Sample records for p-selectin expression platelet

  1. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products (United States)

    Fuentes, Eduardo; Palomo, Iván


    Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists. PMID:24324520

  2. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

    Directory of Open Access Journals (Sweden)

    Eduardo Fuentes


    Full Text Available Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists.

  3. Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro

    Directory of Open Access Journals (Sweden)

    Gröschel Werner


    Full Text Available Abstract Background The effect of non-steroidal anti-inflammatory drugs (NSAIDs for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. Methods Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. Results There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. Conclusions Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.

  4. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression (United States)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.


    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  5. In vitro effect of anti-β2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation

    Directory of Open Access Journals (Sweden)

    A. Hoxha


    Full Text Available Antiphospholipid antibodies (aPL associated with thromboembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS. Beta2glycoprotein I (β2GPI is the main target antigen for aPL, but the pathogenic role of anti-β2GPI antibodies (aβ2GPI is still unclear. Some authors assume they play a role in activating platelets. We evaluated the effects of aβ2GPI antibodies on platelet P-selectin expression. Aβ2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography at two different stages (catastrophic and quiescent of the disease. Gel filtered platelets (100 x 109/L from healthy volunteers were incubated with β2-GPI (20 µg/mL and with different concentrations (5. 25 and 50 µg/mL of aβ2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. Aβ2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6, a platelet agonist, at a subthreshold concentration. Aβ2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47 vs 15%. TRAP-6 dependent platelet activation by aβ2GPI antibodies is consistent with the “two hit” pathogenetic hypothesis for thrombosis. Aβ2GPI antibodies induce higher platelet P-selectin expression during the active rather than the acute phases.

  6. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

    Energy Technology Data Exchange (ETDEWEB)

    Nygaard, Gyrid [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway); Herfindal, Lars; Kopperud, Reidun [Department of Biomedicine, University of Bergen, Bergen (Norway); Aragay, Anna M. [Department of Biomedicine, University of Bergen, Bergen (Norway); Molecular Biology Institute of Barcelona (IBMB, CSIC), Barcelona (Spain); Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune [Department of Biomedicine, University of Bergen, Bergen (Norway); Selheim, Frode, E-mail: [Proteomic Unit at University of Bergen (PROBE), University of Bergen, Bergen (Norway); Department of Biomedicine, University of Bergen, Bergen (Norway)


    Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

  7. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. (United States)

    Palabrica, T; Lobb, R; Furie, B C; Aronovitz, M; Benjamin, C; Hsu, Y M; Sajer, S A; Furie, B


    The glycoprotein P-selectin is a cell adhesion molecule of stimulated platelets and endothelial cells, which mediates the interaction of these cells with neutrophils and monocytes. It is a membrane component of cell storage granules, and is a member of the selectin family which includes E-selectin and L-selectin. P-selectin recognizes both lineage-specific carbohydrate ligands on monocytes and neutrophils, including the Lewis x antigen, sialic acid, and a protein component. In inflammation and thrombosis, P-selectin may mediate the interaction of leukocytes with platelets bound in the region of tissue injury and with stimulated endothelium. To evaluate the role of P-selectin in platelet-leukocyte adhesion in vivo, the accumulation of leukocytes within an experimental thrombus was explored in an arteriovenous shunt model in baboons. A Dacron graft implanted within an arteriovenous shunt is thrombogenic, accumulating platelets and fibrin within its lumen. These bound platelets express P-selectin. Here we show that antibody inhibition of leukocyte binding to P-selectin expressed on platelets immobilized on the graft blocks leukocyte accumulation and inhibits the deposition of fibrin within the thrombus. These results indicate that P-selectin is an important adhesion molecule on platelets, mediating platelet-leukocyte binding in vivo, that the presence of leukocytes in thrombi is mediated by P-selectin, and that these leukocytes promote fibrin deposition.

  8. In vitro effect of anti-β2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation


    Hoxha, A.; M. Tonello; E. Falcinelli; Giannini, S.; A. Ruffatti; A. Bontadi; P. Gresele; L. Punzi


    Antiphospholipid antibodies (aPL) associated with thromboembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (β2GPI) is the main target antigen for aPL, but the pathogenic role of anti-β2GPI antibodies (aβ2GPI) is still unclear. Some authors assume they play a role in activating platelets. We evaluated the effects of aβ2GPI antibodies on platelet P-selectin expression. Aβ2GPI antibodies in the plasma of a pregnant APS patie...

  9. Differing patterns of P-selectin expression in lung injury

    DEFF Research Database (Denmark)

    Bless, N M; Tojo, S J; Kawarai, H


    -selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression...

  10. The human megakaryocytic cell line UT-7/TPO responds to platelet agonists with intracellular Ca2+ elevation and P-selectin expression. (United States)

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Nawa, Katsuhiko; Yokota, Hiroshi


    Megakaryocytes have several signal transduction cascades that are similar, but not identical to platelet activation signals. In order to understand platelet signals in detail, it is useful to compare the similarities and/or differences between platelets and megakaryocytes. We evaluated platelet activation signals related to three kinds of Gq protein-coupled receptors using the megakaryocytic cell line UT-7/TPO. It was found that UT-7/TPO responded to thrombin, resulting in a continuous elevation of the [Ca2+]i (intracellular Ca2+) and P-selectin expression on the surface of the cells. Activation of integrin αIIbβ3 and thromboxane generation was not detected by any of the three stimulations. Taken together, although strong [Ca2+]i elevation by thrombin stimulation caused further P-selection expression, we could detect [Ca2+]i elevation, which is thought to be the individual signals through the thrombin, thromboxane A2 or ADP receptor, without considering the secondary signalling caused by αIIbβ3 activation and the arachidonic acid cascade using UT-7/TPO.

  11. Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets. (United States)

    Bachelet, Laure; Bertholon, Isabelle; Lavigne, Damien; Vassy, Roger; Jandrot-Perrus, Martine; Chaubet, Frédéric; Letourneur, Didier


    P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC(50) of 20 nM as compared to 400 nM for heparin and affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K(D) of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

  12. Differing Patterns of P-Selectin Expression in Lung Injury (United States)

    Bless, Nicolas M.; Tojo, Shinichiro J.; Kawarai, Hiroko; Natsume, Yasuhiro; Lentsch, Alex B.; Padgaonkar, Vaishalee A.; Czermak, Boris J.; Schmal, Hagen; Friedl, Hans P.; Ward, Peter A.


    Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor α. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed. PMID:9777942

  13. Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis. (United States)

    de Stoppelaar, S F; Van't Veer, C; Roelofs, J J T H; Claushuis, T A M; de Boer, O J; Tanck, M W T; Hoogendijk, A J; van der Poll, T


    Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis. © 2015 International Society on Thrombosis and Haemostasis.

  14. In Vivo Tracking of Platelets: Circulating Degranulated Platelets Rapidly Lose Surface P-Selectin but Continue to Circulate and Function

    National Research Council Canada - National Science Library

    Michelson, A


    To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo...

  15. Melanoma cell metastasis via P-selectin-mediated activation of acid sphingomyelinase in platelets. (United States)

    Becker, Katrin Anne; Beckmann, Nadine; Adams, Constantin; Hessler, Gabriele; Kramer, Melanie; Gulbins, Erich; Carpinteiro, Alexander


    Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90 % of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.

  16. P-Selectin (CD62P) Expression in Liver Tissue of Biliary Atresia: A New Perspective in Etiopathogenesis. (United States)

    Sira, Mostafa M; Sira, Ahmad M; Ehsan, Nermine A; Mosbeh, Asmaa


    The etiology of biliary atresia (BA) is still elusive. Inflammation plays a key role in bile duct and liver injury. The recruitment and accumulation of inflammatory cells is largely dependent on adhesion molecules. We aimed to investigate P-selectin (CD62P) expression in liver tissue in patients with BA compared with other neonatal cholestatic disorders. The study included 63 infants with neonatal cholestasis in 2 groups: BA group (n = 32) and non-BA group (n = 31) with non-BA cholestatic disorders as controls. Demographic, clinical, laboratory, ultrasonographic, and histopathological parameters were collected. P-selectin immunostaining was performed. Immunostaining in bile duct epithelium, cellular infiltrate, and vascular endothelial cells were scored as positive or negative. The frequency of P-selectin-positive endothelium, platelets, and bile duct epithelium was significantly higher in the BA group (72%, 72%, and 63%, respectively) than in the non-BA group (32%, 16%, and 13%, respectively) with P of 0.002, thrombocytosis versus 25% in the controls (P = 0.001 for both). The significant expression of P-selectin in endothelium, platelets, and bile duct epithelium in patients with BA suggests a potential role for this adhesion molecule in the pathogenesis of this devastating neonatal hepatic disorder. It further suggests that platelets in BA are activated and may have a role in the inflammatory process in BA.

  17. Levels of platelet-derived microparticles and soluble p-selectin in patients of acute myocardial infarction (case control study). (United States)

    Hameed, Aisha; Rubab, Zille; Abbas Rizvi, Syed Khizar; Hussain, Shabbir; Latif, Waqas; Mohsin, Shahida


    TTo measure levels of platelet-derived microparticles and soluble P-selectin in patients of acute myocardial infarction and their comparison with healthy controls. This case-control study was conducted in Department of Haematology, University of Health Sciences Lahore from April to September 2013, and comprised patients of acute myocardial infarction in group 1 and healthy controls in group 2. Platelet-derived microparticles and soluble P-selectin were measured by enzyme-linked immunosorbent assay. SPSS21 was used for data analysis. Of the 80 participants, 50(62.5%) were patients and 30(37.5%) were controls. The mean levels of platelet-derived microparticles and soluble P-selectin were significantly higher in group 1 compared to group 2 (45.70±10.30 vs 10.60±0.96, and 51.46±9.30 vs 9.16±1.04, respectively) (pderived microparticles and soluble P-selectin in three intervals after acute myocardial infarction (p>0.05). Although levels of platelet-derived microparticles and soluble P-selectin did not correlate to creatinekinase-myocardial band levels (p>0.05), but there was a trend of significant correlation with cardiac troponin T (pderived microparticles and soluble P-selectin can be used as novel early diagnostic marker of acute myocardial infarction.

  18. The significance of diminished sTWEAK and P-selectin content in platelets of patients with pulmonary arterial hypertension. (United States)

    Kazimierczyk, Remigiusz; Błaszczak, Piotr; Kowal, Krzysztof; Jasiewicz, Małgorzata; Knapp, Małgorzata; Szpakowicz, Anna; Ptaszyńska-Kopczyńska, Katarzyna; Sobkowicz, Bożena; Waszkiewicz, Ewa; Grzywna, Ryszard; Musial, Włodzimierz J; Kamiński, Karol A


    Pulmonary arterial hypertension (PAH) is a progressive disease characterized by proliferative changes in pulmonary arteries. There is growing evidence suggesting that soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and P-selectin could be involved in PAH development and progression. Here we investigate whether circulating platelets may be a source of sTWEAK and contribute to diminished availability of sTWEAK and P-selectin in PAH patients. We have prospectively enrolled two independent study groups of stable patients with confirmed PAH and age matched controls: derivation (10 PAH; 15 controls) and validation (20 PAH; 12 controls). P-selectin and sTWEAK concentrations were measured in platelet-poor plasma and platelet lysate. To avoid procedural bias, in each group we employed different protocols for platelet isolation. Consistently, both in derivation and validation groups PAH patients presented significantly lower sTWEAK content in platelets than control group with no significant differences in plasma levels. Similarly, patients presented comparable to controls plasma P-selectin concentrations and lower concentration in platelet lysate. Kaplan-Meier analysis revealed that patients with low platelet sTWEAK/total protein concentration ratio had more frequently detoriation of PAH in the follow-up (16.51 ± 3.32 months), log-rank test, p = .03. Patients diagnosed with pulmonary arterial hypertension present diminished sTWEAK and P-selectin storage capacity in platelets. Thrombocytes appear to be a major source of sTWEAK that could be released upon local injury and its decreased availability could have an impact on pathophysiology and prognosis in PAH. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. [Effects of riboflavin combined with photosensitization on reduction of Gram-positive and Gram-negative indicating germs in plasma and P-selectin expression of apheresis platelet concentrates]. (United States)

    Zhou, Xue-Yin; Xiong, Wen; Kong, Ling-Kui


    This study was purposed to confirm the practical efficacy of reducing indicating germs suspended in plasma by riboflavin and photosensitized inactivation and to evaluate its influence on activation of apheresis platelet concentrates. The synergistic effects of riboflavin combined with ultraviolet irradiation on inactivation of germs were investigated by using Escherichia Coli (E. coli) and Staphylococcus Aureus (S. aureus) as Gram⁻ and Gram(+) indicating germs, respectively. The activation status of apheresis-platelet concentrates treated with riboflavin combined with ultraviolet irradiation was detected by flow cytometry. The results showed that when 50 μmol/L of riboflavin was combined with 6.2 J/ml of ultraviolet irradiation, the T/E ratios reached 1.42 for E. coli and 1.68 for S. Aureus, and reduction of E. Coli and S. Aureus were 3.87 Logs and 3.82 Logs respectively; the CD62p expression level on germ-inactivated platelets stored at 22 degrees C for 0 and 5 days were 4.92% and 36.18% respectively, which slightly increased as compared with controls (3.94% and 32.03)% (p < 0.05). It is concluded that combination of riboflavin with ultraviolet irradiation displays well synergistic effects which can reduce E. Coli and S. Aureus counts, but no significantly influence on platelets. The partial activation of liquid platelets mainly presents metabolism damage during storage, which is found at an acceptable level.

  20. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Samia A. Ebeid


    Sep 19, 2013 ... Abstract Background: Hypercoagulability in thalassemia especially in thalassemia major has emerged as a complication of the disease. There is evidence of increased platelet aggregation and increased proportion of platelets expressing P-selectin in thalassemia. P-selectin is a cell adhesion molecule ...

  1. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Background: Hypercoagulab ility in thalassemia especially in thalassemia major has emerged as a complication of the disease. There is evidence of increased platelet aggregation and increased proportion of platelets expressing P-selectin in thalassemia. P-selectin is a cell adhesion molecule which plays a key role in ...

  2. Isolation and characterization of N-feruloyltyramine as the P-selectin expression suppressor from garlic (Allium sativum) (United States)

    Because garlic (Allium sativum) is believed to have positive health effects on cardiovascular disease, the screening of isolated fractions from a garlic extract against cardiovascular disease related-processes should help identify active compounds. Both P-selectin expression suppressing activity ag...

  3. Activation of platelets by in vitro whole blood contact with materials: increases in microparticle, procoagulant activity, and soluble P-selectin blood levels. (United States)

    Gemmell, C H


    Non-adherent platelets and plasma were analyzed for evidence of platelet activation after whole blood contact with materials under conditions of low shear for one hour at 37 degrees C. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, procoagulant platelet membranes and soluble P-selectin levels. Flow cytometric quantification of microparticles (MPs) was highly sensitive and entailed the direct determination of microparticle concentrations as opposed to the traditional quantification of microparticle percentages (relative to total number of MPs and platelets). Whole blood contact with polypropylene surfaces led to the greatest drops in bulk platelet counts and also to the lowest increases in microparticle concentrations. Flow cytometry was also used to assess procoagulant levels (annexin V binding) within a light scatter region known to contain platelets and some large microparticles. All surfaces were noted to generate a significant procoagulant population that was, based on forward light scatter, mostly very small platelets or large microparticles. In contrast, most of the P-selectin positive platelets were averaged sized. Lastly. all surfaces generated soluble P-selectin levels that were approximately double the level (25 ng ml(-1)) noted in the resting whole blood samples. In addition to our previous reports, these findings support the observation that there is strong evidence of platelet activation in the bulk that we anticipate will ultimately lead to more relevant in vitro testing of the compatibility of platelets towards materials.

  4. The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells.

    LENUS (Irish Health Repository)

    Corcoran, T B


    BACKGROUND: Reperfusion injury is characterized by free radical production and endothelial inflammation. Neutrophils mediate much of the end-organ injury that occurs, requiring P-selectin-mediated neutrophil-endothelial adhesion, and this is associated with decreased endothelial nitric oxide production. Propofol has antioxidant properties in vitro which might abrogate this inflammation. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia and then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg\\/l or propofol 5 microg\\/l for 4 h after re-oxygenation and were then examined for P-selectin expression and supernatant nitric oxide concentrations for 24 h. P-selectin was determined by flow cytometry, and culture supernatant nitric oxide was measured as nitrite. RESULTS: In saline-treated cells, a biphasic increase in P-selectin expression was demonstrated at 30 min (P = 0.01) and 4 h (P = 0.023) after re-oxygenation. Propofol and Diprivan prevented these increases in P-selectin expression (P < 0.05). Four hours after re-oxygenation, propofol decreased endothelial nitric oxide production (P = 0.035). CONCLUSION: This is the first study to demonstrate an effect of propofol upon endothelial P-selectin expression. Such an effect may be important in situations of reperfusion injury such as cardiac transplantation and coronary artery bypass surgery. We conclude that propofol attenuates re-oxygenation-induced endothelial inflammation in vitro.

  5. Role of Soluble P-Selectin Among Type 2 Diabetic Patients with and ...

    African Journals Online (AJOL)

    Type 2 diabetes mellitus (T2DM) is associated with a marked increase in the risk of coronary heart disease. Platelets play a significant role in coronary artery disease (CAD). P-selectin is a component of the platelet granule membrane that is expressed on the platelet surface membrane and shed into the plasma as ...

  6. CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin. (United States)

    Doyle, Emily L; Ridger, Victoria; Ferraro, Francesco; Turmaine, Mark; Saftig, Paul; Cutler, Daniel F


    The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.

  7. CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells. (United States)

    Aigner, S; Sthoeger, Z M; Fogel, M; Weber, E; Zarn, J; Ruppert, M; Zeller, Y; Vestweber, D; Stahel, R; Sammar, M; Altevogt, P


    P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohydrate epitope) but did not react with MoAbs against the sialylLe(x/a) epitope. The carcinoma cells and CD24-beads derived from these cells could bind to activated platelets or P-selectin transfected Chinese hamster ovary cells (P-CHO) in a P-selectin-dependent manner and this binding was blocked by soluble CD24. Transfection of human adenocarcinoma cells with CD24 enhanced the P-selectin-dependent binding to activated platelets. Treatment of the carcinoma cells or the CD24 transfectant with phosphatidylinositol-specific phospholipase C reduced CD24 expression and P-selectin-IgG binding concomitantly. These results establish a role of CD24 as a novel ligand for P-selectin on tumor cells. The CD24/P-selectin binding pathway could be important in the dissimination of tumor cells by facilitating the interaction with platelets or endothelial cells.

  8. Differential expression of genes encoding CD30L and P-selectin in cattle with Johne's disease: Progress toward a diagnostic gene expression signature

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Grell, S. N.; Heegaard, Peter M. H.


    a unique gene expression signature for paraTB infection. In the present study, non-stimulated leukocytes isolated from 10 sub-clinical paraTB infected cows were examined for genes being expressed at significantly different levels than in similar cells from control cows with the same herd background. We...... of the seven genes selected for qRT-PCR, CD30 ligand (CD30L) and P-selectin were consistently differentially expressed in freshly isolated leukocytes from paraTB infected and control animals of both breeds of cattle. Although further work is clearly needed to develop a more complete gene expression signature...

  9. Associations of combined polymorphisms of the platelet membrane glycoproteins Ia and IIIa and the platelet-endothelial cell adhesion molecule-1 and P-Selectin genes with IVF implantation failures. (United States)

    Vlachadis, Nikolaos; Tsamadias, Vasileios; Vrachnis, Nikolaos; Kaparos, Georgios; Vitoratos, Nikolaos; Kouskouni, Evaggelia; Economou, Emmanuel


    The aim of the study was to investigate the combined impact of the genetic heterogeneity of the glycoproteins Ia (GpIa) and IIIa (GpIIIa) and the platelet-endothelial cell adhesion molecule-1 (PECAM-1) and P-Selectin genes on IVF embryo transfer implantation failures (IVF-ET failures). Sixty nulligravida women with previous IVF-ET failures and 60 fertile controls were genotyped for the GpIa-C807T, GpIIIa-PlA1/PA2, PECAM-1-C373G (Leu125Val) and P-Selectin-A37674C (Thr715Pro) polymorphisms by pyrosequencing. Compared with wild-type combined homozygotes, carriers of combinations of risk alleles in two gene loci were at significantly increased risk for IVF-ET failure, whereas carriers of the combination of GpIa-807T, GpIIIa-PlA2 and PECAM-1-373G alleles had OR = 52.50 (95%CI: 4.05-680.95, p IVF-ET failures especially for younger women and provided a genetic risk score with good diagnostic accuracy in the prediction of IVF-ET failures.

  10. Standardized flavonoid-rich Eugenia jambolana seed extract retards in vitro and in vivo LDL oxidation and expression of VCAM-1 and P-selectin in atherogenic rats. (United States)

    Jadeja, Ravirajsinh N; Thouaojam, Menaka C; Sankhari, Jayantha M; Jain, Mahendra; Devkar, Ranjitsinh V; Ramachandran, A V


    The present inventory evaluates anti-atherogenic potential of flavonoid-rich Eugenia jambolana seed extract (EJSE) against in vitro low-density lipoprotein (LDL) oxidation, foam cell formation, and atherogenic (ATH) diet-induced experimental atherosclerosis in rats. EJSE was able to prevent in vitro LDL oxidation and oxidized LDL-induced macrophage foam cell formation. Also, EJSE supplementation to ATH rats significantly minimized increment in serum markers of LDL oxidation. The ex vivo oxidation indices were also minimized in LDL of EJSE-treated animals. Microscopic evaluation of thoracic aorta of ATH + EJSE rats recorded minimal evidence of atheromatous plaque formation, accumulation of lipid laden macrophages, calcium deposition, and expression of cell adhesion molecules (vascular cell adhesion molecule-1 and P-selectin). This is the first scientific report that demonstrates anti-atherogenic potential of EJSE and warrants further evaluation at clinical level.

  11. A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT). (United States)

    Jy, W; Mao, W W; Horstman, L L; Valant, P A; Ahn, Y S


    Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

  12. Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

    Directory of Open Access Journals (Sweden)

    Lubor Borsig


    Full Text Available Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.


    Directory of Open Access Journals (Sweden)

    Samo Zver


    Full Text Available Background. Essential thrombocythemia (ET is a chronic myeloproliferative disease with a platelet count within the range of 400–2000 × 109/L. Higher percentage of platelets in the circulation of patients with ET express also activation markers on their membranes. Two of such markers are P-selectin and activated fibrinogen on platelet membranes. Because of frequent thrombembolic and also bleeding related complications, treatment of ET is mandatory. Patients whose platelet count is less than 1000 × 109/L and who did not suffer any thrombembolic complication during the course of the disease, are ussually treated with an antiaggregation drug, acetylsalicylic acid 100 mg/daily orally. Clopidogrel is an adenosyn-di-phosphate (ADP receptor antagonist in platelets. There is no routine clinical data about clopidogrel treatment in the patients with ET and only sporadic case reports can be find in the literature.Patients and methods. In our clinical study we compared antiaggregational effects of acetylsalicylic acid and clopidogrel, by measuring the P-selectin level and activated fibrinogen expression on platelet membranes.There were 35 ET patients included, within the age range between 21 and 78 years and with platelet counts within 451–952 × 109/L. None of the patients did suffer any thrombembolic complication during the course of the disease. During the sequential 14 day periods, patients received acetylsalicylic acid 100 mg/daily orally, followed by clopidogrel 75 mg/daily orally and ultimativelly, together acetylsalicylic acid 100 mg/daily orally plus clopidogrel 75 mg/daily orally. After each fourteen days period the level of P-selectin and activated fibrinogen activated platelets were determined with monoclonal antibodies on flow cytometer. Statistical evaluation was calculated on the difference of average values between the two small, independent pair groups with the t-test.Results. When the patients stopped with acetylsalicylic acid and

  14. P-selectin, carcinoma metastasis and heparin: novel mechanistic connections with therapeutic implications

    Directory of Open Access Journals (Sweden)

    Varki A.


