40 healthy donors, collected in CPD anticoagulant and PL-146 polyvinylchloride containers, were examined in order to determine the pH value, pCO2 ,pO2 and lactate concentrations. In addition, the aggregation of platelets with thrombin and collagen were examined to evaluate platelet function. A pH increase from 7.07 ± 0.04 to 7.36 ± 0.07 (p < 0.01 was observed. The pCO2 concentration decreased progressively from 69.2 ± 7.7 mmHg to 28.8 ± 6.2 mmHg (p < 0.001 during the storage period. In contrast, pO2 value increase from 103.4 ± 30.6 to 152.3 ± 24.6 mmHg (p < 0.001 was evidenced during the 48 hours of storage. The lactate concentration increased from 17.97 ± 5.2 to 57.21 ± 5.7 mg/dl (p < 0.001. Platelet aggregation using 0.25 U/ml-thrombin and 2.0 µg/ml-collagen showed significant hypofunction from 61.8 ± 2.7% to 24.8 ± 9.8% and 62.7±5.0 to 33.4± 6.2 (p < 0.001, respectively. We concluded that the evaluated biochemical parameters and the platelet function changed significantly when the platelets were kept under routine storage conditions.

  17. Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classes of PDGF receptor.

    Science.gov (United States)

    Bowen-Pope, D F; Hart, C E; Seifert, R A

    1989-02-15

    Platelet-derived growth factor (PDGF) is encoded by separate genes for two possible subunit chains (A-chain and B-chain) which can form three possible dimers (AA, AB, and BB). We have recently presented evidence that multiple forms of PDGF receptor exist which distinguish between these isoforms (Hart, C. H., Forstrom, J. W., Kelley, J. D., Smith, R. A., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1988) Science 240, 1529-1531). We used this specificity to determine the amount of PDGF from different sources which is able to bind to each class of receptor and found that each source had a characteristic isoform composition. Levels of total PDGF activity in sera from different species ranged more than 15-fold, from less than 1 ng/ml in dog, chicken, pig, and calf, to greater than 13 ng/ml in mouse and human. Despite these differences in PDGF content, the total mitogenic activities of the sera were comparable indicating that the relative importance of PDGF as a serum mitogen may vary considerably between species. Analysis of the total PDGF into the amounts of each isoform revealed great differences in composition. PDGF-BB constitutes only about 15% of the total binding activity in human PDGF purified by the method of Raines and Ross (Raines, E. W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160) but is the predominant isoform in whole blood serum from all other species. In contrast to serum, medium conditioned by cultured PDGF-secreting cell types contained no detectable PDGF-BB except in two cases: medium conditioned by vascular endothelial cells and by cells transformed by simian sarcoma virus. The existence of isoform-specific PDGF receptors and the large variation in PDGF isoform composition dependent upon source may provide an important mechanism through which the effects of PDGF can be targeted to different cell types and/or toward eliciting different cell responses.

  18. A novel functional role of collagen glycosylation

    DEFF Research Database (Denmark)

    Jürgensen, Henrik J; Madsen, Daniel H; Ingvarsen, Signe

    2011-01-01

    , the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose...... receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens....... By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate...

  19. Tetrandrine ameliorates collagen-induced arthritis in mice by restoring the balance between Th17 and Treg cells via the aryl hydrocarbon receptor.

    Science.gov (United States)

    Yuan, Xusheng; Tong, Bei; Dou, Yannong; Wu, Xin; Wei, Zhifeng; Dai, Yue

    2016-02-01

    Tetrandrine is an alkaloid constituent of the root of Stephania tetrandra S. Moore. The long-term clinical uses of tetrandrine for treatments of rheumatalgia and arthralgia as well as the inhibition of rat adjuvant-induced arthritis imply that tetrandrine may have therapeutic potential in rheumatoid arthritis (RA). Here, we explored its anti-RA mechanism in collagen-induced arthritis (CIA) in relation to the balance between T helper (Th) 17 cells and regulatory T (Treg) cells. DBA/1 mice were immunized with chicken type II collagen and were orally administered tetrandrine for 14 consecutive days. Then, the mice were sacrificed, their joints were removed for histological analysis, and spleens and mesenteric lymph nodes (MLNs) were removed to examine the Th17 and Treg cells. Tetrandrine markedly alleviated the severity of arthritis, reduced the serum levels of pro-inflammatory cytokines, and restored the Th17/Treg balance, as demonstrated by the serum levels of their related cytokines (IL-17 and IL-10) and the proportion of each cell type. Tetrandrine inhibited Th17 cell differentiation and induced Treg cell differentiation in vitro . Notably, aryl hydrocarbon receptor (AhR) was proven to play a crucial role in tetrandrine-mediated T cell differentiation. The correlation between AhR activation, regulation of Th17/Treg and amelioration of arthritis by tetrandrine was verified in the CIA mice. Moreover, tetrandrine might be a ligand of AhR because it facilitated the expression of the AhR target gene cytochrome P450 1A1 (CYP1A1) and the activation of its downstream signaling pathways. Taken together, tetrandrine exerts its anti-arthritis efficacy by restoring Th17/Treg balance via AhR.

  20. Toll-like receptor 4 is involved in inflammatory and joint destructive pathways in collagen-induced arthritis in DBA1J mice.

    Directory of Open Access Journals (Sweden)

    Matthias Pierer

    Full Text Available In rheumatoid arthritis, a significant proportion of cytokine and chemokine synthesis is attributed to innate immune mechanisms. TLR4 is a prominent innate receptor since several endogenous ligands known to activate the innate immune system bind to it and may thereby promote joint inflammation. We generated TLR4 deficient DBA1J mice by backcrossing the TLR4 mutation present in C3H/HeJ strain onto the DBA1J strain and investigated the course of collagen-induced arthritis in TLR4 deficient mice in comparison to wild type littermates. The incidence of collagen- induced arthritis was significantly lower in TLR4 deficient compared to wild type mice (59 percent vs. 100 percent. The severity of arthritis was reduced in the TLR4 deficient mice compared to wild type littermates (mean maximum score 2,54 vs. 6,25. Mice deficient for TLR4 were virtually protected from cartilage destruction, and infiltration of inflammatory cells was reduced compared to wt mice. In parallel to the decreased clinical severity, lower anti-CCP antibody concentrations and lower IL-17 concentrations were found in the TLR4 deficient mice. The study further supports the role of TLR4 in the propagation of joint inflammation and destruction. Moreover, since deficiency in TLR4 led to decreased IL-17 and anti-CCP antibody production, the results indicate a link between TLR4 stimulation and the adaptive autoimmune response. This mechanism might be relevant in human rheumatoid arthritis, possibly in response to activating endogenous ligands in the affected joints.

  1. A human platelet calcium calculator trained by pairwise agonist scanning.

    Directory of Open Access Journals (Sweden)

    Mei Yan Lee

    2015-02-01

    Full Text Available Since platelet intracellular calcium mobilization [Ca(t]i controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t]i in response to combinatorial agonists. Pairwise Agonist Scanning (PAS deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50; 154 conditions including a null condition to stimulate platelet P2Y1/P2Y12 GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban, diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t]i data for training 10 neural networks (NN, 2-layer/12-nodes per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions as was a sampling of 45 higher ordered combinations (four to six agonists. The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t]i beyond the single and binary training set for trinary stimulations (R = 0.924. The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850 as were the calcium dynamics (R = 0.871 and high-dimensional synergy scores (R = 0.695 for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921. NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.

  2. Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC).

    Science.gov (United States)

    Basabe-Desmonts, L; Ramstrom, S; Meade, G; O'Neill, S; Riaz, A; Lee, L P; Ricco, A J; Kenny, D

    2010-09-21

    We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 μm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (

  3. Analysis of aggregation of platelets in thrombosis

    Science.gov (United States)

    Ahuja, Suresh

    Platelets are key players in thrombus formation by first rolling over collagen bound von Willebrand factor followed by formation of a stable interaction with collagen. The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. The coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place because of morphological changes in platelets. Coagulation takes place by platelet activation and aggregation mainly through fibrinogen polymerization into fibrin fibers. The process includes multiple factors, such as thrombin, plasmin, and local shear-rate which regulate and control the process. Coagulation can be divided into two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by the exposure of a negatively charged. It is able to activate factor XII, using a complex reaction that includes prekallikrein and high-molecular-weight kininogen as cofactors.. Thrombin is the final enzyme that is needed to convert fibrinogen into fibrin. The extrinsic pathway starts with the exposure of tissue factor to the circulating blood, which is the major initiator of coagulation. There are several feedback loops that reinforce the coagulation cascade, resulting in large amounts of thrombin. It is dependent on the presence of pro-coagulant surfaces of cells expressing negatively charged phospholipids--which include phosphatidylserine (PS)--on their outer membrane. PS-bearing surfaces are able to increase the efficiency of the reactions by concentrating and co-localizing coagulation factors.. Aggregation of platelets are analyzed and compared to adhesion of platelet to erythrocyte and to endothelial cells. This abstract is replacing MAR16-2015-020003.

  4. Association Study of a Proliferation-inducing Ligand, Spermatogenesis Associated 8, Platelet-derived Growth Factor Receptor-alpha, and POLB Polymorphisms with Systemic Lupus Erythematosus in Chinese Han Population

    OpenAIRE

    Ping Li; Yuan Li; Ai-Hong Zhou; Si Chen; Jing Li; Xiao-Ting Wen; Zi-Yan Wu; Liu-Bing Li; Feng-Chun Zhang; Yong-Zhe Li

    2016-01-01

    Background: Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. This study was conducted to examine whether the association of a proliferation-inducing ligand (APRIL), spermatogenesis associated 8 (SPATA8), platelet-derived growth factor receptor-alpha (PDGFRA), and DNA polymerase beta (POLB) with SLE can be replicated in a Chinese Han population. Methods: Chinese SLE patients (n = 1247) and ethnically and geographically matched healthy cont...

  5. No changes in lymphocyte muscarinic receptors and platelet monoamine oxidase-B examined as surrogate central nervous system biomarkers in a Faroese children cohort prenatally exposed to methylmercury and polychlorinated biphenyls

    DEFF Research Database (Denmark)

    Coccini, Teresa; Manzo, Luigi; Debes, Frodi

    2009-01-01

    Experimental evidence suggests that monoamine oxidase B (MAO-B) and muscarinic cholinergic receptors (mAChRs) are involved in the pathogenesis of neurotoxicity caused by methylmercury and polychlorinated biphenyls (PCBs). Blood samples from 7-year-old exposed children were analyzed for platelet M....../or PCB exposure, whereas these markers are significantly altered in sustained exposure scenarios, as shown by clinical studies in drug addicts or patients treated with psychopharmacological agents....

  6. Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

    NARCIS (Netherlands)

    van der Wal, E.

    2010-01-01

    Storage of platelets at low temperature reduces bacterial growth and might better preserve the haemostatic function of platelets than current procedures. Incubation at 0C is known to expose ?-N-acetyl-D-glucosamine-residues on glycoprotein (GP)Ibalpha inducing receptor-clustering and platelet destru

  7. Glycoprotein Ibα clustering in platelet storage and function

    NARCIS (Netherlands)

    Gitz, E.