    Full Text Available Metastasis is a multistep cascade initiated when malignant cells penetrate the tissue surrounding the primary tumor and enter the bloodstream. Classic studies indicated that blood platelets form complexes around tumor cells in the circulation and facilitate metastases. In other work, the anticoagulant drug heparin diminished metastasis in murine models, as well is in preliminary human studies. However, attempts to follow up the latter observation using vitamin K antagonists failed, indicating that the primary mechanism of heparin action was unrelated to its anticoagulant properties. Other studies showed that the overexpression of sialylated fucosylated glycans in human carcinomas is associated with a poor prognosis. We have now brought all these observations together into one mechanistic explanation, which has therapeutic implications. Carcinoma cells expressing sialylated fucosylated mucins can interact with platelets, leukocytes and endothelium via the selectin family of cell adhesion molecules. The initial organ colonization of intravenously injected carcinoma cells is attenuated in P-selectin-deficient mice, in mice receiving tumor cells pretreated with O-sialoglycoprotease (to selectively remove mucins from cell surfaces, or in mice receiving a single dose of heparin prior to tumor cell injection. In each case, we found that formation of a platelet coating on cancer cells was impeded, allowing increased access of leukocytes to the tumor cells. Several weeks later, all animals showed a decrease in the extent of established metastasis, indicating a long-lasting effect of the short-term intervention. The absence of obvious synergism amongst the three treatments suggests that they all act via a common pathway. Thus, a major mechanism of heparin action in cancer may be inhibition of P-selectin-mediated platelet coating of tumor cells during the initial phase of the metastatic process. We therefore suggest that heparin use in cancer be re

  15. Relationship between changes in platelet reactivity and changes in platelet receptor expression induced by physical exercise. (United States)

    Aurigemma, Cristina; Fattorossi, Andrea; Sestito, Alfonso; Sgueglia, Gregory A; Farnetti, Sara; Buzzonetti, Alexia; Infusino, Fabio; Landolfi, Raffaele; Scambia, Giovanni; Crea, Filippo; Lanza, Gaetano A


    In previous studies we have consistently shown a significant increase of platelet reactivity after exercise in patients with obstructive coronary artery disease (CAD). We also observed a significant individual variability in the response to exercise of platelet reactivity in these patients. Whether exercise-induced changes in platelet reactivity correlate with changes in platelet membrane receptors in patients with CAD is unknown. We studied 26 patients with stable CAD and 10 matched healthy controls who underwent a symptom-limited treadmill exercise stress test. Venous blood samples were collected at rest and within 5 min of peak exercise. Platelet reactivity was measured by the PFA-100 method as time to occlude (closure time, CT) a ring coated with collagen/adenosine diphosphate (C/ADP). Platelet expression of glycoprotein (GP) IIb/IIIa, in both global (CD41) and active form (PAC-1), and P-selectin (CD62P) and formation of leukocyte-platelet aggregates were assessed by flow cytometry. After exercise CT did not change in controls (85.4+/-12 to 84.0+/-9 s, p=0.37), whereas it decreased in CAD patients (98.8+/-24 to 91.4+/-25 s, p5 s after exercise) and CAD group 2 (10 patients no increase in platelet reactivity after exercise). CD41 and PAC-1 expression increased in CAD group 1 (p=0.008 and p=0.026, respectively) but not in CAD group 2 (p=0.39 and p=0.50, respectively). No significant differences were observed between the 2 groups for changes in CD62P and leukocyte-platelet aggregates. Our data show that, in patients with stable CAD, an increased platelet reactivity to C/ADP stimulation after exercise, as assessed by the PFA-100 method, is specifically associated with an increased expression of platelet GP IIb/IIIa receptor.

  16. The effect of centrifugation speed and time on pre-analytical platelet activation

    DEFF Research Database (Denmark)

    Söderström, Anna Cecilia; Nybo, Mads; Nielsen, Christian


    BACKGROUND: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation....... METHODS: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean...... of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), p...

  17. Material-induced tissue factor expression but not CD11b upregulation depends on the presence of platelets. (United States)

    Gorbet, M B; Sefton, M V


    Biomaterials activate leukocytes as well as platelets when exposed to blood. One feature of leukocyte activation at least at times beyond a few hours is tissue factor expression, contributing to a procoagulant state. We show here that platelet activation and specifically platelet-monocyte aggregate formation appears to be a precondition for tissue factor expression. Material-induced Tissue Factor (TF) expression by isolated leukocytes (6 x 10(6) cells/mL) resuspended in increasing concentrations of platelets in plasma was elevated when the platelet concentration was 50 x 10(6) platelets/mL or more; at lower platelet concentrations (1-25 x 10(6). cells/mL) the TF expression remained at background levels. On the other hand, significant CD11b upregulation was observed on leukocytes, in bulk and adherent to beads, at all platelet concentrations. This platelet effect on material-induced TF expression appeared to be mediated by the formation of platelet-monocyte aggregates. Anti-P-selectin, which blocked the association between platelets and leukocytes, reduced monocyte adhesion and material-induced TF expression for bulk monocytes. Anti-GPIIb/IIIa, a GPIIb/IIIa platelet antagonist, also reduced monocyte adhesion and material-induced TF expression in the bulk, most likely due to its inhibiting effect on the formation of platelet-monocyte aggregates, secondary to platelet activation. However, the antibody-associated reductions for bulk leukocytes (mainly neutrophils) were small and incomplete. Similar levels of TF expression, in the bulk, were observed with both polystyrene (PS), a strong platelet activator, and polyethylene glycol-modified PEG (PS-PEG), a mild platelet activator. The role of platelets in material-induced TF expression appears to be mediated in part via the formation of platelet-monocyte aggregates, although other mechanisms are likely also involved. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 792-800, 2003

  18. Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

    Directory of Open Access Journals (Sweden)

    Andrew D Westmuckett

    Full Text Available We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1 is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST activity.Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6 or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6 which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

  19. Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains. (United States)

    Itoh, Saotomo; Susuki, Chie; Takeshita, Kana; Nagata, Kisaburo; Tsuji, Tsutomu


    P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte interactions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid microdomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8-treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-perturbing agents such as methyl-beta-cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL-8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with antibodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin.

  20. Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

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    Schapira Marc


    Full Text Available Abstract Background P-selectin glycoprotein ligand-1 (PSGL-1 plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog and examined mammalian PSGL-1 interactions with human selectins. Results A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR. Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin

  1. Depression is associated with an increase in the expression of the platelet adhesion receptor glycoprotein Ib. (United States)

    Walsh, Marie-Thérèse; Dinan, Timothy G; Condren, Rita M; Ryan, Martina; Kenny, Dermot


    There is a significant association between cardiovascular disease and depression. Previous studies have documented changes in platelets in depression. It is unknown if depression causes functional changes in platelet surface receptors. Therefore, we analyzed (1) the surface expression of glycoprotein (GP)Ib and the integrin receptor alpha(IIb)beta(IIIa), receptors involved in platelet adhesion and aggregation, (2) CD62 (P-selectin) and CD63, integral granule proteins translocated during platelet activation, (3) platelet aggregation in response to ADP and (4) plasma levels of glycocalicin and von Willebrand factor (vWF), in depressed patients compared to healthy volunteers. Fifteen depressed patients with a Hamilton depression score of at least 22 and fifteen control subjects were studied. Platelets were assessed for surface expression levels of GPIb, alpha(IIb)beta(IIIa), CD62 and CD63 by flow cytometry. Genomic DNA was isolated to investigate a recently described polymorphism in the 5' untranslated region of the GPIbalpha gene. The number of GPIb receptors was significantly increased on the surface of platelets from patients with depression compared to control subjects. Surface expression of CD62 was also significantly increased in the depressed patients versus control subjects. There was no significant difference between depressed patients and healthy volunteers in the surface expression of alpha(IIb)beta(IIIa) or CD63, or in glycocalicin or vWF plasma concentration, or ADP-induced aggregation. There was no difference in allele frequency of the Kozak region polymorphism of the GPIbalpha gene, which can affect GPIb expression. The results of this study demonstrate that the number of GPIb receptors on platelets are increased in depression and suggest a novel risk factor for thrombosis in patients with depression.

  2. P-selectin in preterm infants suffering necrotizing enterocolitis ...

    African Journals Online (AJOL)

    Objective: To study P-selectin, a possible cause of NEC, in the blood of preterm infants. Study design: Twenty-four consecutive preterms, clinically suspected or proven to have NEC, were enrolled in this pilot study. Their weight ranged from 1 to 2.3 Kg (mean ±SD: 1.7±0.5 Kg), age ranged from 2 to 21 days (mean ±SD: ...

  3. Role of Soluble P-Selectin Among Type 2 Diabetic Patients with and ...

    African Journals Online (AJOL)

    ROC curve analysis for hsCRP and sP-selectin indicated that, sP-selectin had higher sensitivity and specificity than hsCRP in diabetic patient with coronary artery disease. In conclusion, measurement of soluble P-selectin seems more helpful marker of impending coronary artery insult in diabetic patients and had higher ...

  4. Development of Polymer Microcapsules Functionalized with Fucoidan to Target P-Selectin Overexpressed in Cardiovascular Diseases. (United States)

    Li, Bo; Juenet, Maya; Aid-Launais, Rachida; Maire, Murielle; Ollivier, Véronique; Letourneur, Didier; Chauvierre, Cédric


    New tools for molecular imaging and targeted therapy for cardiovascular diseases are still required. Herein, biodegradable microcapsules (MCs) made of polycyanoacrylate and polysaccharide and functionalized with fucoidan (Fuco-MCs) are designed as new carriers to target arterial thrombi overexpressing P-selectin. Physicochemical characterizations demonstrated that microcapsules have a core-shell structure and that fucoidan is present onto the surface of Fuco-MCs. Furthermore, their sizes range from 2 to 6 µm and they are stable on storage over 30 d at 4 °C. Flow cytometry experiments evidenced the binding of Fuco-MCs for human activated platelets as compared to MCs (mean fluorescence intensity: 12 008 vs. 9, p microcapsules reveal excellent compatibility with 3T3 cells in cytotoxicity assay. One hour after intravenous injection of microcapsules, histological analysis revealed that Fuco-MCs are localized in the rat abdominal aortic aneurysm thrombotic wall and that the binding in the healthy aorta is low. In conclusion, these microcapsules appear as promising carriers for targeting of tissues characterized by P-selectin overexpression and for their molecular imaging or treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules. (United States)

    Asako, H; Kurose, I; Wolf, R; DeFrees, S; Zheng, Z L; Phillips, M L; Paulson, J C; Granger, D N


    The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

  6. New in vitro effects of clopidogrel on platelets in hyperlipidemic and healthy subjects

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    Derya Özsavcı


    Full Text Available Objective: We aimed to detect novel in vitro effects of clopidogrel on platelets by assessment of the following parameters: malondialdehyde, glutathione, nitrite, aggregation response, and expressions of P-selectin, fibrinogen, apolipoprotein A1, apolipoprotein B, and phosphatidylserine.Materials and Methods: Platelets were obtained from healthy (n: 9 and hyperlipidemic (n: 9 volunteers. Expressions of P-selectin, fibrinogen, apolipoproteins A1/B and phosphatidylserine with and without clopidogrel were assayed by flow cytometry. Malondialdehyde, glutathione, aggregation and nitrite levels were also assayed. Results: Without clopidogrel, the baseline values of platelet aggregation, malondialdehyde, and expressions of P-selectin, fibrinogen and phosphatidylserine were significantly higher, whereas nitrite and expression of apolipoproteins A1/B were significantly lower in hyperlipidemics than in the healthy group. In both groups, clopidogrel significantly reduced aggregation and expression of fibrinogen, but it elevated nitrite levels. Clopidogrel significantly decreased P-selectin and phosphatidylserine expression and malondialdehyde but increased expressions of apolipoproteins A1/B only in hyperlipidemics. Conclusion: It seems that clopidogrel has some new in vitro antiplatelet effects. The present study is a basic in vitro study to suggest new insights into the effects of clopidogrel on platelet functions.

  7. Soluble P-selectin level correlates with acetylsalicylic acid but not with clopidogrel response in patients with stable coronary artery disease after a percutaneous coronary intervention. (United States)

    Kaufmann, Jan; Wellnhofer, Ernst; Kappert, Kai; Urban, Daniel; Meyborg, Heike; Hauptmann, Tobias; Müller, Aline; Meixner, Martin; Graf, Kristof; Fleck, Eckart; Stawowy, Philipp


    Impaired response to dual antiplatelet therapy is associated with worse cardiovascular outcome. Besides antiplatelet effects, there is evidence that both clopidogrel and acetylsalicylic acid (ASA) have anti-inflammatory properties. However, little is known about the relationship between platelet function and inflammation under dual antiplatelet therapy in patients with stable coronary artery disease. The purpose of the study was to investigate the correlation of platelet function with soluble (s)P-selectin and soluble (s)CD40L in patients undergoing elective percutaneous coronary intervention. Poor response to ASA and clopidogrel could lead to increased levels of inflammatory markers. A total of 148 patients were included. Eighty percent of the patients were on 100 mg ASA and all patients were clopidogrel naive. They underwent percutaneous coronary intervention and received a loading dose of 600 mg clopidogrel. Platelet function was assessed by light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein analysis at baseline, 24 h after loading, and after 1 month of maintenance therapy, respectively. Plasma levels of sP-selectin and sCD40L were measured. To classify low responders to clopidogrel, patients were screened for genetic variants determining clopidogrel absorption and metabolization. sP-selectin levels correlated with LTA findings after stimulation with arachidonic acid (P=0.012). Further, in addition to decreased platelet reactivity observed on LTA, lower sP-selectin levels were seen in patients under ASA therapy (P=0.004). CYP2C19*2 allele carriers had a higher platelet reactivity after clopidogrel loading measured by adenosine diphosphate-induced aggregation in LTA (P=0.008) and vasodilator-stimulated phosphoprotein phosphorylation (P=0.035); however, there was no difference in the inflammatory markers. Multiple regression analysis showed that variables significantly related to sP-selectin plasma levels were sCD40L (Pacid (P<0

  8. Equid herpesvirus type 1 activates platelets.

    Directory of Open Access Journals (Sweden)

    Tracy Stokol

    Full Text Available Equid herpesvirus type 1 (EHV-1 causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression and platelet microvesiculation (increased small events double positive for CD41 and Annexin V. Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM. A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis

  9. Pathogen reduction treatment using riboflavin and ultraviolet light impairs platelet reactivity toward specific agonists in vitro

    NARCIS (Netherlands)

    Zeddies, Sabrina; de Cuyper, Iris M.; van der Meer, Pieter F.; Daal, Brunette B.; de Korte, Dirk; Gutiérrez, Laura; Thijssen-Timmer, Daphne C.


    Recent studies showed that Mirasol pathogen reduction treatment (PRT) leads to increased P-selectin expression and increased oxygen and glucose consumption in resting platelets (PLTs). This study investigates the effect of PRT on PLT activation. Untreated or Mirasol-treated PLTs were analyzed at

  10. Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets. (United States)

    Garlichs, C D; Eskafi, S; Raaz, D; Schmidt, A; Ludwig, J; Herrmann, M; Klinghammer, L; Daniel, W G; Schmeisser, A


    To investigate whether CD40L/CD154 on platelets and soluble CD40L/CD154 may play a role in the inflammatory process of acute coronary syndromes. Observational study in a university hospital. 15 patients with acute myocardial infarction, 25 patients with unstable angina, 15 patients with stable angina, and 12 controls. CD40L/CD154 on platelets, P-selectin/CD62P on platelets, soluble CD40L/CD154 serum concentrations. Mean (SD) CD40L/CD154 expression on platelets was 6.2 (2.8) MFI (mean fluorescence intensity) in the infarct group, 11 (3.3) MFI in the unstable angina group (p angina group (p angina), and 3.2 (1.0) MFI in the controls (p angina; NS v stable angina). Soluble CD40L/CD154 concentration was 5.2 (1.1) ng/ml in the infarct group, 4.2 (0.7) ng/ml in the unstable angina group (p angina group (p angina), and 3.0 (0.5) ng/ml in the controls (p angina; NS v stable angina). At a six months follow up, there was lower expression of CD40L/CD154 on platelets in patients with unstable angina (12.3 (3.6) v 3.8 (1.2) MFI, p angina who needed redo coronary angioplasty (PTCA) or who had recurrence of angina were characterised by increased CD40L/CD154 expression on platelets compared with the remainder of the study group (recurrence of angina: 12.7 (3.2) v 9.7 (1.6) MFI, p angina and myocardial infarction. These findings suggest that CD40-CD40L/CD154 interactions may play a pathogenic role in triggering and propagation of acute coronary syndromes.

  11. Characterization of surface antigens of reticulated immature platelets. (United States)

    Lador, Adi; Leshem-Lev, Dorit; Spectre, Galia; Abelow, Aryeh; Kornowski, Ran; Lev, Eli I


    Reticulated platelets (RPs) are immature platelets with high dense granules content and a residual amount of megakaryocyte-derived of mRNA. Increased level of RPs has been found to be an independent predictor of cardiovascular ischemic events, and has been associated with impaired response to various anti-platelet drugs. The study aimed to characterize and compare the surface antigenic properties of reticulated versus mature platelets. Platelets from healthy individuals and diabetic patients were tested at rest and after activation with adenosine diphosphate (ADP). For each patient, we calculated the proportion of RPs and mature platelets using flow cytometry analysis with thiazole orange staining (for RPs) and CD42b platelet-specific antibody. We also tested the surface expression of P-selectin and Annexin V, by double staining flow cytometry in RPs versus mature platelets. A total of 20 subjects were recruited (10 healthy individuals, 10 diabetics). Activation with ADP did not cause a significant change in the proportion of RPs. Following activation, RPs demonstrated a significant increase in the expression of both P-selectin and Annexin V, while mature platelets exhibited a non-significant increase in both markers. These findings were consistent in both healthy subjects and patients with diabetes. In conclusion, RPs have a significantly higher capacity to increase the expression of platelet activation markers compared with mature platelets.

  12. P-selectin glycoprotein ligand-1 in T cells. (United States)

    Abadier, Michael; Ley, Klaus


    We review P-selectin glycoprotein ligand-1 (PSGL-1) as a selectin and chemokine-binding adhesion molecule. PSGL-1 is widely studied in neutrophils. Here, we focus on T cells, because PSGL-1 was recently described as a major immunomodulatory molecule during viral infection. PSGL-1 also plays a crucial role in T-cell homeostasis by binding to lymphoid chemokines, and can induce tolerance by enhancing the functions of regulatory T cells. PSGL-1 was originally described as a leukocyte ligand for P-selectin, but it is actually a ligand for all selectins (P-, L- and E-selectin), binds chemokines, activates integrins and profoundly affects T-cell biology. It has been shown recently that PSGL-1 can modulate T cells during viral infection by acting as a negative regulator for T-cell functions. Absence of PSGL-1 promotes effector CD4 and CD8 T-cell differentiation and prevents T-cell exhaustion. Consistent with this, tumor growth was significantly reduced in PSGL-1-deficient mice because of an enhanced number of effector T cells together with reduced levels of inhibitory receptors that induce T-cell exhaustion. PSGL-1 is the best-studied selectin ligand and has become a posterchild of versatility in leukocyte adhesion, inflammation and immunology. The direct involvement of PSGL-1 in T-cell biology suggests that it might be a drug target. Indeed, PSGL-1 has been tested in some clinical trials and recently, PSGL-1 blockers were proposed as a potential cotherapy in cancer immunotherapy.

  13. Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype. (United States)

    Vajen, Tanja; Benedikter, Birke J; Heinzmann, Alexandra C A; Vasina, Elena M; Henskens, Yvonne; Parsons, Martin; Maguire, Patricia B; Stassen, Frank R; Heemskerk, Johan W M; Schurgers, Leon J; Koenen, Rory R


    Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1-CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.

  14. Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease. (United States)

    Dudley, Alicia; Byron, Julie K; Burkhard, Mary Jo; Warry, Emma; Guillaumin, Julien


    OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbβ3 (αIIbβ3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbβ3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbβ3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbβ3, and P-selectin.

  15. Fetal wound healing using a genetically modified murine model: the contribution of P-selectin (United States)

    During early gestation, fetal wounds heal with paucity of inflammation and absent scar formation. P-selectin is an adhesion molecule that is important for leukocyte recruitment to injury sites. We used a murine fetal wound healing model to study the specific contribution of P-selectin to scarless wo...

  16. Value of plasma P-selectin for vascular complications in liver transplantation. (United States)

    Fábrega, E; Casafont, F; Merino, J; de la Peña, J; Crespo, J; Amado, J A; Pons-Romero, F


    Recent data suggest that plasma P-selectin, an adhesion molecule, may be a clinically useful marker for thrombosis. Hepatic vessel thrombosis is one of the most serious complications following liver transplantation. To assess the contribution of the soluble P-selectin to this complication, we measured plasma P-selectin levels in 32 orthotopic liver transplantations, pre-, intra-, and post-operatively. We found that levels of circulating P-selectin were not different between cirrhotic patients and healthy subjects. Of the 32 patients, 8 had vascular complications. We found a significant increase in the plasma P-selectin concentration in the thrombotic group in early postoperative period compared with the non-thrombotic group (p < 0.002). It measurement may facilitate the diagnosis of thrombosis in the early postoperative period after liver transplantation, and therefore the management of these patients.

  17. Clinical and genetic correlates of soluble P-selectin in the community. (United States)

    Lee, D S; Larson, M G; Lunetta, K L; Dupuis, J; Rong, J; Keaney, J F; Lipinska, I; Baldwin, C T; Vasan, R S; Benjamin, E J


    P-selectin is a cell adhesion molecule that is involved in atherogenesis, and soluble concentrations of this biomarker reflect cardiovascular risk. However, the clinical correlates and genetic characterization of soluble P-selectin have not been clearly elucidated. To describe clinical and genetic correlates of circulating P-selectin in the community. In Framingham Heart Study Offspring (European descent) and Omni (ethnic/racial minority) participants, we examined the association of cardiovascular risk factors with soluble P-selectin concentrations. In Offspring participants, we evaluated heritability, linkage and association of 29 SELP single-nucleotide polymorphisms (SNPs) with adjusted P-selectin concentrations. In multivariable analysis of 3,690 participants (54% women, mean age 60 +/- 10 years), higher log-transformed P-selectin concentrations were inversely associated with female sex and hormone replacement therapy, and positively associated with age, ethnic/racial minority status, cigarette smoking, waist circumference, systolic blood pressure, fasting glucose, and total/high-density lipoprotein cholesterol and triglyceride concentrations. Clinical factors explained 10.4% of the interindividual variability in P-selectin concentrations. In 571 extended pedigrees (n = 1,841) with >or= 2 phenotyped members per family, multivariable-adjusted heritability was 45.4 +/- 5.8%. Among the SELP SNPs examined, a non-synonymous SNP (rs6136) encoding a threonine-to-proline substitution at position 715 was highly significantly associated with decreased P-selectin concentrations (P = 5.2 x 10(-39)), explaining 9.7% of variation after adjustment for clinical factors. Multiple clinical factors and an SNP in the SELP gene were significantly associated with circulating P-selectin concentrations. One SNP in SELP explained significant variation in circulating P-selectin concentrations, even after accounting for known clinical correlates.

  18. Elevated maternal serum sP-selectin levels in preeclamptic pregnancies with and without intrauterine fetal growth restriction, but not in normotensive pregnancies complicated by isolated IUGR. (United States)

    Laskowska, Marzena; Laskowska, Katarzyna; Oleszczuk, Jan


    The aim of this study was to show differences of maternal serum sP-selectin levels in pregnancies complicated by intrauterine fetal growth restriction (IUGR) in the course of preeclampsia and to compare the results with normotensive pregnant women with isolated IUGR. These studies were also conducted on preeclamptic pregnancies with appropriate-for-gestational-age weight infants and on the control normotensive pregnant women. The study was carried out on 55 patients with pregnancy complicated by fetal growth restriction in the course of preeclampsia, 70 normotensive patients with pregnancies complicated by isolated IUGR, 39 preeclamptic patients with appropriate-for-gestational-age weight fetuses and 54 healthy normotensive pregnant patients with normal fetal growth. Maternal serum levels of sP-selectin were determined using the enzyme-linked immunosorbent assay. Levels of sP-selectin were higher in women with pregnancy complicated by preeclampsia with and without IUGR; whereas, in the group of normotensive pregnant women with isolated fetal growth restriction, serum sP-selectin levels tended to be lower than in the control subjects, but this difference was not statistically significant. The mean values were 192.05±70.96 ng/mL in the IUGR group, 293.18±222.92 ng/mL in the PI group, 379.78±353.13 ng/mL in the P group and 227.96±134.04 ng/mL in the healthy controls (p<0.001*). Our findings may suggest that the elevated level of the soluble P-selectin is associated with preeclampsia, and that it may confirm the presence of platelet and endothelial activation, the presence of the hypercoagulant state and may be due to the systemic inflammatory response in this serious pregnancy disorder.