    2013-01-01

    Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

  8. Glycoprotein Ibα clustering in platelet storage and function

    NARCIS (Netherlands)

    Gitz, E.

    2013-01-01

    Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

  9. Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry

    DEFF Research Database (Denmark)

    Rubak, Peter; Villadsen, Kirsten; Hvas, Anne-Mette

    2012-01-01

    Abstract Introduction Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed. Material...... and methods We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow...... reference interval is presented as 95% confidence interval suitable for any age and both sex. Day-to-day variation was

  10. Platelet-rich plasma affects the proliferation and collagen production of mesenchymal stem cells%富血小板血浆与间充质干细胞增殖和胶原的产生

    Institute of Scientific and Technical Information of China (English)

    荣春; 申延清; 吕亚青; 李明玉; 夏长所

    2013-01-01

    BACKGROUND:Mesenchymal stem cel s are the seed cel s for tendon tissue engineering which can be obtained in large quantities, but how to induce in vitro is a key technology. OBJECTIVE:To explore the effect of platelet-rich plasma on the proliferation and col agen production of in vitro cultured mesenchymal stem cel s. METHODS:The rabbit mesenchymal stem cel s were separated ad cultured. The high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group were set to induce the mesenchymal stem cel s, and the blank control group was set as control. RESULTS AND CONCLUSION:The proliferation of mesenchymal stem cel s in the high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group was high, and in rapid growth with big increase amplitude, and there was no significant difference in the proliferation when compared with the blank control group (P  目的:观察富血小板血浆对体外培养间充质干细胞分裂增殖和胶原产生的影响。  方法:分离培养兔间充质干细胞,设立富血小板血浆高、中、低剂量组干预间充质干细胞,并设空白组做对照。  结果与结论:富血小板血浆高、中、低组间充质干细胞均保持较高的增殖活性,呈快速生长,曲线上升幅度大,与空白对照组比较差异有显著性意义(P <0.05)。培养时间越长作用越明显,培养一定时间后其作用具有剂量依赖性,较高剂量的富血小板血浆对细胞的增殖作用较为明显。提示富血小板血浆能明显促进间充质干细胞的Ⅰ和Ⅲ型胶原合成,剂量越大,刺激胶原产生的作用越明显。

  11. Roll, adhere, spread and contract: structural mechanics of platelet function.

    Science.gov (United States)

    Sorrentino, Simona; Studt, Jan-Dirk; Medalia, Ohad; Tanuj Sapra, K

    2015-01-01

    Platelets are involved in life-sustaining processes such as hemostasis, wound healing, atherothrombosis and angiogenesis. Mechanical trauma to blood vessels causes platelet activation resulting in their adherence and clot formation at the damaged site, culminating in clot retraction and tissue repair. Two of the major players underlying this process are the cytoskeleton, i.e., actin and microtubules, and the membrane integrin receptors. Rare congenital bleeding disorders such as Glanzmann thrombasthenia and Bernard-Soulier syndrome are associated with genetic alterations of platelet surface receptors, also affecting the platelet cytoskeletal structure. In this review, we summarize the current knowledge about platelet structure and adhesion, and delve into the mechanical aspects of platelet function. Platelets lack a nucleus, and can thus provide a minimal model of a biological cell. New biophysical tools may help to scrutinize platelets anew and to extend the existing knowledge on cell biology.

  12. Flavan-3-ol-enriched dark chocolate and white chocolate improve acute measures of platelet function in a gender-specific way--a randomized-controlled human intervention trial.

    Science.gov (United States)

    Ostertag, Luisa M; Kroon, Paul A; Wood, Sharon; Horgan, Graham W; Cienfuegos-Jovellanos, Elena; Saha, Shikha; Duthie, Garry G; de Roos, Baukje

    2013-02-01

    We examined whether flavan-3-ol-enriched dark chocolate, compared with standard dark and white chocolate, beneficially affects platelet function in healthy subjects, and whether this relates to flavan-3-ol bioavailability. A total of 42 healthy subjects received an acute dose of flavan-3-ol-enriched dark, standard dark or white chocolate, in random order. Blood and urine samples were obtained just before and 2 and 6 h after consumption for measurements of platelet function, and bioavailability and excretion of flavan-3-ols. Flavan-3-ol-enriched dark chocolate significantly decreased adenosine diphosphate-induced platelet aggregation and P-selectin expression in men (all p ≤ 0.020), decreased thrombin receptor-activating peptide-induced platelet aggregation and increased thrombin receptor-activating peptide-induced fibrinogen binding in women (both p ≤ 0.041), and increased collagen/epinephrine-induced ex vivo bleeding time in men and women (p ≤ 0.042). White chocolate significantly decreased adenosine diphosphate-induced platelet P-selectin expression (p = 0.002) and increased collagen/epinephrine-induced ex vivo bleeding time (p = 0.042) in men only. Differences in efficacy by which flavan-3-ols affect platelet function were only partially explained by concentrations of flavan-3-ols and their metabolites in plasma or urine. Flavan-3-ols in dark chocolate, but also compounds in white chocolate, can improve platelet function, dependent on gender, and may thus beneficially affect atherogenesis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Clearance of Apoptotic Cells by Macrophages Induces Regulatory Phenotype and Involves Stimulation of CD36 and Platelet-Activating Factor Receptor

    Directory of Open Access Journals (Sweden)

    Matheus Ferracini

    2013-01-01

    Full Text Available Phagocytosis of apoptotic cells (efferocytosis induces macrophage differentiation towards a regulatory phenotype (IL-10high/IL-12p40low. CD36 is involved in the recognition of apoptotic cells (AC, and we have shown that the platelet-activating factor receptor (PAFR is also involved. Here, we investigated the contribution of PAFR and CD36 to efferocytosis and to the establishment of a regulatory macrophage phenotype. Mice bone marrow-derived macrophages were cocultured with apoptotic thymocytes, and the phagocytic index was determined. Blockage of PAFR with antagonists or CD36 with specific antibodies inhibited the phagocytosis of AC (~70–80%. Using immunoprecipitation and confocal microscopy, we showed that efferocytosis increased the CD36 and PAFR colocalisation in the macrophage plasma membrane; PAFR and CD36 coimmunoprecipitated with flotillin-1, a constitutive lipid raft protein, and disruption of these membrane microdomains by methyl-β-cyclodextrin reduced AC phagocytosis. Efferocytosis induced a pattern of cytokine production, IL-10high/IL-12p40low, that is, characteristic of a regulatory phenotype. LPS potentiated the efferocytosis-induced production of IL-10, and this was prevented by blocking PAFR or CD36. It can be concluded that phagocytosis of apoptotic cells engages CD36 and PAFR, possibly in lipid rafts, and this is required for optimal efferocytosis and the establishment of the macrophage regulatory phenotype.

  14. Changes in Otx2 and Parvalbumin Immunoreactivity in the Superior Colliculus in the Platelet-Derived Growth Factor Receptor-β Knockout Mice

    Directory of Open Access Journals (Sweden)

    Juanjuan Zhao

    2013-01-01

    Full Text Available The superior colliculus (SC, a relay nucleus in the subcortical visual pathways, is implicated in socioemotional behaviors. Homeoprotein Otx2 and β subunit of receptors of platelet-derived growth factor (PDGFR-β have been suggested to play an important role in development of the visual system and development and maturation of GABAergic neurons. Although PDGFR-β-knockout (KO mice displayed socio-emotional deficits associated with parvalbumin (PV-immunoreactive (IR neurons, their anatomical bases in the SC were unknown. In the present study, Otx2 and PV-immunolabeling in the adult mouse SC were investigated in the PDGFR-β KO mice. Although there were no differences in distribution patterns of Otx2 and PV-IR cells between the wild type and PDGFR-β KO mice, the mean numbers of both of the Otx2- and PV-IR cells were significantly reduced in the PDGFR-β KO mice. Furthermore, average diameters of Otx2- and PV-IR cells were significantly reduced in the PDGFR-β KO mice. These findings suggest that PDGFR-β plays a critical role in the functional development of the SC through its effects on Otx2- and PV-IR cells, provided specific roles of Otx2 protein and PV-IR cells in the development of SC neurons and visual information processing, respectively.

  15. miR-503 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration through targeting the insulin receptor.

    Science.gov (United States)

    Bi, Rui; Ding, Fangbao; He, Yi; Jiang, Lianyong; Jiang, Zhaolei; Mei, Ju; Liu, Hao

    2016-12-01

    Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) is a common feature of disease progression in atherosclerosis. Here, we investigated the potential role of miR-503 in platelet-derived growth factor (PDGF)-induced proliferation and migration of human aortic smooth muscle cells and the underlying mechanisms of action. miR-503 expression was significantly downregulated in a dose- and time-dependent manner following PDGF treatment. Introduction of miR-503 mimics into cultured SMCs significantly attenuated cell proliferation and migration induced by PDGF. Bioinformatics analyses revealed that the insulin receptor (INSR) is a target candidate of miR-503. miR-503 suppressed luciferase activity driven by a vector containing the 3'-untranslated region of INSR in a sequence-specific manner. Downregulation of INSR appeared critical for miR-503-mediated inhibitory effects on PDGF-induced cell proliferation and migration in human aortic SMCs. Based on the collective data, we suggest a novel role of miR-503 as a regulator of VSMC proliferation and migration through modulating INSR.

  16. Malignant melanoma of the nasal cavity: a case report with examination of KIT and platelet derived growth factor receptor-α (PDGFRA

    Directory of Open Access Journals (Sweden)

    Tadashi Terada

    2011-10-01

    Full Text Available Although several clinicopathological studies of malignant melanoma of the nasal cavity have been reported, there are no studies of the expression and gene mutation of KIT and platelet derived growth factor receptor-α (PDGFRA in melanoma of the nasal cavity. A 92-year-old Japanese woman consulted to our hospital because of right nasal obstruction and epistaxis. Physical examination and imaging modalities showed a tumor of the right nasal cavity. A biopsy was taken, and it showed malignant epithelioid cells with melanin deposition. Immunohistochemically, the tumor was positive for S100 protein, HMB45, p53, Ki-67 (labeling=20%, KIT and PDGFRA. The tumor was negative for cytokeratins (AE1/3 and CAM5.2. A genetic analysis using PCR-direct sequencing revealed no mutation of KIT gene (exons 9, 11, 13, and 17 or the PDGFRA gene (exons 12 and 18. The pathological diagnosis was primary malignant melanoma of the nasal cavity. The tumor was reduced in size by local resection and chemotherapy (Darthmose regimen: dacarbazine, carmustine, cisplatine, and tamoxifen, and the patient is now alive and free from metastasis 9 months after the first manifestation. In conclusion, the author reported a case of melanoma of the nasal cavity expressing KIT and PDGFRA without gene mutations of KIT and PDGFRA.

  17. Sestrin 2 protein regulates platelet-derived growth factor receptor β (Pdgfrβ) expression by modulating proteasomal and Nrf2 transcription factor functions.