  19. Soluble P-selectin and matrix metalloproteinase 2 levels are elevated in patients with diastolic dysfunction independent of glucose metabolism disorder or coronary artery disease. (United States)

    Füth, Reiner; Dinh, Wilfried; Nickl, Werner; Bansemir, Lars; Barroso, Michael Coll; Bufe, Alexander; Sause, Armin; Scheffold, Thomas; Krahn, Thomas; Ellinghaus, Peter; Lankisch, Mark


    The development of diastolic dysfunction (DDF) is multifactorial. Possible mechanisms include metabolic disturbances, myocardial fibrosis, chronic inflammation and endothelial dysfunction. Recognizing early stages of DDF may help to identify patients at risk of developing symptomatic DDF. Therefore, biomarkers reflecting pathophysiological changes within the myocardium were investigated in patients with DDF. Seventy-seven patients submitted for coronary angiography with stable or suspected coronary artery disease (CAD) were consecutively enrolled. Those without known diabetes mellitus (DM) underwent a standardized oral glucose tolerance test. Echocardiography for the diagnosis of DDF was performed according to the European Society of Cardiology. Matrix metalloproteinase 2 (MMP-2) and soluble P-selectin (sP-selectin) serum concentrations were analyzed using the ELISA technique. A total of 36% of patients had DM and 74% had CAD. The prevalence of DDF was higher in patients with DM (89% versus 74%) and CAD (84% versus 53%) (PDDF in patients with DM was more severe with a significantly lower mitral annulus velocity of 6.5 cm/s versus 7.8 cm/s (PDDF showed significantly higher sP-selectin (140.3 mug/L versus 107.6 mug/L, PDDF. There was a significant correlation between sP-selectin and MMP-2 (P=0.01), independent of the diagnosis of DM or CAD. sP-selectin as a marker for platelet hyperactivity, inflammation and endothelial dysfunction, and MMP-2 as a marker for extracellular matrix turnover were significantly elevated in patients with DDF. This elevation was independent of coexisting DM or CAD. This observation may help to identify and monitor patients with DDF.

  20. 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


    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...... platelet activation and platelet thrombin receptor expression (SPAN-12 and WEDE-15 expression). Agonists were thrombin, thrombin receptor-activating peptide, protease-activated receptor-4 agonist, collagen, collagen-related peptide, arachidonic acid, and adenosine diphosphate. All concentrations...... of dabigatran fully inhibited platelet aggregation for thrombin up to 2 IU/mL, while dabigatran did not affect platelet aggregation by other agonists. Platelet activation (percentage of platelets positive for activated GPIIb/IIIa, CD63, P-selectin) was reduced after thrombin stimulation in samples...

  1. Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1.

    Directory of Open Access Journals (Sweden)

    Jessica M Banks

    Full Text Available Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1 active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.

  2. Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1. (United States)

    Banks, Jessica M; Herman, Christine T; Bailey, Ryan C


    Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1) active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.

  3. The effect of centrifugation speed and time on pre-analytical platelet activation

    DEFF Research Database (Denmark)

    Söderström, Anna Cecilia; Nybo, Mads; Nielsen, Christian


    . METHODS: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean......BACKGROUND: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation...... platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. RESULTS: The median percentage...

  4. Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation

    DEFF Research Database (Denmark)

    Zingariello, M; Fabucci, M E; Bosco, D


    Willebrand factor are two proteins present in the alpha-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the alpha-granule contents in response to external....... These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the alpha-granule content in response to external stimuli....

  5. Assembly of multimeric von Willebrand factor directs sorting of P-selectin

    NARCIS (Netherlands)

    Hop, C.; Guilliatt, A.; Daly, M.; de Leeuw, H. P.; Brinkman, H. J.; Peake, I. R.; van Mourik, J. A.; Pannekoek, H.


    We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis

  6. Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation. (United States)

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


    [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.

  7. Flavanols and Platelet Reactivity

    Directory of Open Access Journals (Sweden)

    Debra A. Pearson


    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.

  8. Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

    Directory of Open Access Journals (Sweden)

    Shih-Sheng Chang


    Full Text Available Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid.

  9. Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis

    Directory of Open Access Journals (Sweden)

    Guoxing Liu


    Full Text Available Background/Aims: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP, which trigger increase of cytosolic Ca2+-activity ([Ca2+]i, production of reactive oxygen species (ROS, degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i, degranulation (P-selectin exposure, integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. Methods: Platelets from mice lacking Ano6 (ano6-/- were compared to platelets from corresponding wild-type mice (ano6+/+. [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. Results: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml and CRP (2 or 5 µg/ml increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. Conclusions: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis

  10. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro. (United States)

    Mangalpally, Kiran Kumar R; Siqueiros-Garcia, Alan; Vaduganathan, Muthiah; Dong, Jing-Fei; Kleiman, Neal S; Guthikonda, Sasidhar


    Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B(2) (TxB(2)) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin. Platelets exhibit heterogeneity in size and structure. Whether this translates into platelet function and efficacy of antiplatelet therapy is unclear. We evaluated platelet functional properties and the effects of aspirin on separated platelet subpopulations in an in vitro setting. Platelets were sorted into the largest and smallest size quintiles using flow cytometry forward scatter. Alpha-granule protein release, dense granule content

  11. Peptides from multiple regions of the lectin domain of P-selectin inhibiting neutrophil adhesion. (United States)

    Heavner, G A; Falcone, M; Kruszynski, M; Epps, L; Mervic, M; Riexinger, D; McEver, R P


    The selectins are a family of three structurally related glycoproteins that are integral components of leukocyte adhesion to the vascular endothelium. Their involvement in the recruitment and extravasation of neutrophils is critical in mounting an inflammatory reaction. The carbohydrate nature of the selectin ligands suggests that the binding regions of the selectins are contained within the lectin-like domains of the selectins. The synthesis and evaluation for inhibition of selectin binding of overlapping peptides of the lectin and adjacent EGF-like domains of P-selectin have been used to identify small peptides that completely inhibit P-selectin-dependent neutrophil adhesion. These peptides span a region of more than 100 amino acids and may define the carbohydrate recognition domain of P-selectin.

  12. Platelet activation and platelet-leukocyte interaction in dogs naturally infected with Babesia rossi

    DEFF Research Database (Denmark)

    Goddard, Amelia; Leisewitz, Andrew L; Kristensen, Annemarie Thuri


    for regulating innate immunity and systemic inflammation. The objective of this study was to investigate PLA formation in canine babesiosis and to determine whether it was associated with outcome. Blood was collected from 36 client-owned dogs diagnosed with Babesia rossi infection and 15 healthy controls using......Using flow cytometry, platelet-leukocyte aggregate (PLA) formation has previously been documented in dogs with a variety of systemic inflammatory disorders and immune-mediated haemolytic anaemia. Platelet activation and subsequent interaction between platelets and leukocytes are important...... EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia-infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher (P = 0...

  13. Platelet dysfunction contributes to bleeding complications in patients with probable leptospirosis. (United States)

    Tunjungputri, Rahajeng N; Gasem, Muhammad Hussein; van der Does, Willemijn; Sasongko, Pandu H; Isbandrio, Bambang; Urbanus, Rolf T; de Groot, Philip G; van der Ven, Andre; de Mast, Quirijn


    Severe leptospirosis is frequently complicated by a hemorrhagic diathesis, of which the pathogenesis is still largely unknown. Thrombocytopenia is common, but often not to the degree that spontaneous bleeding is expected. We hypothesized that the hemorrhagic complications are not only related to thrombocytopenia, but also to platelet dysfunction, and that increased binding of von Willebrand factor (VWF) to platelets is involved in both platelet dysfunction and increased platelet clearance. A prospective study was carried out in Semarang, Indonesia, enrolling 33 hospitalized patients with probable leptospirosis, of whom 15 developed clinical bleeding, and 25 healthy controls. Platelet activation and reactivity were determined using flow cytometry by measuring the expression of P-selectin and activation of the αIIbβ3 integrin by the binding of fibrinogen in unstimulated samples and after ex vivo stimulation by the platelet agonists adenosine-diphosphate (ADP) and thrombin-receptor activating peptide (TRAP). Platelet-VWF binding, before and after VWF stimulation by ristocetin, as well as plasma levels of VWF, active VWF, the VWF-inactivating enzyme ADAMTS13, thrombin-antithrombin complexes (TAT) and P-selectin were also measured. Bleeding complications were graded using the WHO bleeding scale. Our study revealed that platelet activation, with a secondary platelet dysfunction, is a feature of patients with probable leptospirosis, especially in those with bleeding manifestations. There was a significant inverse correlation of bleeding score with TRAP-stimulated P-selectin and platelet-fibrinogen binding (R = -0.72, P = 0.003 and R = -0.66, P = 0.01, respectively) but not with platelet count. Patients with bleeding also had a significantly higher platelet-VWF binding. Platelet counts were inversely correlated with platelet-VWF binding (R = -0.74; P = 0.0009. There were no correlations between platelet-VWF binding and the degree of platelet dysfunction, suggesting that

  14. Time course of soluble P-selectin and von Willebrand factor levels in trauma patients: a prospective observational study. (United States)

    Tang, Ning; Yin, Shiyu; Sun, Ziyong; Pan, Yingying


    Coagulopathy often develops in patients with serious trauma and is correlated with the clinical outcome. The contribution of platelet activity and endothelial dysfunction to trauma-induced coagulopathy remain to be defined. The purpose of this study was to investigate the time courses of soluble P-selectin (sPsel, an index of platelet activation) and von Willebrand factor (VWF, an index of endothelial dysfunction) in trauma patients and elucidate their relationship to coagulation parameter levels, the presence of coagulopathy, and patient outcome. This prospective observational study, which took place in a university hospital intensive care unit (ICU), included 82 severely injured trauma patients. The sPsel, VWF antigen, protein C, and factor VII levels were measured and routine coagulation tests were performed upon admission to ICU and daily within the first week. The 30-day mortality rate was also determined. Thirty-seven (45.1%) patients developed coagulopathy upon admission to the ICU, and the 30-day mortality rate was 20.7% (n = 17). Both the admission sPsel and VWF levels were lower in patients with coagulopathy than in those without (p trauma patients in the ICU, lower levels of sPsel and VWF on admission were associated with the presence of coagulopathy and might not predict a better outcome. An increase in the VWF level at the end of the first week after admission to ICU was associated with increased 30-day mortality.

  15. Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

    Directory of Open Access Journals (Sweden)

    Monique Ramos de Oliveira Trugilho


    Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

  16. Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue. (United States)

    Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas


    Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue

  17. Early Dynamics of P-selectin and Interleukin 6 Predicts Outcomes in Ischemic Stroke

    DEFF Research Database (Denmark)

    Pusch, Gabriella; Debrabant, Birgit; Molnar, Tihamer


    to poststroke infection, death, and functional outcome, and assessed the ability of the models to predict each outcome. RESULTS: Interleukin 6 (IL-6) levels and change of IL-6 concentrations by 72 hours correlated with the size of tissue damage indicated by S100B titers. Levels of IL-6 and P-selectin at 72...

  18. Association between circulating levels of P-selectins and burden of ...

    African Journals Online (AJOL)

    We tested the hypothesis that, in the acute phase of ST-segment elevation myocardial infarction (STEMI), the circulating level of P-selectin (PS) is predictive of angiographic morphologic features that indicate burden of thrombus formation in the infarct-related artery (IRA). One hundred and ninety-five consecutive patients ...

  19. Platelets of patients with chronic kidney disease demonstrate deficient platelet reactivity in vitro

    Directory of Open Access Journals (Sweden)

    van Bladel Esther R


    Full Text Available Abstract Background In patients with chronic kidney disease studies focusing on platelet function and properties often are non-conclusive whereas only few studies use functional platelet tests. In this study we evaluated a recently developed functional flow cytometry based assay for the analysis of platelet function in chronic kidney disease. Methods Platelet reactivity was measured using flow cytometric analysis. Platelets in whole blood were triggered with different concentrations of agonists (TRAP, ADP, CRP. Platelet activation was quantified with staining for P-selectin, measuring the mean fluorescence intensity. Area under the curve and the concentration of half-maximal response were determined. Results We studied 23 patients with chronic kidney disease (9 patients with cardiorenal failure and 14 patients with end stage renal disease and 19 healthy controls. Expression of P-selectin on the platelet surface measured as mean fluorescence intensity was significantly less in chronic kidney disease patients compared to controls after maximal stimulation with TRAP (9.7 (7.9-10.8 vs. 11.4 (9.2-12.2, P = 0.032, ADP (1.6 (1.2-2.1 vs. 2.6 (1.9-3.5, P = 0.002 and CRP (9.2 (8.5-10.8 vs. 11.5 (9.5-12.9, P = 0.004. Also the area under the curve was significantly different. There was no significant difference in half-maximal response between both groups. Conclusion In this study we found that patients with chronic kidney disease show reduced platelet reactivity in response of ADP, TRAP and CRP compared to controls. These results contribute to our understanding of the aberrant platelet function observed in patients with chronic kidney disease and emphasize the significance of using functional whole blood platelet activation assays.

  20. Calpain Activator Dibucaine Induces Platelet Apoptosis

    Directory of Open Access Journals (Sweden)

    Jun Liu


    Full Text Available Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

  1. Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.

    Directory of Open Access Journals (Sweden)

    Sirada Srihirun

    Full Text Available Nitrite is a nitric oxide (NO metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated.Platelet aggregation was studied in platelet-rich plasma (PRP and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger, suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes.Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.

  2. Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia (United States)

    Baron, Rebecca M.; Lopez-Guzman, Silvia; Riascos, Dario F.; Macias, Alvaro A.; Layne, Matthew D.; Cheng, Guiying; Harris, Cailin; Chung, Su Wol; Reeves, Raymond; von Andrian, Ulrich H.; Perrella, Mark A.


    Background The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight

  3. Distamycin A inhibits HMGA1-binding to the P-selectin promoter and attenuates lung and liver inflammation during murine endotoxemia.

    Directory of Open Access Journals (Sweden)

    Rebecca M Baron


    Full Text Available The architectural transcription factor High Mobility Group-A1 (HMGA1 binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes" that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI. AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs, such as Distamycin A (Dist A, interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting

  4. Enhanced platelet MRP4 expression and correlation with platelet function in patients under chronic aspirin treatment. (United States)

    Massimi, Isabella; Lotti, Lavinia Vittoria; Temperilli, Flavia; Mancone, Massimo; Sardella, Gennaro; Calcagno, Simone; Turriziani, Ombretta; Frati, Luigi; Pulcinelli, Fabio M


    Platelet multidrug resistance protein4 (MRP4)-overexpression has a role in reducing aspirin action. Aspirin in vivo treatment enhances platelet MRP4 expression and MRP4 mediated transport inhibition reduces platelet function and delays thrombus formation. The aim of our work was to verify whether MRP4 expression is enhanced in platelets obtained from patients under chronic aspirin treatment and whether it correlates with residual platelet reactivity. We evaluated changes on mRNA and protein-MRP4 expression and platelet aggregation in four populations: healthy volunteers (HV), aspirin-free control population (CTR), patients who started the treatment less than one month ago (ASAaspirinated patients who started the treatment more than two months ago (ASA>2 months patients). In platelets obtained from ASA>2 months patients, it was found a statistically significant MRP4 enhancement of both mRNA and protein expression compared to HV, CTR and ASA2 months patients that present high levels of platelet MRP4, have higher serum TxB2 levels and collagen-induced platelet aggregation compared to patient with low levels of MRP4 in platelets. In addition collagen induced platelet aggregation is higher in in vitro aspirinated platelets obtained from patients with high levels of MRP4 patients compared to those obtained from patients with low MRP4 levels. We can assert that, in patients under chronic aspirin treatment, platelets that present high MRP4 levels have an increase of residual platelet reactivity, which is due in part to incomplete COX-1 inhibition, and in part to COX-1-independent mechanism.

  5. Protective effects of oligosaccharides in P-selectin-dependent lung injury. (United States)

    Mulligan, M S; Paulson, J C; De Frees, S; Zheng, Z L; Lowe, J B; Ward, P A


    Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules, including selectins (E,P,L), which are reactive with oligosaccharides, and the family of beta 2 integrins which are reactive with endothelial intercellular adhesion molecules. These processes result in the initial rolling of leukocytes along the endothelial surfaces, followed by the firm attachment of leukocytes to the endothelium. The intravenous infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical mediated and P-selectin-dependent. Here we report that infusion of sialyl-Lewis X, a ligand for P-selectin, dramatically reduced lung injury and diminished the tissue accumulation of neutrophils, whereas irrelevant oligosaccharides had no such effects. These results suggest that sialyl-Lewis X carbohydrates may be used as a new strategy for anti-inflammatory therapy.

  6. Glycopeptide Analogues of PSGL-1 Inhibit P-Selectin In Vitro and In Vivo


    Krishnamurthy, Venkata R.; Sardar, Mohammed Y. R.; Yu, Ying; Song, Xuezheng; Haller, Carolyn; Dai, Erbin; Wang, Xiacong; Hanjaya-Putra, Donny; Sun, Lijun; Morikis, Vasilios; Simon, Scott I.; Woods, Robert; Cummings, Richard D.; Chaikof, Elliot L.


    Blockade of P-selectin/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis, and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan bu...

  7. Directed transport of neutrophil-derived extracellular vesicles enables platelet-mediated innate immune response. (United States)

    Rossaint, Jan; Kühne, Katharina; Skupski, Jennifer; Van Aken, Hugo; Looney, Mark R; Hidalgo, Andres; Zarbock, Alexander


    The innate immune response to bacterial infections requires the interaction of neutrophils and platelets. Here, we show that a multistep reciprocal crosstalk exists between these two cell types, ultimately facilitating neutrophil influx into the lung to eliminate infections. Activated platelets adhere to intravascular neutrophils through P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1)-mediated binding, a primary interaction that allows platelets glycoprotein Ibα (GPIbα)-induced generation of neutrophil-derived extracellular vesicles (EV). EV production is directed by exocytosis and allows shuttling of arachidonic acid into platelets. EVs are then specifically internalized into platelets in a Mac1-dependent fashion, and relocated into intracellular compartments enriched in cyclooxygenase1 (Cox1), an enzyme processing arachidonic acid to synthesize thromboxane A2 (TxA2). Finally, platelet-derived-TxA2 elicits a full neutrophil response by inducing the endothelial expression of ICAM-1, intravascular crawling, and extravasation. We conclude that critical substrate-enzyme pairs are compartmentalized in neutrophils and platelets during steady state limiting non-specific inflammation, but bacterial infection triggers regulated EV shuttling resulting in robust inflammation and pathogen clearance.

  8. Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

    NARCIS (Netherlands)

    van Golen, Rowan F.; Stevens, Katarzyna M.; Colarusso, Pina; Jaeschke, Hartmut; Heger, Michal


    Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival.

  9. Therapeutic angiogenesis for the critical limb ischemia decreases platelet activation. (United States)

    Chudý, Peter; Chudá, Daniela; Hudeček, Jan; Fedorová, Jana; Sinák, Igor; Hlinka, Luboš; Talapková, Renáta; Laca, Ludovít; Kubisz, Peter


    Platelets are required for the recruitment of bone marrow-derived mononuclear cells (BMMNC) into ischemia-induced vasculature, which underlines their key role in angiogenesis. The difference in platelet immunophenotype between healthy controls and patients with critical limb ischemia (CLI) treated with therapeutic angiogenesis (TA) using BMMNC was assessed. The impact of TA on the expression of platelet membrane markers was studied as well. CLI patients (N = 26) and blood donors as controls (N = 21) were enrolled. Bone marrow (600 ± 50 ml) was centrifuged (3200 g, 20 min, 22 °C). BMMNC (100-120 ml) were separated by Optipress I and implanted to the ischemic limb using deep intramuscular injections. Flow cytometry was employed for the peripheral blood platelets immunophenotyping. CD41FITC, CD62PE, CD36FITC, CD29FITC antibodies were used. Patients were followed up prior to the procedure and at months 1, 3 and 6. The expression of CD41 was lower in CLI patients than in the controls. P-selectin (CD62P) was higher in CLI patients than in controls at the baseline and at month 6. It was significantly down-regulated at month 3, however not at months 1 and 6 compared to baseline. Platelet GPIV (CD36) was higher at the baseline, but not during the follow-up compared to the controls. β1-integrin (CD29) progressively decreased during the follow-up as compared to the baseline value. Platelets in CLI express P-selectin, GPIV and β1-integrin more abundantly than platelets of healthy subjects. TA down-regulates the expression of the respective markers. Possible mechanism could be higher clearance of the activated platelets in the ischemic tissues during angiogenesis.

  10. Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. II. Tether flow terminated by P-selectin dissociation from PSGL-1. (United States)

    Heinrich, Volkmar; Leung, Andrew; Evans, Evan


    We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip most often by one precipitous step of P-selectin:PSGL-1 dissociation. Analyzing the initial elastic response and membrane unbinding from the cytoskeleton in our companion article I, we focus in this article on the regime of tether extrusion that nearly always occurred before release of the extracellular adhesion bond at pulling speeds > or =1 microm/s. The force during tether growth appeared to approach a plateau at long times. Examined over a large range of pulling speeds up to 150 microm/s, the plateau force exhibited a significant shear thinning as indicated by a weak power-law dependence on pulling speed, f(infinity) = 60 pN(nu(pull)/microm/s)(0.25). Using this shear-thinning response to describe the viscous element in a nonlinear Maxwell-like fluid model, we show that a weak serial-elastic component with a stiffness of approximately 0.07 pN/nm provides good agreement with the time course of the tether force approach to the plateau under constant pulling speed.

  11. Platelet activation and inflammation markers as emerging risk ...

    African Journals Online (AJOL)

    Platelet activation and inflammation markers were assessed by measuring plasma levels of sP-selectin, platelet factor 4 (PF4), IL-6 and tumor necrosis factor alpha (TNF-α). Results: HIV infected patients had higher levels of sP- selectin, PF4 and IL-6 than uninfected controls (p<0.001). ART naïve subjects had higher levels ...

  12. Modified expression of surface glyconjugates in stored human platelets

    Energy Technology Data Exchange (ETDEWEB)

    Dhar, A.; Ganguly, P.


    Platelets are anucleated cells which play an important part in blood coagulation and thrombosis. These cells may be stored in the blood bank for only 4/5 days. In order to improve the storage of platelets, it is essential to first understand the changes in these cells due to storage. In this work, human platelets were stored in autologous plasma at 4/sup 0/ or 22/sup 0/ and their surface changes were monitored with three lectins - wheat germ afflutinin (WGA), concanavalin A (Con A) and lentil lectin (LL). Blood was drawn from healthy donors and platelet rich plasma (PRP) was collected by slow speed centrifugation. Platelets stored at either temperature for different times showed increased sensitivity to agglutination by WGA after 34-48 hrs. Lectins, Con A and LL, which were not agglutinating to fresh platelets readily caused agglutination after 48-72 hrs. The platelets stored for 25 hrs or longer period were insensitive to thrombin but showed enhanced aggregation with WGA. Labelling of surface glycoconjugates of stored platelets with /sup 3/H-boro-hydride revealed progressive loss of a glycoprotein of Mr 150,000 (GPIb infinity) together with the appearance of components of Mr 69,000; Mr 60,000; Mr 25,000. New high molecular weight glycoproteins were also detected only in stored platelets. The author studies clearly indicate that modification or altered expression of platelets surface glycoproteins may be one factor of storage related dysfunction of platelets.

  13. Platelet indexes in relation to target organ damage in high-risk hypertensive patients: a substudy of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT)

    National Research Council Canada - National Science Library

    Nadar, Sunil K; Blann, Andrew D; Kamath, Sridhar; Beevers, D Gareth; Lip, Gregory Y H


    We sought to investigate the relationship between target organ damage (TOD) in hypertension and a prothrombotic/hypercoagulable state, using a new technique of "platelet lysis" to quantify the amount of P-selectin per platelet (pP-sel...