    Science.gov (United States)

    Tomasovic, Ana; Kurrle, Nina; Sürün, Duran; Heidler, Juliana; Husnjak, Koraljka; Poser, Ina; Schnütgen, Frank; Scheibe, Susan; Seimetz, Michael; Jaksch, Peter; Hyman, Anthony; Weissmann, Norbert; von Melchner, Harald

    2015-04-10

    We recently identified the antioxidant protein Sestrin 2 (Sesn2) as a suppressor of platelet-derived growth factor receptor β (Pdgfrβ) signaling and Pdgfrβ signaling as an inducer of lung regeneration and injury repair. Here, we identified Sesn2 and the antioxidant gene inducer nuclear factor erythroid 2-related factor 2 (Nrf2) as positive regulators of proteasomal function. Inactivation of Sesn2 or Nrf2 induced reactive oxygen species-mediated proteasomal inhibition and Pdgfrβ accumulation. Using bacterial artificial chromosome (BAC) transgenic HeLa and mouse embryonic stem cells stably expressing enhanced green fluorescent protein-tagged Sesn2 at nearly endogenous levels, we also showed that Sesn2 physically interacts with 2-Cys peroxiredoxins and Nrf2 albeit under different reductive conditions. Overall, we characterized a novel, redox-sensitive Sesn2/Pdgfrβ suppressor pathway that negatively interferes with lung regeneration and is up-regulated in the emphysematous lungs of patients with chronic obstructive pulmonary disease (COPD).

  18. Optimization of potent DFG-in inhibitors of platelet derived growth factor receptorβ (PDGF-Rβ) guided by water thermodynamics.

    Science.gov (United States)

    Horbert, Rebecca; Pinchuk, Boris; Johannes, Eugen; Schlosser, Joachim; Schmidt, Dorian; Cappel, Daniel; Totzke, Frank; Schächtele, Christoph; Peifer, Christian

    2015-01-08

    In this study we report on the hit optimization of substituted 3,5-diaryl-pyrazin-2(1H)-ones toward potent and effective platelet-derived growth factor receptor (PDGF-R) β-inhibitors. Originally, the 3,5-diaryl-pyrazin-2-one core was derived from the marine sponge alkaloid family of hamacanthins. In our first series compound 2 was discovered as a promising hit showing strong activity against PDGF-Rβ in the kinase assay (IC50 = 0.5 μM). Furthermore, 2 was shown to be selective for PDGF-Rβ in a panel of 24 therapeutically relevant protein kinases. Molecular modeling studies on a PDGF-Rβ homology model using prediction of water thermodynamics suggested an optimization strategy for the 3,5-diaryl-pyrazin-2-ones as DFG-in binders by using a phenolic OH function to replace a structural water molecule in the ATP binding site. Indeed, we identified compound 38 as a highly potent inhibitor with an IC50 value of 0.02 μM in a PDGF-Rβ enzymatic assay also showing activity against PDGF-R dependent cancer cells.

  19. Platelet-derived growth factor receptors form complexes with neuropilin-1 during megakaryocytic differentiation of thrombopoietin-dependent UT-7/TPO cells.

    Science.gov (United States)

    Ohsaka, Akimichi; Hirota-Komatsu, Satoko; Araki, Marito; Komatsu, Norio

    2015-04-10

    Neuropilin-1 (NRP-1) is involved in angiogenesis, but the role of NRP-1 in megakaryocytopoiesis is not yet fully understood. In this study, we investigated whether thrombopoietin (TPO) regulates the expression of platelet-derived growth factor (PDGF) and its receptors (PDGFRs) on TPO-dependent UT-7/TPO cells and whether PDGFRs and NRP-1 on UT-7/TPO cells form complexes during megakaryocytic differentiation. When UT-7/TPO cells were starved of TPO for 24 h and then stimulated with 5 ng/ml TPO, the expression of PDGF-B, PDGFRα, and PDGFRβ were significantly up-regulated after the addition of TPO. TPO also induced tyrosine phosphorylation of PDGFRα but not PDGFRβ, and promoted the formation of PDGFRαβ heterodimer complexes. Furthermore, megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of PDGFRβ and NRP-1 protein expression, complex formation between PDGFRs and NRP-1, PDGFRαβ heterodimer complexes, and an increase in PDGF-BB-binding activity. Immunocytochemistry confirmed complex formation between PDGFRs and NRP-1 and PDGFRαβ heterodimer complexes in PMA-differentiated UT-7/TPO cells. Our observations suggest that NRP-1 is involved in megakaryocytopoiesis through complex formation with PDGFRs, and that NRP-1-PDGFR-complexes may contribute to effective cellular functions mediated by TPO and PDGF in megakaryocytic cells.

  20. Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis.

    Science.gov (United States)

    Koupenova, Milka; Vitseva, Olga; MacKay, Christopher R; Beaulieu, Lea M; Benjamin, Emelia J; Mick, Eric; Kurt-Jones, Evelyn A; Ravid, Katya; Freedman, Jane E

    2014-07-31

    Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival.

  1. [Glycoproteins, inherited diseases of platelets, and the role of platelets in wound healing].

    Science.gov (United States)

    Nurden, Alan T; Nurden, Paquita

    2013-02-01

    Recognition that platelets have a glycocalyx rich in membrane glycoproteins prompted the discovery in France that inherited bleeding syndromes due to defects of platelet adhesion and aggregation were caused by deficiencies in major receptors at the platelet surface. Identification of the alpha IIb beta3 integrin prompted the development of powerful anti-thrombotic drugs that have gained worldwide use. Since these discoveries, the genetic causes of many other defects of platelet function and production have been elucidated, with the identification of an ADP receptor, P2 Y12, another widespread target for anti-thrombotic drugs. Discovery of the molecular basis of a rare disease of storage of biologically active proteins in platelet alpha-granules has been accompanied by the recognition of the roles of platelets in inflammation, the innate immune system and tissue repair, opening new avenues for therapeutic advances.