  14. Interleukin-2 regulatory effect on P-selectin and interleukin-8 production in patients with chronic renal failure. (United States)

    Abou-Shousha, Seham A; Youssef, Amany I


    Patients with chronic renal failure (CRF) show a clinical state of immunodysfunction that occurs in both humoral and cellular immunity as well as inflammatory response. In this study, we investigated the mononuclear-endothelial cells (MCs/ECs) interaction and the possible protective role of IL-2 as the main T lymphocyte activator in CRF patients. The levels of soluble P-selectin (sP-selectin) and interleukin-8 (IL-8) as the two main mediators of MCs/ECs interaction were measured in IL-2 supplemented and non-supplemented peripheral blood mononuclear cells (PBMCs) supernatant of CRF patients. The obtained results were correlated with those of sex and age matched controls. Significantly higher levels of sP-selectin and IL-8 were detected in both IL-2 supplemented and non-supplemented PBMCs culture supernatant of CRF patients than controls (P = 0.000). Those levels were significantly lower in IL-2 supplemented PBMCs culture supernatant than non-supplemented ones of both CRF (P = 0.000) (for both mediators) and normal control groups (P = 0.01, P= 0.04 for sP-selectin and IL-8 respectively). The higher sP-selectin in CRF indicates impairment of MCs/ECs interaction that may be resulted from blockade of P-selectin receptors on PBMCs by P-selectin molecules shedded from ECs to plasma and bind to PBMCs in vivo. The elevated IL-8 level in PBMCs of CRF reflect the imbalance of Thl/Th2 ratio and subsequent impairment of cellular immunity in those patients. The lower level of both sP-selectin and IL-8 in IL-2 supplemented PBMCs supernatant than in non-supplemented one seemed to be due to the IL-2 induced proliferation of Th1 lymphocytes yielding newly in vitro formed T cells which do not carry P-selectin as well as relative increase of Th1/Th2 ratio in both normal and CRF groups. Thus, IL-2 may improve the MCs/ECs interaction and correct the Th1/Th2 ratio in CRF providing a novel promising therapeutic approach to improve the immuno-pathological condition of those patients.

  15. Contribution of the CR domain to P-selectin lectin domain allostery by regulating the orientation of the EGF domain.

    Directory of Open Access Journals (Sweden)

    Shouqin Lü

    Full Text Available The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1' state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain.

  16. Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia


    Olsson, Mattias; Bruhns, Pierre; Frazier, William A.; Ravetch, Jeffrey V.; Oldenborg, Per-Arne


    Interaction between target cell CD47 and the inhibitory macrophage receptor signal regulatory protein α (SIRPα) counteracts macrophage phagocytosis of CD47-expressing host cells. As platelets also express CD47, we asked whether inhibitory CD47/SIRPα signaling regulates normal platelet turnover and clearance of platelets in immune thrombocytopenic purpura (ITP). CD47-/- mice had a mild spontaneous thrombocytopenia, which was not due to a decreased platelet half-life as a result of increased ex...

  17. Allicin and disulfiram enhance platelet integrin alphaIIbbeta3-fibrinogen binding. (United States)

    Manaster, Yoav; Shenkman, Boris; Rosenberg, Nurit; Savion, Naphtali


    Activation of the platelet receptor alphaIIbbeta3 (glycoprotein IIbIIIa) involves a change in the disulfide bonds pattern in the extra-cellular domain of the receptor. The disulfide-bond reducing agent, dithiothreitol (DTT), can increase integrin activity, and point mutations of specific cysteine residues of the integrin can cause its lockage at the high affinity state. The present study is aimed to support the hypothesis that prevention of specific alphaIIbbeta3 intra-molecular disulfide bond formation increases receptor-ligand binding activity. Platelet aggregation was induced by collagen or ADP and epinephrine. Integrin alphaIIbbeta3-fibrinogen binding was evaluated on prostaglandins E(1) (PGE(1))-treated washed platelets or baby hamster kidney (BHK) cells expressing human alphaIIbbeta3. Integrin was directly activated by an anti-ligand induced binding site (LIBS) PT25-2 antibody. The effect of sulfhydryl-reactive agents, such as allicin, glutathione, dithiobis nitrobenzoic acid (DTNB) and disulfiram, was tested on alphaIIbbeta3 activity. Allicin (40 microM) completely inhibited washed platelets agonist-induced aggregation. Both allicin and disulfiram (40 microM) inhibited alphaIIbbeta3-fibrinogen binding and P-selectin expression in washed platelets. However, there was an increase in alphaIIbbeta3-fibrinogen binding but not P-selectin expression in PGE(1)-treated washed platelets activated by PT25-2 antibody. At a high concentration (400 microM) both inhibited alphaIIbbeta3-fibrinogen binding. Similarly, in BHK cells expressing alphaIIbbeta3 activated by PT25-2 antibody, allicin at a low concentration increased alphaIIbbeta3 activity. Allicin and disulfiram inhibit agonist-induced washed platelet activation probably via inhibition of platelet signaling, but enhance PT25-2 antibody-induced alphaIIbbeta3 integrin activity most likely by preventing reformation of disulfide bridges thereby stabilizing the active conformation of the integrin.

  18. Platelet activity and aspirin efficacy after off-pump compared with on-pump coronary artery bypass surgery: results from the prospective randomized trial PRAGUE 11-Coronary Artery Bypass and REactivity of Thrombocytes (CABARET). (United States)

    Bednar, Frantisek; Osmancik, Pavel; Vanek, Tomas; Mocikova, Heidi; Jares, Martin; Straka, Zbynek; Widimsky, Petr


    Changes in hemostasis after off-pump coronary artery bypass grafting are still being discussed. There is a lack of information about platelet activity and aspirin efficacy after coronary artery bypass grafting. The aim of this study was to assess and compare platelet activity and aspirin efficacy early and late after off-pump and on-pump coronary artery bypass grafting. Eighty patients were enrolled in a prospective randomized study. Platelet activity was determined based on membrane expression of antigen CD62P (P-selectin) by means of flow cytometric analysis. Aspirin efficacy was assessed by using arachidonic acid-induced platelet aggregation. Blood samples were collected before the operation, immediately postoperatively, and on days 1, 2, 5, and 30. In the off-pump group expression of P-selectin was markedly increased in comparison with preoperative values, with a maximum difference observed on day 2 (+53%, P = .02), and it was significantly higher compared with that seen in the on-pump group on days 2 and 5 (+53% vs +4%, P = .004, and +20% vs -16%, P = .005). On day 30, P-selectin expression was similar both between the groups and in comparison with the preoperative values. Arachidonic acid-induced platelet aggregation was gradually decreasing until day 30, but on day 2, there was an unexpected increase in aggregation that was more expressed in the off-pump group. The platelet activity is higher in the early postoperative period in off-pump compared with on-pump coronary artery bypass grafting. The present aspirin strategy seems to be insufficient in the early postoperative period, irrespective of the surgical technique used.

  19. Consumption of both low and high (-)-epicatechin apple puree attenuates platelet reactivity and increases plasma concentrations of nitric oxide metabolites: a randomized controlled trial. (United States)

    Gasper, Amy; Hollands, Wendy; Casgrain, Amelie; Saha, Shikha; Teucher, Birgit; Dainty, Jack R; Venema, Dini P; Hollman, Peter C; Rein, Maarit J; Nelson, Rebecca; Williamson, Gary; Kroon, Paul A


    We hypothesised that consumption of flavanol-containing apple puree would modulate platelet activity and increase nitric oxide metabolite status, and that high flavanol apple puree would exert a greater effect than low flavanol apple puree. 25 subjects consumed 230 g of apple puree containing 25 and 100mg epicatechin (low and high flavanol apple puree, respectively) and aspirin (75 mg) in random order. Measurements were made at baseline, acutely after treatment (2, 6 and 24 h), and after 14 d of treatment. Low flavanol apple puree significantly attenuated ADP and epinephrine-induced integrin-β3 expression 2 h and 6 h after consumption and ADP and epinephrine-induced P-selectin expression within 2h of consumption. High flavanol apple puree attenuated epinephrine and ADP-induced integrin-β3 expression after 2 and 6h. ADP and epinephrine-induced integrin-β3 expression was significantly attenuated 2, 6 and 24 h after consumption of aspirin, whilst 14 d aspirin consumption attenuated collagen-induced P-selectin expression only. The plasma total nitric oxide metabolite conc. was significantly increased 6h after consumption of both low and high flavanol apple purees. In conclusion, consumption of apple purees containing ⩾25 or 100 mg flavanols transiently attenuated ex vivo integrin-β3 and P-selectin expression and increased plasma nitric oxide metabolite conc. in healthy subjects, but the effect was not enhanced for the high flavanol apple puree. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. In vitro shear stress-induced platelet activation: sensitivity of human and bovine blood. (United States)

    Lu, Qijin; Hofferbert, Bryan V; Koo, Grace; Malinauskas, Richard A


    As platelet activation plays a critical role in physiological hemostasis and pathological thrombosis, it is important in the overall hemocompatibility evaluation of new medical devices and biomaterials to assess their effects on platelet function. However, there are currently no widely accepted in vitro test methods to perform this assessment. In an effort to develop effective platelet tests for potential use in medical device evaluation, this study compared the sensitivity of platelet responses to shear stress stimulation of human and bovine blood using multiple platelet activation markers. Fresh whole blood samples anticoagulated with heparin or anticoagulant citrate dextrose, solution A (ACDA) were exposed to shear stresses up to 40 Pa for 2 min using a cone-and-plate rheometer model. Platelet activation was characterized by platelet counts, platelet surface P-selectin expression, and serotonin release into blood plasma. The results indicated that exposure to shear stresses above 20 Pa caused significant changes in all three of the platelet markers for human blood and that the changes were usually greater with ACDA anticoagulation than with heparin. In contrast, for bovine blood, the markers did not change with shear stress stimulation except for plasma serotonin in heparin anticoagulated blood. The differences observed between human and bovine platelet responses suggest that the value of using bovine blood for in vitro platelet testing to evaluate devices may be limited. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  1. HuEP5C7 as a humanized monoclonal anti-E/P-selectin neurovascular protective strategy in a blinded placebo-controlled trial of nonhuman primate stroke. (United States)

    Mocco, J; Choudhri, Tanvir; Huang, Judy; Harfeldt, Elisabeth; Efros, Lyubov; Klingbeil, Corine; Vexler, Vladimir; Hall, William; Zhang, Yuan; Mack, William; Popilskis, Sulli; Pinsky, David J; Connolly, E Sander


    Although inhibiting interaction of beta(2) integrins with cognate immunoglobulin class adhesion receptor ligands is an effective neuroprotective strategy in small mammal models of stroke, the strategy has failed in human trials. A completely different antiadhesion receptor strategy was therefore rigorously tested in a model that may more closely approximate human reperfused stroke. Early leukoadhesive events in postischemic cerebral microvessels are mediated by upregulated selectin-class adhesion receptors on endothelial cells. Therefore, a blocking antibody prepared against common P- and E-selectin epitopes was humanized to suppress complement activation and tested in a reperfused hemispheric stroke model in Papio anubis (baboon). Histological examination of postischemic cerebral microvessels revealed a strong upregulation of E-and P-selectin expression. Placebo-blinded administration of the humanized anti-human E- and P-selectin monoclonal antibody (HuEP5C7, 20 mg/kg IV, n=9; placebo, n=9) immediately after the onset of 1 hour of temporary ischemia resulted in trends showing reduced polymorphonuclear leukocyte (PMN) infiltration into ischemic cortex, reduced infarct volumes (by 41%), improved neurological score (by 35%), and improved ability to self-care (by 39%). Importantly, there was no evidence of systemic complement activation, immune suppression, or pathological coagulopathy associated with this therapy. These data suggest that a humanized anti-E/P-selectin antibody approach is safe and may be effective as a clinical treatment for human stroke.

  2. The use of surface immobilization of P-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling (United States)

    Lo, Chi Y.; Antonopoulos, Aristotelis; Dell, Anne; Haslam, Stuart M.; Lee, Techung; Neelamegham, Sriram


    Mesenchymal stem/stromal cells (MSCs) are an important candidate for cell-based therapy since they can be easily isolated and expanded, secrete beneficial paracrine factors, and differentiate into multiple lineages. Since the endothelium at sites of injury and inflammation often express adhesion molecules belonging to the selectin family, methods to endow MSCs with selectin-ligands can enhance the efficacy of cell delivery and tissue engraftment. Here, we describe a construct 19Fc[FUT7+], where the first 19 amino acids of the pan-selectin ligand PSGL-1 (P-selectin glycoprotein ligand-1) was fused to a human IgG1. When expressed in HEK293T cells over-expressing the α(1,3)fucosyltransferase FUT7, 19Fc[FUT7+] is decorated by a core-2 sialyl Lewis-X sialofucosylated O-glycan. The non-covalent coupling of this protein onto MSC surface using palmitated protein G (PPG) enhanced cell binding to E- and P-selectin under hydrodynamic shear, without altering MSC multipotency. MSCs functionalized with 19Fc[FUT7+] were captured/tethered onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm2. Once captured, the cells rolled robustly up to the highest shear stress tested, 10 dyn/cm2. Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a ‘glycan engineering’ strategy to enable leukocyte-like capture and rolling. PMID:23891082

  3. Calcium sensing receptor initiating cystathionine-gamma-lyase/hydrogen sulfide pathway to inhibit platelet activation in hyperhomocysteinemia rat. (United States)

    Wang, Yuwen; Zhao, Ziqing; Shi, Sa; Gao, Fei; Wu, Jichao; Dong, Shiyun; Zhang, Weihua; Liu, Yanhong; Zhong, Xin


    Hyperhomocysteinemia (HHcy, high homocysteine) induces the injury of endothelial cells (ECs). Hydrogen sulfide (H2S) protects ECs and inhibits the activation of platelets. Calcium-sensing receptor (CaSR) regulates the production of endogenous H2S. However, whether CaSR inhibits the injury of ECs and the activation of platelets by regulating the endogenous cystathionine-gamma-lyase (CSE, a major enzyme that produces H2S)/H2S pathway in hyperhomocysteinemia has not been previously investigated. Here, we tested the ultrastructure alterations of ECs and platelets, the changes in the concentration of serum homocysteine and the parameters of blood of hyperhomocysteinemia rats were measured. The aggregation rate and expression of P-selectin of platelets were assessed. Additionally, the expression levels of CaSR and CSE in the aorta of rats were examined by western blotting. The mitochondrial membrane potential and the production of reactive oxygen species (ROS) were measured; the expression of phospho-calmodulin kinases II (p-CaMK II) and Von Willebrand Factor (vWF) of cultured ECs from rat thoracic aortas were measured. We found that the aggregation rate and the expression of P-selectin of platelets increased, and the expression of CaSR and CSE decreased in HHcy rats. In the ECs of HHcy group, the ROS production increased and the mitochondrial membrane potential decreased markedly, the expression of CSE and the p-CaMK II increased after treatment with CaSR agonist while decreased upon administration of U73122 (PLC-specific inhibitor) and 2-APB (IP3 Receptor inhibitor). CaSR agonist or NaHS significantly reversed the ECs injured and platelet aggregation caused by hyperhomocysteinemia. Our results demonstrate that CaSR regulates the endogenous CSE/H2S pathway to inhibit the activation of platelets which concerts the protection of ECs in hyperhomocysteinemia. Copyright © 2017. Published by Elsevier Inc.

  4. Sgk1 Sensitive Pendrin Expression in Murine Platelets

    Directory of Open Access Journals (Sweden)

    Lisann Pelzl


    Full Text Available Background: The anion exchanger pendrin (SLC26A4 is required for proper development of the inner ear, and contributes to iodide organification in thyroid glands as well as anion transport in various epithelia, such as airways and renal tubules. SLC26A4 deficiency leads to Pendred syndrome, which is characterized by hearing loss with enlarged vestibular aqueducts and variable hypothyroidism and goiter. Pendrin expression in kidney, heart, lung and thyroid is up-regulated by the mineralocorticoid deoxycorticosterone (DOCA. Platelets express anion exchangers but virtually nothing is known about the molecular identity and regulation of those carriers. Other carriers such as the Na+/H+ exchanger are regulated by the mineralocorticoid-sensitive serum and glucocorticoid inducible kinase SGK1. Methods: The present study utilized i quantitative reverse transcription polymerase chain reaction (RT-qPCR to quantify the transcript levels of Slc26a4 as compared to Gapdh and ii western blotting to assess Slc26a4 protein abundance in murine platelets from gene-targeted mice lacking Sgk1 (sgk1-/- and respective wild type animals (sgk1+/+ treated without or with a subcutaneous injection of 2.5 mg DOCA for 3 h, or in sgk1+/+ platelets with or without in vitro treatment for 1 h with 10 µg/ml DOCA. Results: Slc26a4 was expressed in platelets, and in vitro DOCA treatment increased Slc26a4 mRNA levels in platelets isolated from sgk1+/+ mice. Moreover, in vivo DOCA treatment significantly up-regulated Slc26a4 mRNA levels in platelets isolated from sgk1+/+ but not sgk1-/- mice. An increase in Sgk1 mRNA levels paralleled that of Slc26a4 mRNA levels in platelets of sgk1+/+ mice. In addition, DOCA treatment further increased Slc26a4 protein abundance in platelets isolated from sgk1+/+ mice. Conclusions: Pendrin is expressed in platelets and is presumably regulated by SGK1 and mineralocorticoids.

  5. 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.

  6. Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates. (United States)

    Kicken, Cécile H; Roest, Mark; Henskens, Yvonne M C; de Laat, Bas; Huskens, Dana


    Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

  7. Targeting P-Selectin by Gallium-68–Labeled Fucoidan Positron Emission Tomography for Noninvasive Characterization of Vulnerable Plaques: Correlation With In Vivo 17.6T MRI

    National Research Council Canada - National Science Library

    Li, Xiang; Bauer, Wolfgang; Israel, Ina; Kreissl, Michael C; Weirather, Johannes; Richter, Dominik; Bauer, Elisabeth; Herold, Volker; Jakob, Peter; Buck, Andreas; Frantz, Stefan; Samnick, Samuel


    .... We proposed a new approach for noninvasive in vivo characterization of P-selectin on active plaques based on Ga-Fucoidan, which is a polysaccharidic ligand of P-selectin with a nanomolar affinity...

  8. Plasma substance P and soluble P-selectin as biomarkers of β ...

    African Journals Online (AJOL)

    Samia A. Ebeid


    Sep 19, 2013 ... molecule which plays a key role in hemostasis and thrombosis, mediating platelet rolling and gen- erating procoagulant molecules. Substance P is one of the tachykinins which constitute a family of neuropeptides. It now appears that platelets contain substance P which is released upon stimulation.

  9. Critical role of P-selectin-dependent leukocyte recruitment in endotoxin-induced intestinal barrier dysfunction in mice.


    Mangell, Peter; Röme, Andrada; Wang, Yusheng; Schramm, R; Jeppsson, Bengt; Thorlacius, Henrik


    Objective: To define the importance of leukocyte recruitment in endotoxin-induced gut permeability. Materials and methods: 31 male C57BL/6 mice were challenged with lipopolysaccharide (LPS). Ileal permeability was measured in Ussing chambers and leukocyte-endothelium interactions studied with intravital fluorescence microscopy after 18 h. Results: LPS caused a clear-cut increase in leukocyte accumulation and intestinal permeability. Immunoneutralisation of P-selectin not only reduced leukocyt...

  10. Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Ikuko, E-mail: [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Cardiovascular Medicine, Saga University, Saga (Japan); Hasegawa, Koki [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Pathology and Experimental Medicine, Kumamoto University, Kumamoto (Japan); Wada, Yasuhiro [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Hirase, Tetsuaki; Node, Koichi [Department of Cardiovascular Medicine, Saga University, Saga (Japan); Watanabe, Yasuyoshi, E-mail: [RIKEN Center for Molecular Imaging Science, Kobe (Japan)


    Highlights: ► P-selectin regulates leukocyte recruitment as an early stage event of atherogenesis. ► We developed an antibody-based molecular imaging probe targeting P-selectin for PET. ► This is the first report on successful PET imaging for delineation of P-selectin. ► P-selectin is a candidate target for atherosclerotic plaque imaging by clinical PET. -- Abstract: Background: Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and results: A {sup 64}Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ({sup 64}Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by {sup 64}Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3 MBq of {sup 64}Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180 min PET scan, autoradiography and biodistribution of {sup 64}Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. {sup 64}Cu-DOTA-anti-P-selectin m

  11. The Effect of Regular Intake of Dry-Cured Ham Rich in Bioactive Peptides on Inflammation, Platelet and Monocyte Activation Markers in Humans (United States)

    Martínez-Sánchez, Sara María; Minguela, Alfredo; Prieto-Merino, David; Zafrilla-Rentero, María Pilar; Abellán-Alemán, José; Montoro-García, Silvia


    Background and aims: Dietary studies have shown that active biopeptides provide protective health benefits, although the mediating pathways are somewhat uncertain. To throw light on this situation, we studied the effects of consuming Spanish dry-cured ham on platelet function, monocyte activation markers and the inflammatory status of healthy humans with pre-hypertension. Methods: Thirty-eight healthy volunteers with systolic blood pressure of >125 mmHg were enrolled in a two-arm crossover randomized controlled trial. Participants received 80 g/day dry-cured pork ham of >11 months proteolysis or 100 g/day cooked ham (control product) for 4 weeks followed by a 2-week washout before “crossing over” to the other treatment for 4 more weeks. Soluble markers and cytokines were analyzed by ELISA. Platelet function was assessed by measuring P-selectin expression and PAC-1 binding after ADP (adenosine diphosphate) stimulation using whole blood flow cytometry. Monocyte markers of the pathological status (adhesion, inflammatory and scavenging receptors) were also measured by flow cytometry in the three monocyte subsets after the interventional period. Results: The mean differences between dry-cured ham and cooked ham followed by a time period adjustment for plasmatic P-selectin and interleukin 6 proteins slightly failed (p = 0.062 and p = 0.049, respectively), notably increased for MCP-1 levels (p = 0.023) while VCAM-1 was not affected. Platelet function also decreased after ADP stimulation. The expression of adhesion and scavenging markers (ICAM1R, CXCR4 and TLR4) in the three subsets of monocytes was significantly higher (all p < 0.05). Conclusions: The regular consumption of biopeptides contained in the dry-cured ham but absent in cooked ham impaired platelet and monocyte activation and the levels of plasmatic P-selectin, MCP-1 and interleukin 6 in healthy subjects. This study strongly suggests the existence of a mechanism that links dietary biopeptides and beneficial

  12. Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions. (United States)

    Li, Suping; Shi, Quanwei; Liu, Guanglei; Zhang, Weilin; Wang, Zhicheng; Wang, Yuedan; Dai, Kesheng


    Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

  13. Pathogen-Reduced, Extended Platelet Storage in Platelet Additive Solution (PAS) (United States)


    evaluated, as a potentially preferred product for battlefield polytrauma. This was once standard-of-care in transfusion medicine , but was abandoned...Sound Blood Center, led by Dr. Sherrill J. Slichter, have extensive experience in studying platelet biology and transfusion medicine . Dr. Slichter’s...unit Platelet Concentration   Volume   Platelet yield   Blood Gases (pH and pCO2, PO2, HC03)   Glucose and Lactate   P-selectin

  14. Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis. (United States)

    Hohlfeld, T; Zimmermann, N; Weber, A-A; Jessen, G; Weber, H; Schrör, K; Höltje, H-D; Ebel, R


    Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

  15. Metamizole and Platelet Inhibition by Aspirin Following On-Pump Coronary Artery Bypass Grafting. (United States)

    Wilczyński, Mirosław; Wybraniec, Maciej T; Sanak, Marek; Góral, Joanna; Mizia-Stec, Katarzyna


    The purpose of the study was to evaluate the impact of intravenous metamizole on platelet inhibition by aspirin in patients with coronary artery disease early after on-pump coronary artery bypass grafting (CABG). Prospective, single-blind, randomized trial. Tertiary referal hospital. The study comprised 43 patients with multivessel coronary artery disease undergoing CABG. Patients were randomized to postoperative intravenous metamizole ± opioids (study group; n = 23) or opioids alone (control group; n = 20). Aspirin was withheld at least 7 days before the surgery and reinitiated (300 mg) immediately after the procedure prior to metamizole use, and continued daily thereafter (150 mg). Platelet function was evaluated using multielectrode impedance aggregometry (acid-induced platelet activation [ASPI] and collagen-induced platelet activation [COL] test), P-selectin expression and urinary 11-dehydro-thromboxane B2 (11-DTXB2) level at baseline, postoperative day (POD) 0, POD 1, POD 2, and POD 6. Residual platelet reactivity (RPR) was defined as ASPI test >400 AU*min. In all study participants, postoperative ASPI test value moderately decreased (1058.2 v 966.6 AU*min, p = 0.047), urinary 11-DTXB2 level increased (923.4 v 4367.3 pg/mg, p metamizole use did not predict the fluctuations of ASPI and COL test values and P-selectin expression, yet it independently predicted postoperative change of 11-DTXB2 level (b = -0.518, p = 0.001). Intravenous metamizole preceded by a loading dose of aspirin did not modify platelet response to aspirin in the postoperative period after CABG. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. All-trans-Retinoic Acid Ameliorated High Fat Diet-Induced Atherosclerosis in Rabbits by Inhibiting Platelet Activation and Inflammation

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    Birong Zhou


    Full Text Available Background. All-trans-retinoic acid (atRA is effective for many proliferative diseases. We investigated the protective effects of atRA against atherosclerosis. Methods. Rabbits were randomly allocated to receive basal diet or an HFD for 4 weeks. HFD group then received rosuvastatin (3 mg/day, atRA (5 mg/kg/day, or the same volume of vehicle, respectively, for next 8 weeks. Results. HFD group showed increases in plasma lipids and aortic plaque formation. P-selectin expression and fibrinogen binding on platelets or deposition on the intima of the aorta also increased significantly as did the levels of TNF-α, IL-6, and fibrinogen in plasma. After 8 weeks of treatment with atRA, there was a significant decrease in plasma lipids and improvement in aortic lesions. AtRA also inhibited the expression of P-selectin and fibrinogen binding on platelets and deposition on the intima of the aorta. Conclusion. AtRA can ameliorate HFD-induced AS in rabbits by inhibiting platelet activation and inflammation.

  17. Platelet dynamics during natural and pharmacologically induced torpor and forced hypothermia.

    Directory of Open Access Journals (Sweden)

    Edwin L de Vrij

    Full Text Available Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster and non-hibernating (rat and mouse species without changing platelet function. Pharmacological torpor induced by injection of 5'-AMP in mice did not induce thrombocytopenia, possibly because 5'-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of

  18. Platelet-Derived MRP-14 Induces Monocyte Activation in Patients With Symptomatic Peripheral Artery Disease. (United States)

    Dann, Rebecca; Hadi, Tarik; Montenont, Emilie; Boytard, Ludovic; Alebrahim, Dornaszadat; Feinstein, Jordyn; Allen, Nicole; Simon, Russell; Barone, Krista; Uryu, Kunihiro; Guo, Yu; Rockman, Caron; Ramkhelawon, Bhama; Berger, Jeffrey S


    Peripheral artery disease (PAD), a diffuse manifestation of atherothrombosis, is a major cardiovascular threat. Although platelets are primary mediators of atherothrombosis, their role in the pathogenesis of PAD remains unclear. The authors sought to investigate the role of platelets in a cohort of symptomatic PAD. The authors profiled platelet activity, mRNA, and effector roles in patients with symptomatic PAD and in healthy controls. Patients with PAD and carotid artery stenosis were recruited into ongoing studies (NCT02106429 and NCT01897103) investigating platelet activity, platelet RNA, and cardiovascular disease. Platelet RNA sequence profiling mapped a robust up-regulation of myeloid-related protein (MRP)-14 mRNA, a potent calcium binding protein heterodimer, in PAD. Circulating activated platelets were enriched with MRP-14 protein, which augmented the expression of the adhesion mediator, P-selectin, thereby promoting monocyte-platelet aggregates. Electron microscopy confirmed the firm interaction of platelets with monocytes in vitro and colocalization of macrophages with MRP-14 confirmed their cross talk in atherosclerotic manifestations of PAD in vivo. Platelet-derived MRP-14 was channeled to monocytes, thereby fueling their expression of key PAD lesional hallmarks and increasing their directed locomotion, which were both suppressed in the presence of antibody-mediated blockade. Circulating MRP-14 was heightened in the setting of PAD, significantly correlated with PAD severity, and was associated with incident limb events. The authors identified a heightened platelet activity profile and unraveled a novel immunomodulatory effector role of platelet-derived MRP-14 in reprograming monocyte activation in symptomatic PAD. (Platelet Activity in Vascular Surgery and Cardiovascular Events [PACE]; NCT02106429; and Platelet Activity in Vascular Surgery for Thrombosis and Bleeding [PIVOTAL]; NCT01897103). Copyright © 2018 American College of Cardiology Foundation

  19. Response to antiplatelet therapy and platelet reactivity to thrombin receptor activating peptide-6 in cardiovascular interventions: Differences between peripheral and coronary angioplasty. (United States)

    Gremmel, Thomas; Xhelili, Endri; Steiner, Sabine; Koppensteiner, Renate; Kopp, Christoph W; Panzer, Simon


    The long-term prognosis of patients with peripheral arterial disease (PAD) is significantly worse than the prognosis of coronary artery disease (CAD) patients. Detrimental platelet activation could contribute to the increased rate of adverse cardiovascular events in PAD. We therefore investigated whether response to antiplatelet therapy and thrombin inducible platelet activation differ between patients with best medical therapy undergoing angioplasty and stenting for symptomatic PAD (n = 166) or CAD (n = 104). Adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin receptor activating peptide (TRAP)-6 inducible platelet reactivity was measured by multiple electrode aggregometry (MEA). Platelet surface expression of P-selectin and activated glycoprotein IIb/IIIa (GPIIb/IIIa) in response to ADP, AA, and TRAP-6, and the formation of monocyte-platelet aggregates (MPA) in response to ADP and TRAP-6 were assessed by flow cytometry. Patients with PAD had significantly higher platelet reactivity in response to ADP and AA by MEA compared to CAD patients. Likewise, the expression of P-selectin and GPIIb/IIIa following stimulation with ADP and AA, and MPA formation in response to ADP were significantly higher in PAD patients than in CAD patients. In response to TRAP-6, patients with PAD showed a significantly increased platelet aggregation by MEA, higher expression of activated GPIIb/IIIa, and more pronounced formation of MPA than CAD patients. Following angioplasty and stenting, PAD patients exhibit a significantly diminished response to dual antiplatelet therapy and an increased susceptibility to TRAP-6 inducible platelet activation compared to CAD patients. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. Dietary α-linolenic acid increases the platelet count in ApoE-/- mice by reducing clearance. (United States)

    Stivala, Simona; Reiner, Martin F; Lohmann, Christine; Lüscher, Thomas F; Matter, Christian M; Beer, Juerg H


    Previously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE(-/-)) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbβ3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α-converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

  1. platelets

    Directory of Open Access Journals (Sweden)

    Joanna Saluk


    Full Text Available Platelets are the smallest, depleted of nucleus blood cells which contain a typical cellular organelles including the mitochondria, so that have active metabolism. Platelets possess the highly organized cytoskeleton, specific secretory granules and unique membrane receptors system responsible for their high reactivity. The key role of blood platelets is to maintain normal hemostasis, but they also play important roles in inflammation, immune processes and the cancer progression. The anucleated, small platelets occur in representatives of all clusters of mammals, so it seems to be an adaptation feature. In other vertebrates similar hemostatic functions are played by large nucleated platelets, which are much more weakly reactive. Small, reactive platelets, appearing in the evolution of mammals, allowed the formation of clots faster and slower blood loss in case of injury, but also increased the risk of thromboembolic and cardiovascular diseases. Daily the human body forms about 1x1011 platelets, which are produced by a process of differentiation, maturation and fragmentation of the cytoplasm of mature megakaryocytes. The emergence of platelets is the final stage of megakaryocyte differentiation and is followed by formation of the direct precursors called proplatelets. The anucleated platelets are regarded as terminally differentiated cells, which are not capable of further cell division. However, despite the absence of a nucleus, in blood platelets the synthesis and transcription of mitochondrial DNA and protein synthesis occurring on the basis of mRNA from megakaryocytes has been confirmed. However, recent studies published in 2012 show that the platelets are capable not only of the process of protein synthesis, but also of generation of new cells, which are functionally and structurally similar to the parent platelets.

  2. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

    Directory of Open Access Journals (Sweden)

    Barbara Muz


    Full Text Available Multiple myeloma (MM is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  3. Binding of Platelets to Lymphocytes: A Potential Anti-Inflammatory Therapy in Rheumatoid Arthritis. (United States)

    Zamora, Carlos; Cantó, Elisabet; Nieto, Juan C; Bardina, Jorge; Diaz-Torné, Cesar; Moya, Patricia; Magallares, Berta; Ortiz, M Angels; Julià, Germà; Juarez, Candido; Llobet, Josep M; Vidal, Silvia


    Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-μm pore size filter. The binding of platelets to lymphocytes was blocked with anti-P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses. Copyright © 2017 by The American Association of Immunologists, Inc.

  4. Human Platelets Express Functional Thymic Stromal Lymphopoietin Receptors: a Potential Role in Platelet Activation in Acute Coronary Syndrome

    Directory of Open Access Journals (Sweden)

    Boyuan Wang


    Full Text Available Background: Thymic stromal lymphopoietin (TSLP has been shown to be expressed in various inflammatory tissues, such as human atherosclerotic plaques. Many types of myeloid cells involved in atherosclerosis, including mast cells, lymphocytes, dendritic cells and monocytes/macrophages, present TSLP receptors (TSLPR. However, it is unknown whether platelets, which also play important roles in atherothrombosis, express TSLPR. Methods and Results: We applied flow cytometry and western blotting to show that TSLPR was expressed on the surface of human platelets. Following the addition of TSLP to platelets, the expression of CD62P, CD63, PAC-1 and p-Akt as well as aggregation and ATP release were increased significantly. A TSLPR antibody and a PI3K (phosphatidylinositol 3-kinase enzyme inhibitor (LY294002 significantly inhibited the platelet activation induced by TSLP. The expression of TSLPR, CD62P and CD63 and the increment of the expression of CD62P and CD63 induced by TSLP in the acute coronary syndrome (ACS group were markedly higher than those in the control group and the stable angina pectoris (SAP group. The expression and the increment of the expression of CD62P and CD63 induced by TSLP were positively correlated with the expression of TSLPR. Conclusion: Human platelets express functional TSLPR, which can be activated by TSLP to promote platelet activation. TSLP/TSLPR functions via activating the PI3K/AKT pathway, and this signalling pathway may be one of the mechanisms involved in thrombosis in ACS. In coronary disease patients, the determination of TSLPR in platelets may help to identify the risk of ACS.

  5. Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

    DEFF Research Database (Denmark)

    Evans, Evan; Heinrich, Volkmar; Leung, Andrew


    , and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane...... unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential...

  6. Oxidative Versus Thrombotic Stimulation of Platelets Differentially activates Signalling Pathways

    Directory of Open Access Journals (Sweden)

    Pouran Karimi


    Conclusion: Our results indicated that MAPK inhibitors can reduce atherothrombotic events via alterations in P-selectin expression suggesting that these inhibitors may be useful in the inhibition of atheroma development.

  7. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI. (United States)

    Gardiner, Elizabeth E; Andrews, Robert K


    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. FcgammaRII mediates platelet aggregation caused by disintegrins and GPIIb/IIIa monoclonal antibody, AP2. (United States)

    Huang, Tur-Fu; Chang, Chien-Hsin; Ho, Pei-Ling; Chung, Ching-Hu


    Disintegrins, snake venom-derived Arg-Gly-Asp (RGD)-containing polypeptides, and GPIIb/IIIa antagonist (AP2) block fibrinogen binding to GPIIb/IIIa of activated platelets, however, the combination of these two agents caused platelet aggregation. We hypothesize that disintegrin initially binds to specific epitope of GPIIb/IIIa, causing conformational change, and the recruitment of FcgammaRII, which can be bound by AP2, and finally triggering platelet aggregation. We prepared human platelet suspensions and measured platelet aggregation, Ca2+ mobilization, thromboxane B2 formation, and signal transduction. Disintegrin (e.g., accutin) and AP2 (a monoclonal antibody [mAb]-raised against GPIIb/IIIa) individually inhibited human platelet aggregation caused by collagen. However, as both accutin and AP2 were sequentially added into platelet suspension, platelet aggregation occurred. Accutin/AP2 caused shape change, cytosolic Ca2+ mobilization, P-selectin expression, and thromboxane A2 formation. Tirofiban, FcgammaRII mAb, or indomethacin completely inhibited platelet aggregation caused by accutin/AP2. Accutin/AP2 also caused tyrosine phosphorylation of signal molecules. Disintegrins enhanced AP2 binding to platelets, and AP2 also promoted disintegrin binding to platelets. FcgammaRII mAb inhibited the enhanced fluorescein isothiocyanate-disintegrin binding to platelet caused by AP2. Immunoprecipitation of the lysates of disintegrin/AP2-treated platelets using FcgammaRII Ab showed complex formation of GPIIb/IIIa and FcgammaRII. FcgammaRII mediates platelet aggregation caused by disintegrin and AP2, triggering a phospholipase C, phospholipase A2, Src-, Syk kinases, and Ca2+-dependent activation process. AP2 triggers platelet aggregation via binding to accessible FcgammaRII and the conformation-altered GPIIb/IIIa caused by disintegrin.

  9. High-risk type-2 diabetes mellitus patients, without prior ischemic events, have normal blood platelet functionality profiles: a cross-sectional study. (United States)

    Shlomai, Gadi; Haran-Appel, Tal; Sella, Tal; Grossman, Yoni; Hauschner, Hagit; Rosenberg, Nurit; Grossman, Ehud


    Patients with type 2 diabetes mellitus (DM) display a predisposition for vascular disease. Platelets taken from vasculopathic diabetic patients, show enhanced stimuli-induced activation and aggregation responses. Aspirin remains the cornerstone antiplatelet agent for secondary prevention of vascular complications among diabetic patients, yet evidence of its efficacy and safety in primary prevention are conflicting. Our aim was to assess whether high risk diabetic patients, without previous ischemic events, have abnormal platelet functionality profiles. The study included 82 diabetic patients and 86 matched non-diabetic patients without prior ischemic events nor treatment with anti-platelet medications. Blood samples were analyzed for platelet markers of activation, turnover and leukocyte-platelet interactions. Our final analysis included 122 males (74 %), with a mean age of 61 years. Mean platelet volume (MPV) was similar between the diabetic patients and controls (9.2 fL for both). Following activation, PAC-1 binding and P-selectin expression were found comparable between the diabetic patients and controls (83 % versus 81 % and 76 % versus 74 %, respectively). Leukocyte-platelet aggregates (LPAs) were similar between the diabetic patients and controls (18 % versus 17 %, respectively). Neutrophil-platelet aggregates (NPAs) and monocyte-platelet aggregates (MPAs) were also found similar in the diabetic patients and controls. Elevated fasting plasma glucose was associated with increased LPAs rates. High risk type-2 diabetes mellitus patients, without prior ischemic events, have normal blood platelet functionality profiles.

  10. Effect of thrombopoietin receptor agonists on markers of coagulation and P-selectin in patients with immune thrombocytopenia

    DEFF Research Database (Denmark)

    Garabet, Lamya; Ghanima, Waleed; Monceyron Jonassen, Christine


    Thrombopoietin-receptor-agonists (TPO-RA) are effective treatments of immune thrombocytopenia (ITP). Previous long-term TPO-RA clinical trials have shown that thrombotic events occurred in 6% of TPO-RA-treated ITP patients. To explore the increased risk of thrombosis, the effects of TPO......-RA on markers of coagulation and P-selectin were studied. The study comprised two ITP cohorts and controls. Cohort 1 included 26 patients with sequential samples acquired before and during treatment with TPO-RA. Cohort 2 included a single sample in 18 patients on TPO-RA for more than one year. Thrombin...... patients in cohort 1. Significantly higher levels of F1+2, D-dimer, and PAI-1 were found in ITP patients before TPO-RA treatment and in patients on long-term TPO-RA treatment than in controls. Pre-treatment levels of sP-selectin did not differ from controls. Analysis of longitudinal trends showed...

  11. Platelet CD40 Mediates Leukocyte Recruitment and Neointima Formation after Arterial Denudation Injury in Atherosclerosis-Prone Mice. (United States)

    Jin, Rong; Xiao, Adam Y; Song, Zifang; Yu, Shiyong; Li, Jarvis; Cui, Mei-Zhen; Li, Guohong


    The role of platelets in the development of thrombosis and abrupt closure after angioplasty is well recognized. However, the direct impact of platelets on neointima formation after arterial injury remains undetermined. Herein, we show that neointima formation after carotid artery wire injury reduces markedly in CD40-/- apolipoprotein E-deficient (apoE-/-) mice but only slightly in CD40 ligand-/-apoE-/- mice, compared with apoE-/- mice. Wild-type and CD40-deficient platelets were isolated from blood of apoE-/- and CD40-/-apoE-/- mice, respectively. The i.v. injection of thrombin-activated platelets into CD40-/-apoE-/- mice was performed every 5 days, starting at 2 days before wire injury. Injection of wild-type platelets promoted neointima formation, which was associated with increased inflammation by stimulating leukocyte recruitment via up-regulation of circulating platelet surface P-selectin expression and the formation of platelet-leukocyte aggregates. It was also associated with further promoting the luminal deposition of platelet-derived regulated on activation normal T cell expressed and secreted/chemokine (C-C motif) ligand 5 and expression of monocyte chemoattractant protein-1 and vascular cell adhesion molecule 1 in wire-injured carotid arteries. Remarkably, all these inflammatory actions by activated platelets were abrogated by lack of CD40 on injected platelets. Moreover, injection of wild-type platelets inhibited endothelial recovery in wire-injured carotid arteries, but this effect was also abrogated by lack of CD40 on injected platelets. Results suggest that platelet CD40 plays a pivotal role in neointima formation after arterial injury and might represent an attractive target to prevent restenosis after vascular interventions. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  12. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    Directory of Open Access Journals (Sweden)

    Amal J. Ali


    Full Text Available Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1 and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL. Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  13. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    KAUST Repository

    Ali, Amal J.


    Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  14. Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity. (United States)

    Lee, Wonhwa; Kim, Mi-Ae; Park, InWha; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup


    Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo(L-Pro-L-Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1-2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1-2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.

    LENUS (Irish Health Repository)

    McCabe, Dominick J H


    Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte-platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P-, CD63-, and PAC1-binding, and the percentages of leucocyte-platelet complexes in acute (1-27 d, n = 79) and convalescent (79-725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P-selectin, soluble E-selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and\\/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte-platelet complexes were higher in both acute and convalescent CVD patients than controls (P <\\/= 0.02). The mean white cell count and mean VWF:Ag levels were significantly elevated in the acute and convalescent phases after ischaemic stroke or TIA (P <\\/= 0.02). Otherwise, there was no significant increase in any other marker of platelet or endothelial activation in CVD patients. There was a positive correlation between the percentage expression of CD62P and the percentages of both neutrophil-platelet and monocyte-platelet complexes in the acute phase, and the percentages of all leucocyte-platelet complexes in the convalescent phase after ischaemic CVD. This study provides evidence for ongoing excessive platelet and\\/or endothelial activation in ischaemic CVD patients despite treatment with antithrombotic therapy.

  16. Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice. (United States)

    Pérez-Frías, A; González-Tajuelo, R; Núñez-Andrade, N; Tejedor, R; García-Blanco, M J; Vicente-Rabaneda, E; Castañeda, S; Gamallo, C; Silván, J; Esteban-Villafruela, A; Cubero-Rueda, L; García-García, C; Muñoz-Calleja, C; García-Diez, A; Urzainqui, A


    To define and characterize the progression of the spontaneous autoimmune disease that develops in mice in the absence of the leukocyte adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1). Skin-resident immune cells from PSGL-1-deficient mice and C57BL/6 control mice of different ages were isolated and analyzed by flow cytometry. Biochemical parameters were analyzed in mouse serum and urine, and the presence of serum autoantibodies was investigated. Skin and internal organs were extracted, and their structure was analyzed histologically. Skin-resident innate and adaptive immune cells from PSGL-1(-/-) mice had a proinflammatory phenotype with an imbalanced T effector cell:Treg cell ratio. Sera from PSGL-1(-/-) mice had circulating autoantibodies commonly detected in connective tissue-related human autoimmune diseases. Biochemical and histologic analysis of skin and internal organs revealed skin fibrosis and structural and functional abnormalities in the lungs and kidneys. Furthermore, PSGL-1(-/-) mice exhibited vascular alterations, showing loss of dermal vessels, small vessel medial layer remodeling in the lungs and kidneys, and ischemic processes in the kidney that promote renal infarcts. Our study demonstrates that immune system overactivation due to PSGL-1 deficiency triggers an autoimmune syndrome with characteristics similar to systemic sclerosis, including skin fibrosis, vascular alterations, and systemic organ involvement. These results suggest that PSGL-1 expression contributes to the maintenance of the homeostasis of the immune system and could act as a barrier for autoimmunity in mice. Copyright © 2014 by the American College of Rheumatology.

  17. Expression of surface platelet receptors (CD62P and CD41/61) in horses with recurrent airway obstruction (RAO). (United States)

    Iwaszko-Simonik, Alicja; Niedzwiedz, Artur; Graczyk, Stanislaw; Slowikowska, Malwina; Pliszczak-Krol, Aleksandra


    Recurrent airway obstruction (RAO) is an allergic disease of horses similar to human asthma, which is characterized by airway inflammation and activation of neutrophils, lymphocytes and platelets. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to investigate platelet status in RAO-affected horses based on the platelet morphology and platelet surface expression of CD41/61 and CD62P. Ten RAO-affected horses and ten healthy horses were included in this study. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62P and CD41/61 was detected by flow cytometry on activated platelets. The median PLT was significantly reduced in horses with RAO compared to the controls. The MPV and the P-LCR values were significantly higher in RAO horses than controls. Expression of CD41/61 on platelets was increased in RAO horses, while CD62P expression was reduced. This study demonstrated the morphological changes in platelets and expression of platelet surface receptors. Despite the decrease of CD62P expression, the observed increased surface expression of CD41/61 on platelets in horses with RAO may contribute to the formation of platelet aggregates in their respiratory system. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. [Influence of exam stress on the development of endothelial dysfunction and the formation of platelet-leukocyte coagregates]. (United States)

    Fefelova, E V; Tereshkov, P P; Plotnikova, O K; Stafeev, A N; Semenov, A V; Svistunova, N M; Ivanov, M O


    The aim of this work was to study the qualitative and quantitative composition of circulating endotheliocytes, P-selectin-associated leukocyte and platelet of coogregation blood levels of endothelin, interleukin-6 students on the background of exam stress. The research was made on the group conditionally healthy volunteers aged MT 18.3 ± 1 years that before participating in the study had a medical examination (n = 15). Blood sampling was made three times: 1--three months before the session (in a normal school day, two hours after training sessions), 2--for 25 ± 10 min before the exam, 3 - 15 ± 10 min after the exam. Approved the development of endothelial dysfunction in the background of exam stress, manifested by increased levels of circulating endothelial cells and endothelin. The total number of leukocyte and platelet of coogregation during the semester, before and after the exam has not changed, while there was an increase outlets, due to the expression of P-selectin on red blood cells before the exam and precipitous decline after the exam.

  19. Increased platelet expression of FcGammaRIIa and its potential impact on platelet reactivity in patients with end stage renal disease

    Directory of Open Access Journals (Sweden)

    Sobel Burton E


    Full Text Available Abstract Background Increased platelet reactivity has been implicated in cardiovascular disease – the major cause of death in patients with end stage renal disease (ESRD. FcGammaRIIA is a component of glycoprotein VI and Ib-IX-V that mediate activation of platelets by collagen and von Willebrand factor. To determine whether expression of FcGammaRIIA impacts platelet reactivity we quantified its expression and platelet reactivity in 33 patients with ESRD who were undergoing hemodialysis. Methods Blood samples were obtained from patients immediately before hemodialysis and before administration of heparin. Platelet expression of FcGammaRIIA and the activation of platelets in response to low concentrations of convulxin (1 ng/ml, selected to mimic effects of collagen, thrombin (1 nM, adenosine diphosphate (ADP, 0.2 uM, or platelet activating factor (PAF, 1 nM were determined with the use of flow cytometry in samples of whole blood anticoagulated with corn trypsin inhibitor (a specific inhibitor of Factor XIIa. Results Patients were stratified with respect to the median expression of FcGammaRIIA. Patients with high platelet expression of FcGammaRIIA exhibited 3-fold greater platelet reactivity compared with that in those with low expression in response to convulxin (p Conclusion Increased platelet reactivity in response to low concentrations of diverse agonists is associated with high expression of FcGammaRIIA and may contribute to an increased risk of thrombosis in patients with ESRD.

  20. 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. (United States)

    Ostertag, Luisa M; Kroon, Paul A; Wood, Sharon; Horgan, Graham W; Cienfuegos-Jovellanos, Elena; Saha, Shikha; Duthie, Garry G; de Roos, Baukje


    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.