  2. Collagenous gastritis.

    Science.gov (United States)

    Jin, Xiaoyi; Koike, Tomoyuki; Chiba, Takashi; Kondo, Yutaka; Ara, Nobuyuki; Uno, Kaname; Asano, Naoki; Iijima, Katsunori; Imatani, Akira; Watanabe, Mika; Shirane, Akio; Shimosegawa, Tooru

    2013-09-01

    In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis. © 2012 The Authors. Digestive Endoscopy © 2012 Japan Gastroenterological Endoscopy Society.

  3. Neutrophils accentuate renal cold ischemia-reperfusion injury. Dose-dependent protective effect of a platelet-activating factor receptor antagonist.

    Science.gov (United States)

    Riera, M; Torras, J; Herrero, I; Valles, J; Paubert-Braquet, M; Cruzado, J M; Alsina, J; Grinyo, J M

    1997-02-01

    This study was undertaken to evaluate whether the renal damage induced by cold ischemia-reperfusion was worsened by neutrophils (PMN), and if blockade of platelet-activating factor (PAF) could effectively decrease this injury. After flushing with EuroCollins, 85 kidneys from Sprague-Dawley rats underwent either no cold ischemia or a 4-h cold ischemia, and then were reperfused for 75 min at 37 degrees C and 100 mm Hg in an isolated perfusion circuit. Reperfusion was performed with a Krebs-Henseleit solution containing 4.5% albumin, with and without human PMN (7.5 x 10(5) cells/ml) and with and without addition of a PAF receptor antagonist (BN 52021). Hemodynamic and functional parameters were continuously assessed during reperfusion. At end of the study, PAF production was evaluated. Presence of PMN during reperfusion of nonischemic kidneys produced no alteration of functional parameters or PAF production. After 4-h cold ischemia, the presence of PMN during reperfusion produced a significant worsening of plasma flow rate, glomerular filtration rate and sodium reabsorption in comparison with kidneys reperfused without PMN. Also, higher production of PAF was observed in the kidneys reperfused with PMN than in the kidneys reperfused without PMN. After 4-h cold ischemia, addition of BN 52021 during reperfusion in the presence of PMN significantly increased the plasma flow rate, glomerular filtration rate and sodium reabsorption in comparison with kidneys reperfused without this PAF antagonist. This effect was dose dependent. After 4-h cold ischemia, addition of BN 52021 during reperfusion in the absence of PMN produced no significant effect on functional parameters in comparison with kidneys reperfused without this PAF antagonist. These results indicate that PMN contribute to renal cold ischemia-reperfusion injury evaluated in the isolated perfused kidney. Treatment with a PAF receptor antagonist attenuated this injury in a dose-dependent manner, which suggests that it

  4. Mapuche herbal medicine inhibits blood platelet aggregation.

    Science.gov (United States)

    Falkenberg, Susan Skanderup; Tarnow, Inge; Guzman, Alfonso; Mølgaard, Per; Simonsen, Henrik Toft

    2012-01-01

    12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM) and collagen- (2.0 μg/mL) induced aggregations in human blood. These four species in respective extracts (in brackets) were Blechnum chilense (MeOH), Luma apiculata (H(2)O), Amomyrtus luma (DCM : MeOH 1 : 1) and Cestrum parqui (DCM : MeOH 1 : 1). The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1), and L. apiculata (H(2)O) were substantial and confirmed by inhibition of platelet surface activation markers.

  5. Mapuche Herbal Medicine Inhibits Blood Platelet Aggregation

    Directory of Open Access Journals (Sweden)

    Susan Skanderup Falkenberg

    2012-01-01

    Full Text Available 12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM and collagen- (2.0 μg/mL induced aggregations in human blood. These four species in respective extracts (in brackets were Blechnum chilense (MeOH, Luma apiculata (H2O, Amomyrtus luma (DCM : MeOH 1 : 1 and Cestrum parqui (DCM : MeOH 1 : 1. The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1, and L. apiculata (H2O were substantial and confirmed by inhibition of platelet surface activation markers.

  6. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Seishiro, E-mail: seishiro@nies.go.jp [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Fujitani, Yuji; Furuyama, Akiko [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Kanno, Sanae [Department of Legal Medicine, St. Marianna School of Medicine (Japan)

    2012-02-15

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC{sub 50} value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  7. Understanding platelet function through signal transduction.

    Science.gov (United States)

    Lazarus, Alan H; Song, Seng; Crow, Andrew R

    2003-01-01

    Platelets are activated by a number of stimuli resulting in the expression and/or activation of surface receptors, secretion of vasoactive substances, adhesion, aggregation, and finally thrombus formation. These events are propagated by a process known as transmembrane signaling, which relays the activating signal from the platelet membrane (eg, von Willebrand Factor binding to glycoprotein Ib) to the inside of the platelet which then serves to activate the platelet via a cascade of biochemical interactions. Inhibition of these transmembrane signaling molecules with a variety of available inhibitors or antagonists can in many cases prevent the platelet from becoming activated. An awareness of the mechanisms involved in platelet transmembrane signaling and the recent availability of new reagents to inhibit signaling may provide us with additional means to prevent platelet activation and perhaps even ameliorate the platelet storage lesion. This review will provide an introduction to the field of platelet transmembrane signaling and give an overview of some of the platelet signaling mechanisms that are relevant to transfusion medicine. Copyright 2003, Elsevier Science (USA). All rights reserved.

  8. Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling

    Science.gov (United States)

    Nikolakopoulou, Angeliki Maria; Zhao, Zhen; Montagne, Axel

    2017-01-01

    Pericytes regulate key neurovascular functions of the brain. Studies in pericyte-deficient transgenic mice with aberrant signaling between endothelial-derived platelet-derived growth factor BB (PDGF-BB) and platelet-derived growth factor receptor β (PDGFRβ) in pericytes have contributed to better understanding of the role of pericytes in the brain. Here, we studied PdgfrβF7/F7 mice, which carry seven point mutations that disrupt PDGFRβ signaling causing loss of pericytes and vascular smooth muscle cells (VSMCs) in the developing brain. We asked whether these mice have a stable or progressive vascular phenotype after birth, and whether both pericyte and VSMCs populations are affected in the adult brain. We found an early and progressive region-dependent loss of brain pericytes, microvascular reductions and blood-brain barrier (BBB) breakdown, which were more pronounced in the cortex, hippocampus and striatum than in the thalamus, whereas VSMCs population remained unaffected at the time when pericyte loss was already established. For example, compared to age-matched controls, PdgfrβF7/F7 mice between 4–6 and 36–48 weeks of age developed a region-dependent loss in pericyte coverage (22–46, 24–44 and 4–31%) and cell numbers (36–49, 34–64 and 11–36%), reduction in capillary length (20–39, 13–46 and 1–30%), and an increase in extravascular fibrinogen-derived deposits (3.4–5.2, 2.8–4.1 and 0–3.6-fold) demonstrating BBB breakdown in the cortex, hippocampus and thalamus, respectively. Capillary reductions and BBB breakdown correlated with loss of pericyte coverage. Our data suggest that PdgfrβF7/F7 mice develop an aggressive and rapid vascular phenotype without appreciable early involvement of VSMCs, therefore providing a valuable model to study regional effects of pericyte loss on brain vascular and neuronal functions. This model could be a useful tool for future studies directed at understanding the role of pericytes in the pathogenesis

  9. A Histologically Distinctive Interstitial Pneumonia Induced by Overexpression of the Interleukin 6, Transforming Growth Factor β1, or Platelet-Derived Growth Factor B Gene

    Science.gov (United States)

    Yoshida, Mitsuhiro; Sakuma, Junko; Hayashi, Seiji; Abe, Kin'ya; Saito, Izumu; Harada, Shizuko; Sakatani, Mitsunoir; Yamamoto, Satoru; Matsumoto, Norinao; Kaneda, Yasufumi; Kishmoto, Tadamitsu

    1995-10-01

    Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor β1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor β1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

  10. 807C/T polymorphism of platelet glycoprotein Ia gene is associated with cerebral hemorrhage in a Chinese population.

    Science.gov (United States)

    Zeng, Yi; Zhang, Le; Hu, Zhiping; Yang, Qidong; Ma, Mingming; Liu, Baoqiong; Xia, Jian; Xu, Hongwei; Liu, Yunhai; Du, Xiaoping

    2016-08-01

    Platelet glycoprotein (GP) mediated the role of platelet in coagulation. Platelet GP Ia 807C/T is the only GP polymorphism associated with the expression levels of GP Ia/IIa (the platelet collagen receptor). Recently, the GP Ia 807C/T polymorphism has been reported to have no association with cerebral hemorrhage (CH) in two studies pertained to Caucasian populations. The purpose of this study is to evaluate the association between platelet GP Ia 807C/T polymorphism and CH in a Han Chinese population. We performed genotype analysis for platelet GP Ia 807C/T polymorphism in a case-control study involving 195 patients with CH and 116 age- and sex-matched controls. In contrast to previous reports, we found that the frequencies of GP Ia 807C/T T allele, CT and TT genotype were much higher in CH patients than in controls (33.9% vs. 22.8%, p = 0.004; 45.5% and 11.1% vs. 40.4% and 2.6%, p = 0.022). Logistic regression analysis revealed that the presence of GP Ia 807C/T C allele and CC genotype were both associated with a decreased risk of CH compared with T allele, CT and TT genotypes, respectively (adjusted odds ratio [OR] = 0.565, 95% CI: 0.384-0.887, p = 0.005; adjusted OR = 0.172, 95% CI: 0.043-0.639, p = 0.009; adjusted OR = 0.254, 95% CI: 0.085-0.961, p = 0.041, respectively). These findings indicated that platelet GP Ia 807C/T polymorphism could be a protective factor of CH in the Chinese population.

  11. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

    Science.gov (United States)

    Rowley, Jesse W; Chappaz, Stéphane; Corduan, Aurélie; Chong, Mark M W; Campbell, Robert; Khoury, Amanda; Manne, Bhanu Kanth; Wurtzel, Jeremy G T; Michael, James V; Goldfinger, Lawrence E; Mumaw, Michele M; Nieman, Marvin T; Kile, Benjamin T; Provost, Patrick; Weyrich, Andrew S

    2016-04-07

    Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband β3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband β3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

  12. Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Nielsen, Christian; Söderström, Anna Cecilia

    2017-01-01

    Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor...

  13. Platelet-rich fibrinversus collagen membrane in the repair of gingival defects%富血小板纤维蛋白与胶原膜修复牙龈缺损创面的能力

    Institute of Scientific and Technical Information of China (English)