  1. Storage of apheresis platelet concentrates after manual replacement of >95% of plasma with PAS 5. (United States)

    Morrison, A; McMillan, L; Radwanski, K; Blatchford, O; Min, K; Petrik, J


    Recently, a glucose- and bicarbonate-containing additive solution termed PAS 5 demonstrated acceptable 7-day platelet storage after >95% plasma replacement with PAS on the day of collection (Day 0). In this study, we examined platelet storage in >95% PAS 5 after manual washing of Day 1 apheresis platelets in plasma collected using either the Amicus or Trima plateletpheresis devices. Triple platelet donations in plasma were obtained from Amicus (n = 10) and Trima (n = 10) plateletpheresis devices and stored overnight before being centrifuged and manually processed into three units with the following storage media: 100% plasma, >95% PAS 5 or 65% PAS 5/35% plasma. Platelet units were sampled on Days 1, 5 and 7 of storage using a range of tests recommended by the UK guidelines. The majority of in vitro assay results for platelets in PAS 5 were similar to results in paired 100% plasma platelets (controls). The pH of PAS 5 stored platelet units was above the UK recommended guidelines of 7·4 by Day 5. PAS 5 platelets were no more activated than controls as evidenced by comparable soluble P-selectin levels and CD62p and CD42b expression. PAS 5 platelets also exhibited adhesion and aggregation profiles higher than (Day 1) or comparable to (Days 5 and 7) controls as measured by Impact R. The 7-day in vitro storage parameters investigated were comparable between >95% PAS 5 and 100% plasma platelets derived from both Amicus and Trima plateletpheresis devices, with the exception that lactose dehydrogenase release rate and pH were significantly higher in PAS 5 units. © 2014 International Society of Blood Transfusion.

  2. Assessment of the influence of the inflammatory process on the activation of blood platelets and morphological parameters in patients with ulcerative colitis (colitis ulcerosa

    Directory of Open Access Journals (Sweden)

    Beata Polińska


    Full Text Available Ulcerative colitis (colitis ulcerosa is a non-specific inflammatory bowel disease of unknown etiology. Thesymptoms which are observed in the course of ulcerative colitis are: an increase in the number of leukocytes andblood platelets, an increase in the concentration of IL-6 and anemia. Blood platelets are the key element, linkingthe processes of hemostasis, inflammation and the repair of damaged tissues. Activation of blood platelets is connectedwith changes in their shape and the occurrence of the reaction of release. P-selectin appears on the surfacesof activated blood platelets and the concentration level of soluble P-selectin increases in the blood plasma. The aimof this study was to define whether the increased number of blood platelets in patients with ulcerative colitisaccompanies changes in their activation and morphology. A total of 16 subjects with ulcerative colitis and 32healthy subjects were studied. Mean platelet count, morphological parameters of platelets and MPC were measuredusing an ADVIA 120 hematology analyzer. Concentrations of sP-selectin and IL-6 in serum were marked byimmunoassay (ELISA. MPC, concentration of sP-selectin and IL-6 were significantly higher in subjects with ulcerativecolitis compared to those in the healthy group. There was a decrease of MPV in patients with ulcerativecolitis, which is statistically significant. Chronic inflammation in patients with ulcerative colitis causes an increase inthe number of blood platelets, a change in their morphology and activation. Decreased MPV value reflects activationand the role blood platelets play in the inflammatory process of the mucous membrane of the colon. A highconcentration of sP-selectin, which is a marker of blood platelet activation, demonstrates their part in the inflammatoryprocess. The increase in the concentration of sP-selectin correlated positively with the increase in concentrationof IL-6. This is why it may be a useful marker of the activity of

  3. Platelets Express Activated P2Y12Receptor in Patients With Diabetes Mellitus. (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


    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 P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 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 P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 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, P diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P diabetes mellitus. Platelet P2Y 12 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.

  4. Effect of marathon run and air travel on pre- and post-run soluble d-dimer, microparticle procoagulant activity, and p-selectin levels. (United States)

    Parker, Beth A; Augeri, Amanda L; Capizzi, Jeffrey A; Ballard, Kevin D; Kupchak, Brian R; Volek, Jeffrey S; Troyanos, Christopher; Kriz, Peter; D'Hemecourt, Pierre; Thompson, Paul D


    D-dimer, microparticles, and p-selectin are venous thrombotic risk markers. Elevated p-selectin is associated with increased cardiovascular events. We examined the effects of exercise and air travel on the markers of vascular risk in marathon runners. Forty-one persons participating in the 114th Boston Marathon (April 19, 2010) were divided into travel (n = 23) and nontravel "control" (n = 18) groups according to whether they lived more than a 4-hour plane flight or less than a 2-hour car trip from Boston. The subjects provided venous blood samples the day before, immediately after, and after returning home the day after the marathon. The blood was analyzed for soluble d-dimer, microparticle procoagulant activity, and p-selectin. D-dimer levels increased more before to immediately after (142 ± 83 to 387 ± 196 ng/mL) in the travel group than in the controls (85 ± 26 to 233 ± 95 ng/mL; p = 0.02). Moreover, 6 travel subjects versus 0 controls had d-dimer values >500 ng/mL after returning home the day after the marathon, the clinical threshold for excluding venous thrombosis (p = 0.03). P-selectin increased with exercise (p marathon (r(2) = 0.16, p = 0.01). In conclusion, the combination of exercise and travel increases venous and arterial thrombotic risk. Moreover, the p-selectin levels at rest and after exercise were greater with age. These results might explain the reports of venous thrombosis with air travel after athletic events and the reports of cardiac events in older participants running marathons. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Delayed inhibition of agonist-induced granulocyte-platelet aggregation after low-dose sevoflurane inhalation in humans. (United States)

    Wacker, Johannes; Lucchinetti, Eliana; Jamnicki, Marina; Aguirre, José; Härter, Luc; Keel, Marius; Zaugg, Michael


    Sevoflurane can be used as sedative-analgesic drug with endothelial protective properties. We tested whether low-dose sevoflurane inhalation provides sustained inhibition of detrimental granulocyte-platelet aggregation in humans. Ten healthy male volunteers were enrolled in this crossover study. Each subject inhaled sevoflurane for 1 h at 0.5-1 vol % end-tidal concentration in oxygen (50 vol %). Inhaling oxygen (50 vol %) alone served as control. Venous blood samples were collected at baseline before inhalation, immediately after inhalation, and 24 h thereafter, and were used for flow cytometry to determine platelet surface marker (CD41, CD42b, CD62P/P-selectin, and PAC-1) on platelets and granulocytes and for kaolin-induced clot formation, as assessed by thromboelastography. In flow cytometry experiments, platelets were stimulated with arachidonic acid (AA, 30 microM), adenosine diphosphate (ADP, 1 microM), and thrombin receptor agonist peptide-6 (TRAP-6, 6 microM). AA, ADP, and TRAP-6 markedly increased the expression of CD62P on platelets, whereas CD42b (shedding) and PAC-1 (heterotypic conjugates) expression decreased. The amount of granulocyte-platelet aggregates increased upon agonist stimulation. Low-dose sevoflurane inhalation reduced ADP-induced CD62P expression on platelets 24 h after inhalation, and inhibited the formation of granulocyte-platelet aggregates under stimulation with AA and ADP after 1 and 24 h, and with TRAP-6 after 24 h compared with control. Inhibition of granulocyte-platelet aggregates was accompanied by reduced clot firmness 24 h after sevoflurane inhalation compared with control. We demonstrated for the first time that inhaling low-dose sevoflurane (<1 vol % end-tidal) inhibits agonist-induced granulocyte-platelet interactions 24 h after administration and thus counteracts thromboinflammatory processes.

  6. Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice. (United States)

    Baumgartner, C K; Mattson, J G; Weiler, H; Shi, Q; Montgomery, R R


    Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of

  7. Platelet interaction with activated endothelium: mechanistic insights from microfluidics. (United States)

    Coenen, Daniëlle M; Mastenbroek, Tom G; Cosemans, Judith M E M


    Traditionally, in vitro flow chamber experiments and in vivo arterial thrombosis studies have been proven to be of vital importance to elucidate the mechanisms of platelet thrombus formation after vessel wall injury. In recent years, it has become clear that platelets also act as modulators of inflammatory processes, such as atherosclerosis. A key element herein is the complex crosstalk between platelets, the coagulation system, leukocytes and the activated endothelium. This review provides insight into the platelet-endothelial interface, based on in vitro flow chamber studies and cross referenced with in vivo thrombosis studies. The main mechanisms of platelet interaction with the activated endothelium encompass i) platelet rolling via interaction of platelet glycoprotein Ib-IX-V with endothelial-released von Willebrand factor with a supporting role for the P-selectin - P-selectin glycoprotein ligand 1 axis, followed by ii) firm platelet adhesion to the endothelium via interaction of platelet αIIbβ3 with endothelial αvβ3 and intercellular adhesion molecule 1, and iii) a stimulatory role for thrombin, the thrombospondin-1 - CD36 axis and cyclooxygenase 1 in subsequent platelet activation and stable thrombus formation. In addition, the molecular mechanisms underlying the stimulatory effect of platelets on leukocyte transendothelial migration, a key mediator of atheroprogression, are discussed. Throughout the review emphasis is placed on recommendations for setting up, reporting, interpreting and comparing endothelial-lined flow chamber studies and on suggestions for future studies. Copyright © 2017 American Society of Hematology.

  8. Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode. (United States)

    Pourtau, Line; Sellal, Jean Marc; Lacroix, Romaric; Poncelet, Philippe; Bernus, Olivier; Clofent-Sanchez, Gisèle; Hocini, Mélèze; Haïssaguerre, Michel; Dignat-George, Françoise; Sacher, Frédéric; Nurden, Paquita


    Thrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode. Blood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, αIIbβ3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays. A specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed. Acute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Agent-based model of therapeutic adipose-derived stromal cell trafficking during ischemia predicts ability to roll on P-selectin.

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    Alexander M Bailey


    , or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.

  10. Agent-based model of therapeutic adipose-derived stromal cell trafficking during ischemia predicts ability to roll on P-selectin. (United States)

    Bailey, Alexander M; Lawrence, Michael B; Shang, Hulan; Katz, Adam J; Peirce, Shayn M


    vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 microm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.

  11. Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling.

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    Maria E Lopes Pires

    Full Text Available Lipopolysaccharide (LPS from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4 which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619 or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation.

  12. Down-regulation of platelet surface CD47 expression in Escherichia coli O157:H7 infection-induced thrombocytopenia.

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    Ya-Lan Guo

    Full Text Available BACKGROUND: Platelet depletion is a key feature of hemolytic uremic syndrome (HUS caused by Shiga toxin-producing Escherichia coli (STEC infection. The mechanism underlying STEC-induced platelet depletion, however, is not completely understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrated for the first time that platelet surface expression of CD47 was significantly decreased in C57BL6 mice treated with concentrated culture filtrates (CCF from STEC O157:H7. STEC O157:H7 CCF treatment also led to a sharp drop of platelet counts. The reduction of cell surface CD47 was specific for platelets but not for neutrophil, monocytes and red blood cells. Down-regulation of platelet surface CD47 was also observed in isolated human platelets treated with O157:H7 CCF. Platelet surface CD47 reduction by O157:H7 CCF could be blocked by anti-TLR4 antibody but not anti-CD62 antibody. Down-regulation of platelet surface CD47 was positively correlated with platelet activation and phagocytosis by human monocyte-derived macrophages. Furthermore, the enhanced phagocytosis process of O157:H7 CCF-treated platelets was abolished by addition of soluble CD47 recombinants. CONCLUSIONS/SIGNIFICANCE: Our results suggest that platelet CD47 down-regulation may be a novel mechanism underneath STEC-induced platelet depletion, and that the interactions between CD47 and its receptor, signal regulatory protein alpha (SIRPalpha, play an essential role in modulating platelet homeostasis.

  13. Changes in platelet functional parameters and CD62 P expression ...

    African Journals Online (AJOL)


    5-6, the risk of operation is low; Grade B, 7-9, the risk of operation is higher than Grade A; Grade C, e”10, the risk .... of BPC reflects the generation and decay of platelets. MPV reflects the metabolization of bone marrow ... more powerful adhesive capacity, and more bleeding. They found that MPV and CD62P were higher in.

  14. Changes in platelet functional parameters and CD62 P expression ...

    African Journals Online (AJOL)


    Center Department of Clinical Laboratory Medicine, Linyi People's Hospital, Linyi 276003, China. Abstract. Background: Hepatic impairment, portal hypertension, and multi-systemic ... thrombocytopenia3-6, and consumption of blood coagulation factors and antibodies directed against platelets7-9 could lead to bleeding ...

  15. Lipopolysaccharide and platelet-activating factor stimulate expression of platelet-activating factor acetylhydrolase via distinct signaling pathways (United States)

    Abdel-al, Mohammed; Ditmyer, Marcia; Patel, Nipa


    Objectives This study was designed to investigate and characterize the ability of platelet-activating factor (PAF) to induce the expression of platelet-activating factor acetylhydrolase (PAF-AH). Methods Ribonuclease protection assays and quantitative real-time PCR were used to investigate the ability of lipopolysaccharide (LPS) and PAF to regulate PAF-AH mRNA expression in human monocyte–macrophage 6 (MM6) cells. Pharmacological inhibitors of mitogen activated protein kinases (MAPK) and PAF receptor antagonists were used to investigate the mechanism of regulation of PAF-AH. Results PAF-AH mRNA levels were increased upon exposure to LPS or PAF in a dose-dependent manner. LPS elicited a more potent and rapid increase in PAF-AH expression than the PAF-stimulated response. However, when administered concomitantly, PAF augmented the LPS-stimulated response. LPS-stimulated PAF-AH expression was susceptible to partial inhibition by a p38 MAPK inhibitor and PAF receptor antagonists. PAF-induced up-regulation of PAF-AH levels was solely mediated via the PAF receptor and was p38 MAPK-independent. Conclusion The proinflammatory mediators, LPS and PAF, increased levels of PAF-AH mRNA via distinct signaling pathways. PMID:21432021

  16. Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes--A new assay suitable for bench and bedside conditions. (United States)

    Granja, Tiago; Schad, Jessica; Schüssel, Patricia; Fischer, Claudius; Häberle, Helene; Rosenberger, Peter; Straub, Andreas


    Platelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood. Platelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively. The sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-α and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support. Using the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Incomplete reversibility of platelet inhibition following prolonged exposure to ticagrelor. (United States)

    Gerrits, A J; Jakubowski, J A; Sugidachi, A; Michelson, A D; Frelinger, A L


    Essentials Irreversible platelet inhibition persists after reversibly-binding ticagrelor is discontinued. Reversibility of platelet inhibition by ticagrelor and its active metabolite was assessed. Incomplete recovery was observed after prolonged exposure to ticagrelor. Activated GPIIb-IIIa and P-selectin, not platelet reactivity index, showed irreversibility. Introduction Ticagrelor is described as a reversible P2Y12 antagonist. However, residual platelet inhibition persists after discontinuation of ticagrelor when plasma levels are undetectable. We assessed the reversibility of platelet inhibition by ticagrelor and its active metabolite (T-AM) in comparison with cangrelor and prasugrel's active metabolite (P-AM). Methods Whole blood was treated in vitro with ~ 50% inhibitory concentrations of ticagrelor, T-AM, cangrelor, P-AM and assessed for ADP-stimulated activated GPIIb-IIIa and P-selectin and vasodilator-stimulated phosphoprotein (VASP) platelet reactivity index (PRI) before and after 100-fold dilution. Results Platelets exposed for 30 min to ticagrelor, T-AM or cangrelor showed full recovery of activated GPIIb-IIIa but only partial recovery of P-selectin. Longer exposure (24 h) to the drug decreased reversibility of activated GPIIb-IIIa by ticagrelor (65.1% [49.5-80.6], % of vehicle with 95% confidence interval [CI]) and T-AM (88.8% [79.2-98.3]), but not by cangrelor (101.4% [96.4-106.4]). Compared with 30 min exposure, the reversibility of P-selectin further decreased after 24 h exposure to ticagrelor (from 91.8% [82.1-101.5] to 51.8% [45.5-85.0]), but not T-AM (from 79.0% [67.8-90.3] to 77.4% [61.8-93.1]) or cangrelor (from 76.0% [67.6-84.4] to 76.2% [70.6-81.8]). In contrast, 24 h exposure to ticagrelor, T-AM and cangrelor resulted in full recovery of platelet reactivity as measured by PRI. Platelets exposed to P-AM showed no recovery of ADP reactivity. Conclusions Incomplete recovery after prolonged exposure to ticagrelor, observed by activated GPIIb

  18. SIRT1 prevents pulmonary thrombus formation induced by arachidonic acid via downregulation of PAF receptor expression in platelets. (United States)

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


    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 NH 4 Cl (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.

  19. Expression of Angiogenesis Regulatory Proteins and Epithelial-Mesenchymal Transition Factors in Platelets of the Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Hui Han


    Full Text Available Platelets play a role in tumor angiogenesis and growth and are the main transporters of several angiogenesis regulators. Here, we aimed to determine the levels of angiogenesis regulators and epithelial-mesenchymal transition factors sequestered by circulating platelets in breast cancer patients and age-matched healthy controls. Platelet pellets (PP and platelet-poor plasma (PPP were collected by routine protocols. Vascular endothelial growth factor (VEGF, platelet-derived growth factor BB (PDGF-BB, thrombospondin-1 (TSP-1, platelet factor 4 (PF4, and transforming growth factor-β1 (TGF-β1 were measured by enzyme-linked immunosorbent assay. Angiogenesis-associated expression of VEGF (2.1 pg/106 platelets versus 0.9 pg/106 platelets, P < 0.001, PF4 (21.2 ng/106 platelets versus 10.2 ng/106 platelets, P < 0.001, PDGF-BB (42.9 pg/106 platelets versus 19.1 pg/106 platelets, P < 0.001, and TGF-β1 (15.3 ng/106 platelets versus 4.3 ng/106 platelets, P < 0.001 differed in the PP samples of cancer and control subjects. In addition, protein concentrations were associated with clinical characteristics (P<0.05. Circulating platelets in breast cancer sequester higher levels of PF4, VEGF, PDGF-BB, and TGF-β1, suggesting a possible target for early diagnosis. VEGF, PDGF, and TGF-β1 concentrations in platelets may be associated with prognosis.

  20. HPW-RX40 prevents human platelet activation by attenuating cell surface protein disulfide isomerases

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    Po-Hsiung Kung


    Full Text Available Protein disulfide isomerase (PDI present at platelet surfaces has been considered to play an important role in the conformational change and activation of the integrin glycoprotein IIb/IIIa (GPIIb/IIIa and thus enhances platelet aggregation. Growing evidences indicated that platelet surface PDI may serve as a potential target for developing of a new class of antithrombotic agents. In the present study, we investigated the effects of HPW-RX40, a chemical derivative of β-nitrostyrene, on platelet activation and PDI activity. HPW-RX40 inhibited platelet aggregation, GPIIb/IIIa activation, and P-selectin expression in human platelets. Moreover, HPW-RX40 reduced thrombus formation in human whole blood under flow conditions, and protects mice from FeCl3-induced carotid artery occlusion. HPW-RX40 inhibited the activity of recombinant PDI family proteins (PDI, ERp57, and ERp5 as well as suppressed cell surface PDI activity of platelets in a reversible manner. Exogenous addition of PDI attenuated the inhibitory effect of HPW-RX40 on GPIIb/IIIa activation. Structure-based molecular docking simulations indicated that HPW-RX40 binds to the active site of PDI by forming hydrogen bonds. In addition, HPW-RX40 neither affected the cell viability nor induced endoplasmic reticulum stress in human cancer A549 and MDA-MB-231 cells. Taken together, our results suggest that HPW-RX40 is a reversible and non-cytotoxic PDI inhibitor with antiplatelet effects, and it may have a potential for development of novel antithrombotic agents.

  1. The effect of dabigatran and rivaroxaban on platelet reactivity and inflammatory markers. (United States)

    Zemer-Wassercug, Noa; Haim, Moti; Leshem-Lev, Dorit; Orvin, Katia L; Vaduganathan, Muthiah; Gutstein, Ariel; Kadmon, Ehud; Mager, Aviv; Kornowski, Ran; Lev, Eli I; Lev, Eli L


    The new oral anticoagulants (NOACs) reduce stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF), but dabigatran may increase risk of coronary ischemic events for unclear reasons. Thus, this study assessed the effects of dabigatran and rivaroxaban on platelet reactivity and inflammatory markers in patients with non-valvular AF. Patients with non-valvular AF planned to begin treatment with NOACs were included. Seventeen patients were prescribed dabigatran and ten rivaroxaban. Platelet function (as assessed by multiple-electrode aggregometry, Impact-R shear-induced platelet deposition, P-selectin expression and plasma RANTES levels) and high-sensitivity C-reactive protein (hs-CRP) were measured at enrollment (prior to initiation of NOAC treatment) and at least 7 days into treatment with either dabigratran or rivaroxaban. Seventeen patients treated with dabigatran (mean age 69 ± 7 years, 35 % women, mean CHADS2 score 2.6 ± 1.2), and ten patients treated with rivaroxaban (mean age 73 ± 9 years, 20 % women, mean CHADS2 score 2.7 ± 1.6) completed the study. In both groups, there were no significant differences in platelet reactivity between the baseline and on-anticoagulant treatment time-points, as measured by each of the platelet-specific assays. There was a trend towards increased platelet reactivity in response to arachidonic acid from baseline to on-treatment in both groups, probably as a result of aspirin discontinuation in 33 % of patients. No significant differences were noted between baseline and on-treatment in hs-CRP in both anticoagulant groups. Treatment with dabigatran and rivaroxaban does not appear to be associated with changes in markers of platelet reactivity or systemic inflammation.

  2. Gene deletion of P-Selectin and ICAM-1 does not inhibit neutrophil infiltration into peritoneal cavity following cecal ligation-puncture

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    Hess Karen


    Full Text Available Abstract Background Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis. Methods Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null mice by cecal ligation-puncture (CLP. Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis. Results CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis. Conclusions The data suggest that alternative adhesion pathway(s independent of P-selectin and ICAM

  3. Thrombopoietin and platelet aggregation in patients with stable coronary artery disease. (United States)

    Larsen, Sanne Bøjet; Grove, Erik Lerkevang; Neergaard-Petersen, Søs; Würtz, Morten; Hvas, Anne-Mette; Kristensen, Steen Dalby


    Thrombopoietin (TPO) may facilitate platelet activation and aggregation. However, data on the impact of TPO on platelet aggregation in patients with stable coronary artery disease (CAD) are scarce. We aimed to investigate associations between TPO and platelet aggregation and activation in patients with stable coronary artery disease (CAD). We studied 900 stable CAD patients. Serum TPO was assessed by ELISA. Platelet aggregation was evaluated using the Multiplate Analyzer (agonists: arachidonic acid [AA] and collagen) and the VerifyNow Aspirin Assay. Platelet activation was evaluated by soluble (s)P-selectin. Cyclooxygenase-1 inhibition was evaluated by serum thromboxane B2 (TXB2). We found that TPO correlated weakly with platelet aggregation evaluated by Multiplate using AA (r = -0.09, p = 0.01) and collagen as agonists (r = -0.03, p = 0.43) and by VerifyNow (r = 0.07, p = 0.03). We found no correlation between TPO and sP-selectin (r = -0.01, p = 0.70). Independent predictors of AA-induced platelet aggregation by Multiplate included high levels of sP-selectin and serum TXB2, high platelet count, increasing age and body mass index, female sex, and active smoking. Independent predictors of TPO included low AA-induced platelet aggregation by Multiplate, high levels of hs-CRP, active smoking, and high platelet aggregation evaluated by VerifyNow. In conclusion, TPO levels did not correlate with platelet activation and only weak associations were found between TPO and platelet aggregation, suggesting that TPO did not substantially facilitate platelet aggregation in stable CAD patients.

  4. Efficient removal of platelets from peripheral blood progenitor cell products using a novel micro-chip based acoustophoretic platform.

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    Josefina Dykes

    Full Text Available BACKGROUND: Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties. STUDY DESIGN AND METHODS: PBPC samples were obtained from patients (n = 15 and healthy donors (n = 6 and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality. RESULTS: The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P expression revealed a significant increase of CD62P+ platelets in the target (19% and waste fractions (20%, respectively, compared to the PBPC input samples (9%. However, activation was lower when compared to stored blood bank platelet concentrates (48%. CONCLUSION: Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability

  5. Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis. (United States)

    Li, Chun-Hong; Chen, Cen; Zhang, Qian; Tan, Chen-Ning; Hu, Yuan-Jia; Li, Peng; Wan, Jian-Bo; Feng, Gang; Xia, Zhi-Ning; Yang, Feng-Qing


    Corydalis yanhusuo W.T. Wang (Papaveraceae) (Rhizoma Corydalis) showed inhibitory effects on rabbit platelet aggregation induced by ADP, thrombin (THR) or arachidonic acid (AA). This study separates and identifies the possible target-related platelet proteins and suggests possible signal cascades of RC antiplatelet aggregation. Based on comparative proteomics, the differentially expressed platelet proteins treated before and after with 50 mg/mL RC 90% ethanol extract (for 15 min at 37 °C) were analyzed and identified by two dimensional gel electrophoresis (2-DE) and MALDI-TOF-MS/MS. To further verify the possible signalling pathways of RC antiplatelet aggregation function, the concentration of calcium (Ca2+) was measured by Fura-2/AM fluorescence (Ex 340/380 nm, Em 500 nm) (RC final concentrations of 0.0156-0.1563 mg/mL), the levels of P-selectin and cyclic guanosine monophosphate (cGMP) were quantified by ELISA (OD. 450 nm) (RC final concentrations of 0.0156-1.5625 mg/mL), and the 5-hydroxytryptamine (5-HT) level was measured using ortho-phthalaldehyde (OPT) fluorescence (Ex 340 nm, Em 470 nm) (RC final concentrations of 0.3125-1.5625 mg/mL). The expression of 52 proteins were altered in rabbit platelets after the treatment and the MALDI-TOF-MS analysis indicated that those proteins include 12 cytoskeleton proteins, 7 cell signalling proteins, 3 molecular chaperone proteins, 6 proteins related to platelet function, 16 enzymes and 7 other related proteins. Furthermore, RC extract could decrease the levels of 5-HT [inhibition rate of 96.80% (p rabbit platelets. The present study indicated that P2Y12 receptor might be one of the direct target proteins of RC in platelets. The signal cascades network of RC after binding with P2Y12 receptor is mediating Gαi proteins to activate downstream signalling pathways (AC and/or PI3K signalling pathways) for the inhibition of platelet aggregation.

  6. Gene Expression Profiles Link Respiratory Viral Infection, Platelet Response to Aspirin, and Acute Myocardial Infarction. (United States)

    Rose, Jason J; Voora, Deepak; Cyr, Derek D; Lucas, Joseph E; Zaas, Aimee K; Woods, Christopher W; Newby, L Kristin; Kraus, William E; Ginsburg, Geoffrey S


    Influenza infection is associated with myocardial infarction (MI), suggesting that respiratory viral infection may induce biologic pathways that contribute to MI. We tested the hypotheses that 1) a validated blood gene expression signature of respiratory viral infection (viral GES) was associated with MI and 2) respiratory viral exposure changes levels of a validated platelet gene expression signature (platelet GES) of platelet function in response to aspirin that is associated with MI. A previously defined viral GES was projected into blood RNA data from 594 patients undergoing elective cardiac catheterization and used to classify patients as having evidence of viral infection or not and tested for association with acute MI using logistic regression. A previously defined platelet GES was projected into blood RNA data from 81 healthy subjects before and after exposure to four respiratory viruses: Respiratory Syncytial Virus (RSV) (n=20), Human Rhinovirus (HRV) (n=20), Influenza A virus subtype H1N1 (H1N1) (n=24), Influenza A Virus subtype H3N2 (H3N2) (n=17). We tested for the change in platelet GES with viral exposure using linear mixed-effects regression and by symptom status. In the catheterization cohort, 32 patients had evidence of viral infection based upon the viral GES, of which 25% (8/32) had MI versus 12.2% (69/567) among those without evidence of viral infection (OR 2.3; CI [1.03-5.5], p=0.04). In the infection cohorts, only H1N1 exposure increased platelet GES over time (time course p-value = 1e-04). A viral GES of non-specific, respiratory viral infection was associated with acute MI; 18% of the top 49 genes in the viral GES are involved with hemostasis and/or platelet aggregation. Separately, H1N1 exposure, but not exposure to other respiratory viruses, increased a platelet GES previously shown to be associated with MI. Together, these results highlight specific genes and pathways that link viral infection, platelet activation, and MI especially in the

  7. Gene Expression Profiles Link Respiratory Viral Infection, Platelet Response to Aspirin, and Acute Myocardial Infarction.

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    Jason J Rose

    Full Text Available Influenza infection is associated with myocardial infarction (MI, suggesting that respiratory viral infection may induce biologic pathways that contribute to MI. We tested the hypotheses that 1 a validated blood gene expression signature of respiratory viral infection (viral GES was associated with MI and 2 respiratory viral exposure changes levels of a validated platelet gene expression signature (platelet GES of platelet function in response to aspirin that is associated with MI.A previously defined viral GES was projected into blood RNA data from 594 patients undergoing elective cardiac catheterization and used to classify patients as having evidence of viral infection or not and tested for association with acute MI using logistic regression. A previously defined platelet GES was projected into blood RNA data from 81 healthy subjects before and after exposure to four respiratory viruses: Respiratory Syncytial Virus (RSV (n=20, Human Rhinovirus (HRV (n=20, Influenza A virus subtype H1N1 (H1N1 (n=24, Influenza A Virus subtype H3N2 (H3N2 (n=17. We tested for the change in platelet GES with viral exposure using linear mixed-effects regression and by symptom status.In the catheterization cohort, 32 patients had evidence of viral infection based upon the viral GES, of which 25% (8/32 had MI versus 12.2% (69/567 among those without evidence of viral infection (OR 2.3; CI [1.03-5.5], p=0.04. In the infection cohorts, only H1N1 exposure increased platelet GES over time (time course p-value = 1e-04.A viral GES of non-specific, respiratory viral infection was associated with acute MI; 18% of the top 49 genes in the viral GES are involved with hemostasis and/or platelet aggregation. Separately, H1N1 exposure, but not exposure to other respiratory viruses, increased a platelet GES previously shown to be associated with MI. Together, these results highlight specific genes and pathways that link viral infection, platelet activation, and MI especially in the

  8. Meal-induced platelet activation in diabetes mellitus type 1 or type 2 is related to postprandial insulin rather than glucose levels. (United States)

    Spectre, Galia; Stålesen, Ragnhild; Östenson, Claes-Göran; Hjemdahl, Paul


    Postprandial platelet activation was related to postprandial insulin rather than glucose levels in a previous meal insulin study in type 2 diabetes mellitus (T2DM). We therefore compared postprandial platelet activation in type 1 (T1DM) patients without insulin secretion and T2DM patients with high postprandial insulin levels. Patients with T1DM (n=11) and T2DM (n=12) were studied before and 90min after a standardized meal without premeal insulin. Five T1DM patients volunteered for a restudy with their regular premeal insulin. Platelet activation was assessed by flow cytometry, with and without the thromboxane analogue U46619 or ADP, and by whole blood aggregometry (Multiplate®). Effects of insulin (100μU/mL) in vitro were also studied. Before the meal, glucose, insulin and platelet activation markers other than platelet-leukocyte aggregates (PLAs) were similar in T1DM and T2DM; PLAs were higher in T1DM. Postprandial glucose levels increased more markedly in T1DM (to 22.1±1.4 vs. 11.2±0.6mmol/L) while insulin levels increased only in T2DM (from 24.4±4.4 to 68.8±12.3μU/mL). Platelet P-selectin expression, fibrinogen binding and PLA formation stimulated by U46619 were markedly enhanced (approximately doubled) and whole blood aggregation stimulated by U46619 was increased (ppostprandial platelet activation when postprandial insulin levels increased. In vitro insulin mildly activated platelets in both groups. Postprandial platelet activation via the thromboxane pathway is related to postprandial hyperinsulinemia and not to postprandial hyperglycaemia in patients with diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Platelet activation determines angiopoietin-1 and VEGF levels in malaria: implications for their use as biomarkers.

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    Judith Brouwers

    Full Text Available INTRODUCTION: The angiogenic proteins angiopoietin (Ang-1, Ang-2 and vascular endothelial growth factor (VEGF are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7. METHODS: Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls. RESULTS: Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls. CONCLUSION: In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.

  10. Staphylococcus aureus α-hemolysin promotes platelet-neutrophil aggregate formation. (United States)

    Parimon, Tanyalak; Li, Zhi; Bolz, Devin D; McIndoo, Eric R; Bayer, Clifford R; Stevens, Dennis L; Bryant, Amy E


    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes severe hemorrhagic necrotizing pneumonia associated with high mortality. Exotoxins have been implicated in the pathogenesis of this infection; however, the cellular mechanisms responsible remain largely undefined. Because platelet-neutrophil aggregates (PNAs) can dysregulate inflammatory responses and contribute to tissue destruction, we investigated whether exotoxins from MRSA could stimulate formation of PNAs in human whole blood. Strong PNA formation was stimulated by toxins from stationary phase but not log phase CA-MRSA, and α-hemolysin was singularly identified as the mediator of this activity. MRSA exotoxins also caused neutrophil (polymorphonuclear leukocyte) activation, as measured by increased CD11b expression, although platelet binding was not driven by this mechanism; rather, α-hemolysin-induced PNA formation was solely platelet P-selectin dependent. These findings suggest a role for S. aureus α-hemolysin-induced PNA formation in alveolar capillary destruction in hemorrhagic/necrotizing pneumonia caused by CA-MRSA and offer novel targets for intervention.

  11. Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

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    Mathieu Barbier

    Full Text Available Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC and human brain microvascular endothelial cells (HBEC and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

  12. Sodium Bicarbonate Facilitates Hemostasis in the Presence of Cerebrospinal Fluid Through Amplification of Platelet Aggregation. (United States)

    Kozuma, Yukinori; Yamamoto, Tetsuya; Ishikawa, Eiichi; Yoshida, Fumiyo; Akutsu, Hiroyoshi; Matsuda, Masahide; Nakai, Kei; Tsuruta, Wataro; Takano, Shingo; Matsumura, Akira; Ninomiya, Haruhiko


    Appropriate hemostasis is essential for clear visualization of the neural structures and cleavage planes. It is also essential for avoiding heat-induced injury, minimizing blood loss, and reducing operative time. To determine the role of cerebrospinal fluid (CSF) in platelet-dependent hemostasis during neurosurgery. The amplification of aggregation, activation of integrin αIIbβ3, intrinsic and extrinsic coagulation pathways, and activation of signaling cascades in platelets were evaluated. For comparison, various concentrations of a commercially available artificial CSF solution (aCSF), an artificial CSF solution prepared by the authors, and normal saline (NS) were used. Differences between aCSF and NS in obtaining in vivo hemostasis were assessed by measuring the tail vein bleeding time in C57BL/6N mice. Platelet aggregation was directly amplified by the addition of aCSF through increased activation of integrin αIIbβ3, phosphatidylserine exposure, and P-selectin expression. However, the prothrombin time and activated partial thromboplastin time were not primarily related to coagulation activity with the addition of aCSF. Activation of Src kinase was related to platelet activation by aCSF. The elimination of sodium bicarbonate from aCSF and the addition of the selective inhibitor of the HCO3/Cl exchanger, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, significantly inhibited platelet aggregation. The bleeding time in aCSF-treated mice was significantly shorter than that in NS-treated mice. Sodium bicarbonate facilitates hemostasis through the amplification of platelet aggregation function. The existence of CSF and irrigation with aCSF provide better conditions for physiological hemostasis and they have the potential of improving hemostasis by bipolar coagulation or with irrigation during neuroendoscopic procedures.

  13. Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion. (United States)

    Plenchette, S; Moutet, M; Benguella, M; N'Gondara, J P; Guigner, F; Coffe, C; Corcos, L; Bettaieb, A; Solary, E


    Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progressively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet caspase activation during storage, an effect that is partially prevented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of caspase inhibitors may not improve the quality of platelet concentrates stored in standard conditions.

  14. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A. (United States)

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H


    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

  15. Associations between circulating proteins and corresponding genes expressed in coronary thrombi in patients with acute myocardial infarction. (United States)

    Helseth, Ragnhild; Weiss, Thomas W; Opstad, Trine Baur; Siegbahn, Agneta; Solheim, Svein; Freynhofer, Matthias K; Huber, Kurt; Arnesen, Harald; Seljeflot, Seljeflot


    Several genes are expressed in aspirated coronary thrombi in acute myocardial infarction (AMI), exhibiting dynamic changes along ischemic time. Whether soluble biomarkers reflect the local gene environment and ischemic time is unclear. We explored whether circulating biomarkers were associated with corresponding coronary thrombi genes and total ischemic time. In 33 AMI patients undergoing percutaneous coronary intervention (PCI), blood samples were collected within 6-24h for markers related to plaque rupture (metalloproteinase 9, tissue inhibitor of metalloproteinases 1), platelet and endothelial cell activation (P-selectin, CD40 ligand, PAR-1), hemostasis (tissue factor, tissue plasminogen activator, plasminogen activator inhibitor 1, free and total tissue factor pathway inhibitor, D-dimer, prothrombin fragment 1+2), inflammation (interleukin 8 and 18, fractalkine, monocyte chemoattractant protein 1 (MCP-1), CXCL1, pentraxin 3, myeloperoxidase) and galectin 3, caspase 8 and epidermal growth factor (EGF). Laboratory analyses were performed by Proximity Extension Assay (Proseek Multiplex CVD I(96 × 96)), ELISAs and RT-PCR. Only circulating P-selectin correlated to the corresponding P-selectin gene expression in thrombi (r=0.530, p=0.002). Plasma galectin 3, fractalkine, MCP-1 and caspase 8 correlated inversely to ischemic time (r=-0.38-0.50, all p 4h) ischemic time (all p <0.05). The dynamic changes in circulating mediators along ischemic time were not reflected in the profile of locally expressed genes. These observations indicate a locally confined milieu within the site of atherothrombosis, which may be important for selective therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Platelet expression of stromal cell-derived factor-1 is associated with the degree of valvular aortic stenosis.

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    Thomas Wurster

    Full Text Available BACKGROUND AND PURPOSE: Platelet surface expression of stromal-cell-derived factor-1 (SDF-1 is increased during platelet activation and constitutes an important factor in hematopoetic progenitor cell trafficking at sites of vascular injury and ischemia. Enhanced platelet SDF-1 expression has been reported previously in patients suffering from acute coronary syndrome (ACS. We hypothesized that expression of platelet associated SDF-1 may also be influenced by calcified valvular aortic stenosis (AS. METHODS: We consecutively evaluated 941 patients, who were admitted to the emergency department with dyspnea and chest pain. Platelet surface expression of SDF-1 was determined by flow cytometry, AS was assessed using echocardiography and hemodynamic assessment by heart catheterization. A 1∶1 propensity score matching was implemented to match 218 cases with 109 pairs adjusting for age, sex, cardiovascular risk factors, and medication including ACE inhibitors, angiotensin receptor blockers, beta blockers, statins, aspirin, clopidogrel, GPIIb/IIIa antagonists, and vitamin K antagonists. RESULTS: Patients with valvular AS showed enhanced platelet SDF-1 expression compared to patients without AS (non-valvular disease, NV independent of ACS and stable coronary artery disease (SAP [mean fluorescence intensity (MFI for ACS (AS vs. NV: 75±40.4 vs. 39.5±23.3; P = 0.002; for SAP (AS vs. NV: 54.9±44.6 vs. 24.3±11.2; P = 0.008]. Moreover, the degree of AS significantly correlated with SDF-1 platelet surface expression (r = 0.462; P = 0.002. CONCLUSIONS: Valvular AS is associated with enhanced platelet-SDF-1 expression; moreover the degree of valvular AS correlates with SDF-1 platelet surface expression. These findings may have clinical implications in the future.

  17. Platelet expression of stromal cell-derived factor-1 is associated with the degree of valvular aortic stenosis. (United States)

    Wurster, Thomas; Tegtmeyer, Roland; Borst, Oliver; Rath, Dominik; Geisler, Tobias; Gawaz, Meinrad; Bigalke, Boris


    Platelet surface expression of stromal-cell-derived factor-1 (SDF-1) is increased during platelet activation and constitutes an important factor in hematopoetic progenitor cell trafficking at sites of vascular injury and ischemia. Enhanced platelet SDF-1 expression has been reported previously in patients suffering from acute coronary syndrome (ACS). We hypothesized that expression of platelet associated SDF-1 may also be influenced by calcified valvular aortic stenosis (AS). We consecutively evaluated 941 patients, who were admitted to the emergency department with dyspnea and chest pain. Platelet surface expression of SDF-1 was determined by flow cytometry, AS was assessed using echocardiography and hemodynamic assessment by heart catheterization. A 1∶1 propensity score matching was implemented to match 218 cases with 109 pairs adjusting for age, sex, cardiovascular risk factors, and medication including ACE inhibitors, angiotensin receptor blockers, beta blockers, statins, aspirin, clopidogrel, GPIIb/IIIa antagonists, and vitamin K antagonists. Patients with valvular AS showed enhanced platelet SDF-1 expression compared to patients without AS (non-valvular disease, NV) independent of ACS and stable coronary artery disease (SAP) [mean fluorescence intensity (MFI) for ACS (AS vs. NV): 75±40.4 vs. 39.5±23.3; P = 0.002; for SAP (AS vs. NV): 54.9±44.6 vs. 24.3±11.2; P = 0.008]. Moreover, the degree of AS significantly correlated with SDF-1 platelet surface expression (r = 0.462; P = 0.002). Valvular AS is associated with enhanced platelet-SDF-1 expression; moreover the degree of valvular AS correlates with SDF-1 platelet surface expression. These findings may have clinical implications in the future.

  18. Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study. (United States)

    George, Magdalene M; Li, S David; Mindikoglu, Ayse L; Baluch, Mehdi H; Dhillon, Sonu; Farr, Deborah; Van Thiel, David H


    The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN naïve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.

  19. Changes in platelet morphology and function during 24 hours of storage. (United States)

    Braune, S; Walter, M; Schulze, F; Lendlein, A; Jung, F


    For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet

  20. Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease. (United States)

    Patzelt, J; Mueller, K A L; Breuning, S; Karathanos, A; Schleicher, R; Seizer, P; Gawaz, M; Langer, H F; Geisler, T


    Inhibition of components of the complement system or of its receptors has been postulated as a concept for primary and secondary prevention in atherosclerosis and was applied in clinical trials. Although the anaphylatoxin-receptors C3aR and C5aR are commonly associated with inflammatory cells, in vitro studies suggested their expression also on platelets. Expression levels of C3aR and C5aR were measured by flow cytometry in a collective of 302 patients with documented coronary artery disease (CAD) including patients with stable CAD (n = 152), unstable angina (n = 54), acute myocardial infarction (AMI; Non-ST elevation myocardial infarction, n = 70, ST elevation MI, n = 26) or healthy controls (n = 21). Patients with stable CAD, unstable angina or AMI had significantly higher expression of C5aR on platelets in comparison to healthy controls (MFI 14.68 (5.2), 14.56 (5.18) and 13.34 (4.52) versus 10.68 (3.1)); p  0.001 for C3aR, r = 0.76 for C5aR, p < 0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1. In summary, we observed a positive correlation between the expression of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with CAD. Further investigations are needed to study the clinical and mechanistic relevance of these findings. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Clinical Safety of a High Dose of Phycocyanin-Enriched Aqueous Extract from Arthrospira (Spirulina) platensis: Results from a Randomized, Double-Blind, Placebo-Controlled Study with a Focus on Anticoagulant Activity and Platelet Activation. (United States)

    Jensen, Gitte S; Drapeau, Cassandra; Lenninger, Miki; Benson, Kathleen F


    The goal for this study was to evaluate safety regarding anticoagulant activity and platelet activation during daily consumption of an aqueous cyanophyta extract (ACE), containing a high dose of phycocyanin. Using a randomized, double-blind, placebo-controlled study design, 24 men and women were enrolled after informed consent, and consumed either ACE (2.3 g/day) or placebo daily for 2 weeks. The ACE dose was equivalent to ∼1 g phycocyanin per day, chosen based on the highest dose Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration. Consuming ACE did not alter markers for platelet activation (P-selectin expression) or serum P-selectin levels. No changes were seen for activated partial thromboplastin time, thrombin clotting time, or fibrinogen activity. Serum levels of aspartate transaminase (AST) showed a significant reduction after 2 weeks of ACE consumption (P < .001), in contrast to placebo where no changes were seen; the difference in AST levels between the two groups was significant at 2 weeks (P < .02). Reduced levels of alanine transaminase (ALT) were also seen in the group consuming ACE (P < .08). Previous studies showed reduction of chronic pain when consuming 1 g ACE per day. The higher dose of 2.3 g/day in this study was associated with significant reduction of chronic pain at rest and when physically active (P < .05). Consumption of ACE showed safety regarding markers pertaining to anticoagulant activity and platelet activation status, in conjunction with rapid and robust relief of chronic pain. Reduction in AST and ALT suggested improvement in liver function and metabolism.

  2. Assessment of the influence of the inflammatory process on the activation of blood platelets and morphological parameters in patients with ulcerative colitis (colitis ulcerosa

    Directory of Open Access Journals (Sweden)

    Halina Kemona


    Full Text Available Ulcerative colitis (colitis ulcerosa is a non-specific inflammatory bowel disease of unknown etiology. The symptoms which are observed in the course of ulcerative colitis are: an increase in the number of leukocytes and blood platelets, an increase in the concentration of IL-6 and anemia. Blood platelets are the key element, linking the processes of hemostasis, inflammation and the repair of damaged tissues. Activation of blood platelets is connected with changes in their shape and the occurrence of the reaction of release. P-selectin appears on the surfaces of activated blood platelets and the concentration level of soluble P-selectin increases in the blood plasma. The aim of this study was to define whether the increased number of blood platelets in patients with ulcerative colitis accompanies changes in their activation and morphology. A total of 16 subjects with ulcerative colitis and 32 healthy subjects were studied. Mean platelet count, morphological parameters of platelets and MPC were measured using an ADVIA 120 hematology analyzer. Concentrations of sP-selectin and IL-6 in serum were marked by immunoassay (ELISA. MPC, concentration of sP-selectin and IL-6 were significantly higher in subjects with ulcerative colitis compared to those in the healthy group. There was a decrease of MPV in patients with ulcerative colitis, which is statistically significant. Chronic inflammation in patients with ulcerative colitis causes an increase in the number of blood platelets, a change in their morphology and activation. Decreased MPV value reflects activation and the role blood platelets play in the inflammatory process of the mucous membrane of the colon. A high concentration of sP-selectin, which is a marker of blood platelet activation, demonstrates their part in the inflammatory process. The increase in the concentration of sP-selectin correlated positively with the increase in concentration of IL-6. This is why it may be a useful marker

  3. Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression. (United States)

    McCarrel, Taralyn; Fortier, Lisa


    Platelet-rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet alpha-granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF-beta1 and PDGF-BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT-PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases-3 and 13 (MMP-3, MMP-13) was performed. TGF-beta1 and PDGF-BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF-beta1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP-13 expression. BMA resulted in decreased COMP and increased MMP-3 and MMP-13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP-13, and MMP-3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. Copyright 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  4. Relevance of angiopoietin-2 and soluble P-selectin levels in patients with pulmonary arterial hypertension receiving combination therapy with oral treprostinil: a FREEDOM-C2 biomarker substudy (United States)

    Schermuly, Ralph; Seeger, Werner; Rao, Youlan; Ghofrani, Hossein A.; Gall, Henning


    Abstract Studies have suggested roles for angiopoietin-2 (Ang-2) and soluble P-selectin (sP-selectin) as biomarkers of disease severity and treatment response in pulmonary arterial hypertension (PAH), but additional data are required for validation. We evaluated these biomarkers using data from FREEDOM-C2, in which patients with PAH receiving stable monotherapy or combination therapy were randomized to receive additional treatment with oral treprostinil (up-titrated from 0.25 mg twice daily) or placebo for 16 weeks. Biomarker analysis was optional in FREEDOM-C2. We measured plasma Ang-2 and sP-selectin levels at baseline and at week 16, and we assessed their association with predefined outcomes (6-minute walk distance [6MWD] change from baseline >40 m, 6MWD >380 m, functional class I/II, and/or N-terminal pro-brain natriuretic peptide [NT-proBNP] <1,800 pg/mL at week 16) using Spearman correlation, receiver operating characteristics, and logistic regression. Biomarker data were available for 83 of 157 and 95 of 153 patients in the oral treprostinil and placebo groups, respectively. In the oral treprostinil group, baseline Ang-2 levels correlated with week 16 NT-proBNP levels (P < 0.0001). Baseline Ang-2 ≥12 ng/mL was associated with a reduced likelihood of having NT-proBNP <1,800 pg/mL at week 16 (multivariate odds ratio: 0.08; 95% confidence interval: 0.02–0.32). However, Ang-2 showed no significant association with the other assessed outcomes, and sP-selectin was not associated or correlated with any of the outcomes. These data suggest that Ang-2 and sP-selectin are not associated with response to oral treprostinil in patients already receiving stable PAH therapy. Trial registration: identifier NCT00887978. PMID:28090293

  5. Effects of omega-3 polyunsaturated fatty acids on platelet function in healthy subjects and subjects with cardiovascular disease. (United States)

    McEwen, Bradley J; Morel-Kopp, Marie-Christine; Chen, Walter; Tofler, Geoffrey H; Ward, Christopher M


    Hyperactivation and aggregation of platelets play a major role in thrombosis and hemostasis. The aims of this study were to investigate the effects of omega-3 polyunsaturated fatty acids (PUFAs) on platelet function. Light transmission aggregometry and flow cytometric analyses of platelet activation and platelet-leukocyte aggregates were determined at baseline and after 4 weeks of omega-3 (docosahexaenoic acid 520 mg and eicosapentaenoic acid 120 mg) supplementation. In total, 40 healthy subjects and 16 patients with a history of cardiovascular disease (CVD) completed the study. In healthy subjects, omega-3 PUFA significantly reduced adenosine diphosphate (ADP)-induced (maximum amplitude, 77.0% ± 3.2% vs. 71.6% ± 3.4%, p = 0.036; maximum slope, 86.3 ± 1.8 vs. 80.7 ± 2.1, p = 0.014) and adrenaline-induced platelet aggregation (maximum slope, 42.8 ± 2.7 vs. 37.4 ± 3.0, p = 0.013; lag time, 00:21 ± 00:02 vs. 00:31 ± 00:03 s, p = 0.002). Omega-3 PUFA also reduced P-selectin expression (40.5% ± 2.9% vs. 34.4% ± 2.4%, p = 0.049) on platelets and platelet-monocyte aggregates (38.5% ± 2.6% vs. 31.4% ± 2.5%, p = 0.022) after activation with ADP 0.5 µM. There were fewer changes in platelet aggregation and activation found in subjects with CVD. Nevertheless, there was a reduction in the slope of arachidonic acid-induced platelet aggregation (13.21 ± 6.41 vs. 4.88 ± 3.01, p = 0.009) and increased lag time for U46619 (00:16 ± 00:00 vs. 00:29 ± 00:07 s, p = 0.018) induced platelet aggregation. Thus, 4-week supplementation of 640 mg omega-3 PUFA reduced measures of platelet aggregation and activation in healthy subjects but effects were less evident in patients with existing CVD. Our findings support the recommendation that the omega-3 PUFA dose be higher in CVD than among healthy subjects. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  6. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

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    Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)


    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  7. Morphological and Functional Platelet Abnormalities in Berkeley Sickle Cell Mice (United States)

    Shet, Arun S.; Hoffmann, Thomas J.; Jirouskova, Marketa; Janczak, Christin A.; Stevens, Jacqueline R.M.; Adamson, Adewole; Mohandas, Narla; Manci, Elizabeth A.; Cynober, Therese; Coller, Barry S.