    董露; 肖琼; 杨琴秋; 孙勇; 陈红亮

    2016-01-01

    背景:前期实验中发现富血小板纤维蛋白具有良好的诱导牙龈软组织修复再生的能力。  目的:观察富血小板纤维蛋白膜与胶原膜对牙龈软组织愈合的影响,评价富血小板纤维蛋白膜诱导牙龈软组织缺损修复再生的能力。  方法:选因各种原因需要拔除前磨牙或磨牙同期进行位点保存的患者22例(前磨牙2颗,磨牙20颗),随机分为2组,22个软组织缺损区域,拔牙窝内植入Bio-Oss ,表面覆盖富血小板纤维蛋白膜或海奥胶原膜。通过追踪植入Bio-Oss后牙龈创面的愈合时间和植入Bio-Oss后1,2周的创面愈合率,评价富血小板纤维蛋白促进牙龈组织愈合的能力。  结果与结论:①富血小板纤维蛋白组愈合时间为(12.17±2.25) d,胶原膜组愈合时间为(17.30±2.58) d;植入Bio-Oss后1,2周富血小板纤维蛋白组愈合率明显高于胶原膜组。②结果说明,相同时间节点时富血小板纤维蛋白促进牙龈软组织的愈合能力明显优于胶原膜,达到相同愈合状态时富血小板纤维蛋白组所需时间明显短于胶原膜组。%BACKGROUND:Previous studies have found that platelet-rich fibrin has a good ability to induce gingival soft tissue repair and regeneration. OBJECTIVE:To observe the effects of platelet-rich fibrinversus colagen membrane on gingival soft tissue healing, and to evaluate the ability of platelet-rich fibrin to repair gingival defects. METHODS:Twenty-two patients (2 premolar teeth and 20 molars) scheduled for premolar or molar removal or ridge preservation due to various reasons were selected and randomized into two groups. Bio-Oss was implanted into the extraction socket folowed by covering with platelet-rich fibrin or colagen membrane. Healing time and healing rate of gingival defects were detected to evaluate the ability of platelet-rich fibrin to promote gingival tissue healing at 1-2 weeks after Bio-Oss implantation

  14. The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

    Science.gov (United States)

    Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

    2015-01-16

    Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.

  15. Platelet derivatives in regenerative medicine: an update.

    Science.gov (United States)

    De Pascale, Maria Rosaria; Sommese, Linda; Casamassimi, Amelia; Napoli, Claudio

    2015-01-01

    Prior preclinical and clinical studies support the use of platelet-derived products for the treatment of soft and hard tissue lesions. These regenerative effects are controlled by autocrine and paracrine biomolecules including growth factors and cytokines contained in platelet alpha granules. Each growth factor is involved in a phase of the healing process, such as inflammation, collagen synthesis, tissue granulation, and angiogenesis collectively promoting tissue restitution. Platelet derivatives have been prepared as platelet-rich plasma, platelet gel, platelet-rich fibrin, and platelet eye drops. These products vary in their structure, growth factors, composition, and cytokine concentrations. Here, we review the current use of platelet-derived biological products focusing on the rationale for their use and the main requirements for their preparation. Variation in the apparent therapeutic efficacy may have resulted from a lack of reproducible, standardized protocols for preparation. Despite several individual studies showing favorable treatment effects, some randomized controlled trials as well as meta-analyses have found no constant clinical benefit from the application of platelet-derived products for prevention of tissue lesions. Recently, 3 published studies in dentistry showed an improvement in bone density. Seven published studies showed positive results in joint regeneration. Five published studies demonstrated an improvement in the wound healing, and an improvement of eye epithelial healing was observed in 2 reports. Currently, at least 14 ongoing clinical trials in phase 3 or 4 have been designed with large groups of treated patients (n > 100). Because the rationale of the therapy with platelet-derived compounds is still debated, a definitive insight can be acquired only when these large randomized trials will be completed.

  16. Enhanced platelet adhesion in essential thrombocythemia after in vitro activation

    Directory of Open Access Journals (Sweden)

    Andreas C. Eriksson

    2010-06-01

    Full Text Available Objective: Essential thrombocythemia (ET is a chronic myeloproliferative disorder characterized by elevated platelet counts and increased risk of thrombosis. Ex vivo data suggest increased platelet reactivity in agreement with the increased thrombosis risk, while in vitro tests often detect decreased platelet activity. The present study aimed to investigate adhesion of ET-platelets in vitro, which is an aspect of platelet function that has been addressed in only a few studies on ET patients. Material and Methods: The study included 30 ET patients and 14 healthy controls. Platelet adhesion was measured with a static platelet adhesion assay. Results: The main finding was that ET-platelets were more readily activated by adhesion-inducing stimuli in vitro than control platelets. This was particularly evident in elderly patients and when using multiple stimuli, such as surfaces of collagen or fibrinogen combined with addition of adenosine 5’-diphosphate or ristocetin. Such multiple stimuli resulted in adhesion above the control mean +2 standard deviations for approximately 50% of the patients.Conclusion: The results are in accordance with the concept of increased platelet activity in ET, but opposite to most other in vitro studies. We suggest that the conditions in the adhesion assay might mimic the in vivo situation regarding the presence of chronic platelet activation.

  17. Regulation of platelet-activating factor (PAF) receptor and PAF receptor-mediated cellular response in Kupffer cells: Effect of vanadate

    Energy Technology Data Exchange (ETDEWEB)

    Chao, W.; Liu, H.; Hanahan, D.J.; Olson, M.S. (Univ. of Texas, San Antonio (United States))

    1991-03-11

    Vanadate is a phosphate analogue which affects phosphate transfer reactions which may be involved in regulatory processes in which tyrosine phosphorylation or dephosphorylation may be an important component. In the present study vanadate decreased the surface expression of PAF receptors and caused tyrosine-phosphorylation in numerous proteins in intact Kupffer cells. The vanadate-induced tyrosine-phosphorylation was inhibited by genistein, a specific tyrosine kinase inhibitor. The EC{sub 50} for the vanadate-initiated decrease in the surface expression of PAF receptors was approximately 0.25 mM, 0.65 mM, and 2 mM, respectively, when the vanadate exposure time was 3 h, 2h, and 1h. As a consequence, PAF-stimulated prostaglandin E{sub 2} (PGE{sub 2}) formation was attenuated in vanadate-treated Kupffer cells.