    Berkeley sickle cell mice are used as an animal model of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37 ± 3.2 vs. 27 ± 1.4, mean ± SD; p Howell-Jolly bodies and “pocked” erythrocytes (p <0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5 ± 1 % vs. 1 ± 1%; p <0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease. PMID:18374611

  8. Effects of intensive glucose control on platelet reactivity in patients with acute coronary syndromes. Results of the CHIPS Study ("Control de Hiperglucemia y Actividad Plaquetaria en Pacientes con Sindrome Coronario Agudo"). (United States)

    Vivas, David; García-Rubira, Juan C; Bernardo, Esther; Angiolillo, Dominick J; Martín, Patricia; Calle-Pascual, Alfonso; Núñez-Gil, Iván; Macaya, Carlos; Fernández-Ortiz, Antonio


    Hyperglycaemia has been associated with increased platelet reactivity and impaired prognosis in patients with acute coronary syndrome (ACS). Whether platelet reactivity can be reduced by lowering glucose in this setting is unknown. The aim of this study was to assess the functional impact of intensive glucose control with insulin on platelet reactivity in patients admitted with ACS and hyperglycaemia. This is a prospective, randomised trial evaluating the effects of either intensive glucose control (target glucose 80-120 mg/dl) or conventional control (target glucose 180 mg/dl or less) with insulin on platelet reactivity in patients with ACS and hyperglycaemia. The primary endpoint was platelet aggregation following stimuli with 20 μM ADP at 24 h and at hospital discharge. Aggregation following collagen, epinephrine and thrombin receptor-activated peptide, as well as P2Y₁₂ reactivity index and surface expression of glycoprotein IIb/IIIa and P-selectin were also measured. Of the 115 patients who underwent random assignment, 59 were assigned to intensive and 56 to conventional glucose control. Baseline platelet functions and inhospital management were similar in both groups. Maximal aggregation after ADP stimulation at hospital discharge was lower in the intensive group (47.9 ± 13.2% vs 59.1 ± 17.3%; p=0.002), whereas no differences were found at 24 h. Similarly all other parameters of platelet reactivity measured at hospital discharge were significantly reduced in the intensive glucose control group. In this randomised trial, early intensive glucose control with insulin in patients with ACS presenting with hyperglycaemia was found to decrease platelet reactivity. Clinical Trial Registration Number

  9. Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects (United States)

    Floyd, Christopher N; Goodman, Timothy; Becker, Silke; Chen, Nan; Mustafa, Agnesa; Schofield, Emma; Campbell, James; Ward, Malcolm; Sharma, Pankaj; Ferro, Albert


    Aims Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. Methods Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. Results In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. Conclusions In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker. PMID:25099258

  10. Localization of T and B Lymphocytes to the White Pulp of the Spleen is Independent of L-, E-, and P-Selectin

    Directory of Open Access Journals (Sweden)

    Mitchell H. Grayson


    Full Text Available T and B cell interactions are thought to be of prime importance in the generation of a humoral immune response. These interactions are thought to take place in the secondary lymphoid organs. The largest of which is the spleen. While the pathways involved in lymphocyte migration into other secondary lymphoid organs have been unraveled, very little is understood about T and B cell migration to the spleen. We report that adoptively transferred T lymphocytes appear more rapidly within the lymphoid compartment of the spleen than do B lymphocytes. Indeed, half of the transferred T lymphocytes in the spleen appear within the white pulp by 1.4 hours. B lymphocytes take nearly 4.3 hours to achieve the same level of accumulation. In addition, T lymphocyte arrival is fucoidan sensitive, while B cells are not affected by this polysaccharide. Finally, we show that neither L-, E-, or P-selectin appears to play a significant role in the accumulation of lymphocytes in the white pulp.

  11. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease. (United States)

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K; Gordeuk, Victor R; Cho, Jaehyung


    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease. Copyright© Ferrata Storti Foundation.

  12. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  13. Rebound platelet activation after termination of prasugrel and aspirin therapy due to confirmed non-compliance in patient enrolled in the JUMBO Trial. (United States)

    Serebruany, V L; Midei, M G; Meilman, H; Malinin, A I; Lowry, D R


    )Ib, GPIIb/IIIa activity, P-selectin, protease activated receptor (PAR)-1 thrombin receptor (activated and intact epitopes), and thrombospondin expression. The clinical benefits of antiplatelet agents are not only denied in NC outpatients, but may put them at additional risk for worsened vascular outcomes due to the rebound platelet activation. Proclaimed 'resistance' to antiplatelet agents may at least in part be a result of NC, especially in the chronic uncontrolled setting. Enforcing compliance will improve outcomes in the clinical trials, and save lives of patients really receiving antiplatelet therapy.

  14. Treatment with a histone deacetylase inhibitor, valproic acid, is associated with increased platelet activation in a large animal model of traumatic brain injury and hemorrhagic shock

    DEFF Research Database (Denmark)

    Dekker, Simone E; Sillesen, Martin; Bambakidis, Ted


    left in shock for 2 h before resuscitation with either FFP or FFP + VPA (300 mg/kg). Serum levels of platelet activation markers transforming growth factor beta, CD40 L, P-selectin, and platelet endothelial cell adhesion molecule (PECAM) 1 were measured at baseline, postresuscitation, and after a 6-h......, immunohistochemistry data showed elevated P-selectin in the VPA treatment group (22.30 ± 10.39% versus 8.125 ± 3.94%, P ....05). Circulating transforming growth factor beta levels were elevated in the FFP + VPA group, but this did not reach statistical significance (11.20 ± 1.46 versus 8.09 ± 1.41 ng/mL; P = 0.17). Brain platelet endothelial cell adhesion molecule 1 levels were significantly lower in the FFP + VPA group compared...

  15. The Effect of a Simulated Commercial Flight Environment with Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants with Type 2 Diabetes – A Cross-over Study

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    Judit Konya


    Full Text Available AimsTo determine if clotting, platelet, and endothelial function were affected by simulated short-haul commercial air flight conditions (SF in participants with type 2 diabetes (T2DM compared to controls.Methods10 participants with T2DM (7 females, 3 males and 10 controls (3 females, 7 males completed the study. Participants were randomized to either spend 2 h in an environmental chamber at sea level conditions (temperature: 23°C, oxygen concentration 21%, humidity 45%, or subject to a simulated 2-h simulated flight (SF: temperature: 23°C, oxygen concentration 15%, humidity 15%, and crossed over 7 days later. Main outcome measures: clot formation and clot lysis parameters, functional platelet activation markers, and endothelial function measured by reactive hyperemia index (RHI by EndoPAT and serum microparticles.ResultsComparing baseline with SF conditions, clot maximal absorption was increased in controls (0.375 ± 0.05 vs. 0.39 ± 0.05, p < 0.05 and participants with T2DM (0.378 ± 0.089 vs. 0.397 ± 0.089, p < 0.01, while increased basal platelet activation for both fibrinogen binding and P-selectin expression (p < 0.05 was seen in participants with T2DM. Parameters of clot formation and clot lysis, stimulated platelet function (stimulated platelet response to ADP and sensitivity to prostacyclin, and endothelial function were unchanged.ConclusionWhile SF resulted in the potential of denser clot formation with enhanced basal platelet activation in T2DM, the dynamic clotting, platelet, and endothelial markers were not affected, suggesting that short-haul commercial flying adds no additional hazard for venous thromboembolism for participants with T2DM compared to controls.

  16. Mechanisms of aggregation inhibition by aspirin and nitrate-aspirin prodrugs in human platelets. (United States)

    Harmon, Shona; Inkielewicz-Stepniak, Iwona; Jones, Michael; Ledwidge, Mark; Santos-Martinez, Maria Jose; Medina, Carlos; Radomski, Marek W; Gilmer, John F


    Aspirin is the mainstay of anti-platelet therapy in the secondary prevention of cardiovascular disease. However, problems with aspirin safety and resistance demand clinical strategies based on multiple pharmacological approaches. Prodrugs of aspirin may offer beneficial effects in terms of gastro-intestinal safety and multiple pharmacological approaches. However, the pharmacological profile of aspirin prodrugs in human platelets has not been completed yet. We aimed to compare the effects of aspirin and prodrugs of aspirin (1-5) on human platelet aggregation stimulated by ADP and collagen and associated receptor expression (GPIIb/IIIa and P-selectin) in platelet-rich plasma (PRP) and washed platelets (WP). As aspirin is released from prodrugs following esterase hydrolysis we studied the expression and activity of butyrylcholineterase (BuChE) and carboxyesterase (CE) in plasma and platelets. The mechanism of prodrug-induced platelet aggregation inhibition was explored by studying the effects of plasma and purified human BuChE on aggregation. Finally, the relative contribution of nitric oxide (NO) bioactivity to nitrate-containing prodrugs of aspirin-induced inhibition of aggregation was determined using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ,) a selective inhibitor of the soluble guanylyl cyclase. ST0702, 2, a nicotinic acid-aspirin codrug was equipotent with aspirin with respect to inhibition of collagen-induced platelet aggregation. Compound 4, a NO releasing aspirin was the most potent inhibitor of ADP-induced platelet aggregation, an effect partially reversed by ODQ. The platelet inhibitory effects of aspirin prodrugs were time-dependent as the maximal inhibitory effects against collagen-induced aggregation were achieved by aspirin at 2 min, 1 at 5 min and ST0702 at 15 min. The aspirin prodrugs were significantly less potent in WP than in PRP and the reverse was true of aspirin. In the presence of complete BuChE inhibition in PRP, there was almost

  17. Up-Regulated Expression of Matrix Metalloproteinases in Endothelial Cells Mediates Platelet Microvesicle-Induced Angiogenesis. (United States)

    Sun, Cheng; Feng, Shi-Bin; Cao, Zheng-Wang; Bei, Jun-Jie; Chen, Qiang; Zhao, Wei-Bo; Xu, Xian-Jie; Zhou, Zhou; Yu, Zheng-Ping; Hu, Hou-Yuan


    Platelet microvesicles (PMVs) contribute to angiogenesis and vasculogenesis, but the mechanisms underlying these contributions have not been fully elucidated. In the present study, we investigated whether PMVs regulate the angiogenic properties of endothelial cells (ECs) via mechanisms extending beyond the transport of angiogenic regulators from platelets. In vitro Matrigel tube formation assay and in vivo Matrigel plug assay were used to evaluate the pro-angiogenic activity of PMVs. The effects of PMVs on the migration of human umbilical vein endothelial cells (HUVECs) were detected by transwell assay and wound-healing assay. Real-time PCR and western blot were conducted to examine mRNA and protein expression of pro-angiogenic factors in HUVECs. Matrix metalloproteinase (MMP) activity was assayed by gelatin zymography. Moreover, the effects of specific MMP inhibitors were tested. PMVs promoted HUVEC capillary-like network formation in a dose-dependent manner. Meanwhile, PMVs dose-dependently facilitated HUVEC migration. Levels of MMP-2 and MMP-9 expression and activity were up-regulated in HUVECs stimulated with PMVs. Inhibition of MMPs decreased their pro-angiogenic and pro-migratory effects on HUVECs. Moreover, we confirmed the pro-angiogenic activity of PMVs in vivo in mice with subcutaneous implantation of Matrigel, and demonstrated that blockade of MMPs attenuated PMV-induced angiogenesis. The findings of our study indicate that PMVs promote angiogenesis by up-regulating MMP expression in ECs via mechanism extending beyond the direct delivery of angiogenic factors. © 2017 The Author(s). Published by S. Karger AG, Basel.

  18. Airway epithelial platelet-activating factor receptor expression is markedly upregulated in chronic obstructive pulmonary disease (United States)

    Shukla, Shakti Dhar; Sohal, Sukhwinder Singh; Mahmood, Malik Quasir; Reid, David; Muller, Hans Konrad; Walters, Eugene Haydn


    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 in both COPD-smokers (P<0.005) and COPD-ex-smokers (P<0.002) compared to smokers with normal lung function. There was little evidence that PAFr expression was affected by ICS therapy over 6 months. Conclusion Epithelial PAFr expression is upregulated in smokers, especially in those with COPD, and is not obviously affected by ICS therapy. PMID:25143722

  19. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients. (United States)

    Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang


    Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

  20. Platelet-mediated mesenchymal stem cells homing to the lung reduces monocrotaline-induced rat pulmonary hypertension. (United States)

    Jiang, Lei; Song, Xing Hui; Liu, Pu; Zeng, Chun Lai; Huang, Zhang Sen; Zhu, Lin Jing; Jiang, Yang Zi; Ouyang, Hong Wei; Hu, Hu


    Bone marrow mesenchymal stem cell (BM-MSC) transplantation has been suggested to be a promising method for the treatment of pulmonary arterial hypertension (PAH), a fatal disease currently without effective preventive/therapeutic strategies. However, the detailed mechanisms underlying BM-MSC therapy are largely unknown. We designed the present study to test the hypothesis that circulating platelets facilitate BM-MSC homing to the lung vasculature in a rat model of PAH induced by monocrotalin (MCT). A single subcutaneous administration of MCT induced a marked rise in right ventricular systolic pressure (RVSP) and the weight ratio of right to left ventricle plus septum (RV/LV+S) 3 weeks after injection. The injection of MSCs via tail vein 3 days after MCT significantly reduced the increase of RVSP and RV/LV+S. The fluorescence-labeled MSCs injected into the PAH rat circulation were found mostly distributed in the lungs, particularly on the pulmonary vascular wall, whereas cell homing was abolished by an anti-P-selectin antibody and the GPIIb/IIIa inhibitor tirofiban. Furthermore, using an in vitro flow chamber, we demonstrated that MSC adhesion to the major extracellular matrix collagen was facilitated by platelets and their P-selectin and GPIIb/IIIa. Therefore, the current study suggested that platelet-mediated MSC homing prevented the aggravation of MCT-induced rat PAH, via P-selectin and GPIIb/IIIa-mediated mechanisms.

  1. Platelet bound oxLDL shows an inverse correlation with plasma anaphylatoxin C5a in patients with coronary artery disease. (United States)

    Nording, Henry; Giesser, Annika; Patzelt, Johannes; Sauter, Reinhard; Emschermann, Frederic; Stellos, Konstantinos; Gawaz, Meinrad; Langer, Harald F


    Both oxidized lipids as well as the complement system contribute to atherothrombosis. The expression of complement receptors correlates with the expression of platelet activation markers, and platelet bound oxidized low-density lipoprotein (oxLDL) modulates platelet function. In the present study, we investigated the relationship of markers of complement activation, the anaphylatoxins C5a and C3a, and oxidized low-density lipoprotein. Two hundred and seven patients with coronary artery disease (CAD) were analyzed in this study. Using enzyme-linked immunosorbent assays, plasma levels of oxLDL, C3a, and C5a were measured. Moreover, we assessed platelet bound oxLDL by flow cytometry. The overall level of C5a in the troponin negative group (stable angina (SA) and unstable angina (UA)) compared to the troponin positive group (non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI)) did not differ significantly (62.7 ± 32.4 ng/ml versus 65.8 ± 40.3 ng/ml). While C5a and C3a showed a significant correlation with each other (r = 0.25, p < 0.001), there was no statistically significant relationship between C3a and platelet bound oxLDL (r = 0.06, p = 0.37). Furthermore, plasma oxLDL did not correlate with either C3a or C5a. However, we observed a moderate, yet significant negative correlation between plasma C5a and platelet bound oxLDL (r = -0.15, p = 0.04). Partial correlation analysis correcting for the presence of acute coronary syndrome (ACS), troponin status or the subgroups SA, UA, NSTEMI, or STEMI did not alter this correlation substantially. Interestingly, flow cytometric analysis of human platelets showed increased expression of C5aR and P-selectin after in vitro stimulation with oxLDL. In conclusion, the complement anaphylatoxin C5a shows an inverse correlation with platelet bound oxLDL. The relationship of oxidized lipids to particular complement components may add to the platelet-lipid interplay in atherogenesis and

  2. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15 mRNA in Various Human Cell Types

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    Sang Mee Hwang


    Full Text Available CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC, two peripheral bloods (PB, 12 granulocyte products, natural killer (NK-92, B-lymphocyte (CO88BV59-1, K-562 leukemia cell line, human embryonic stem cell (hESC, and human fibroblasts (HF. HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

  3. ExoU-induced vascular hyperpermeability and platelet activation in the course of experimental Pseudomonas aeruginosa pneumosepsis. (United States)

    Machado, Gloria-Beatriz S; de Assis, Maria-Cristina; Leão, Robson; Saliba, Alessandra M; Silva, Mauricio C A; Suassuna, Jose H; de Oliveira, Albanita V; Plotkowski, Maria-Cristina


    To address the question whether ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, can induce hemostatic abnormalities during the course of pneumosepsis, mice were instilled i.t. with the ExoU-producing PA103 P. aeruginosa or with a mutant obtained by deletion of the exoU gene. Control animals were instilled with sterile vehicle. To assess the role of ExoU in animal survival, mice were evaluated for 72 h. In all the other experiments, animals were studied at 24 h after infection. PA103-infected mice showed significantly higher mortality rate, lower blood leukocyte concentration, and higher platelet concentration and hematocrit than animals infected with the bacterial mutant, as well as evidences of increased vascular permeability and plasma leakage, which were confirmed by our finding of higher protein concentration in bronchoalveolar lavage fluids and by the Evans blue dye assay. Platelets from PA103-infected mice demonstrated features of activation, assessed by the flow cytometric detection of higher percentage of P-selectin expression and of platelet-derived microparticles as well as by the enzyme immunoassay detection of increased thromboxane A2 concentration in animal plasma. Histopathology of lung and kidney sections from PA103-infected mice exhibited evidences of thrombus formation that were not detected in sections of animals from the other groups. Our results demonstrate the ability of ExoU to induce vascular hyperpermeability, platelet activation, and thrombus formation during P. aeruginosa pneumosepsis, and we speculate that this ability may contribute to the reported poor outcome of patients with severe infection by ExoU-producing P. aeruginosa.

  4. Analysis of platelet-derived extracellular vesicles in plateletpheresis concentrates: a multicenter study. (United States)

    Black, Anne; Orsó, Evelyn; Kelsch, Reinhard; Pereira, Melanie; Kamhieh-Milz, Julian; Salama, Abdulgabar; Fischer, Michael B; Meyer, Eduardo; Frey, Beat M; Schmitz, Gerd


    Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers. Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry. During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p < 0.001) and PL-EVs increased in 74 PCs (A, C-E; p < 0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage. PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques. © 2017 AABB.

  5. Proteomic discovery of 21 proteins expressed in human plasma-derived but not platelet-derived microparticles. (United States)

    Smalley, David M; Root, Karen E; Cho, Hyungjun; Ross, Mark M; Ley, Klaus


    Microparticles (MPs) are small membrane vesicles generated by essentially all cell types. In the plasma, most MPs are derived from platelets, but those from other sources, particularly leukocytes (macrophages, lymphocytes, and neutrophils), endothelial cells, and even smooth muscle cells can be detected and appear to play an important role in normal physiology and various diseases. In previous work we analyzed the proteome of MPs generated from isolated platelets (platelet MPs). Here, we report on a comparative analysis of microparticles isolated from plasma (plasma MPs) versus platelet MP using two complementary methods of comparative analysis. The first method, spectral count analysis, yielded 21 proteins detected in plasma MPs (with a total spectral count of 10 or greater) that were essentially absent in platelet MPs (with a total spectral count of 1 or 0). An additional two proteins (von Willebrand Factor, albumin) were present in both types of MPs but enriched in the plasma MPs. The second method, isotope-coded affinity tag (ICAT) labeling of proteins, supported the spectral count results for the more abundant proteins and provided better relative quantitation of differentially expressed proteins. Proteins present only in the plasma MPs include several associated with apoptosis (CD5-like antigen, galectin 3 binding protein, several complement components), iron transport (transferrin, transferrin receptor, haptoglobin), immune response (complement components, immunoglobulin J and kappa chains), and the coagulation process (protein S, coagulation factor VIII).

  6. Leukocyte-Reduced Platelet-Rich Plasma Alters Protein Expression of Adipose Tissue-Derived Mesenchymal Stem Cells. (United States)

    Loibl, Markus; Lang, Siegmund; Hanke, Alexander; Herrmann, Marietta; Huber, Michaela; Brockhoff, Gero; Klein, Silvan; Nerlich, Michael; Angele, Peter; Prantl, Lukas; Gehmert, Sebastian


    Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue-derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. Adipose tissue-derived mesenchymal stem cells were cultured in α-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum. Cell proliferation was assessed with cell cycle kinetics using flow cytometric analyses after 48 hours. Differences in proteome expression of the adipose tissue-derived mesenchymal stem cells were analyzed using a reverse-phase protein array to quantify 214 proteins. Complementary Ingenuity Pathways Analysis and gene set enrichment analysis were performed using protein data, and confirmed by Western blot analysis. A higher percentage of adipose tissue-derived mesenchymal stem cells in the S phase in the presence of platelet-rich plasma advocates the proliferation stimulation. Ingenuity Pathways Analysis and gene set enrichment analysis confirm the involvement of the selected proteins in the process of cell growth and proliferation. Ingenuity Pathways Analysis revealed a participation in the top-ranked canonical pathways PI3K/AKT, PTEN, ILK, and IGF-1. Gene set enrichment analysis identified the authors' protein set as being part of significantly regulated protein sets with the focus on cell cycle, metabolism, and the Kyoto Encyclopedia of Genes and Genomes transforming growth factor-β signaling pathway. The present study provides evidence that platelet-rich plasma stimulates proliferation and induces a unique change in the proteomic profile of adipose tissue-derived mesenchymal stem cells. The interpretation of altered expression of regulatory proteins represents a step forward toward achieving good manufacturing practice-compliant criteria

  7. Unfractionated and low-molecular-weight heparin and the phosphodiesterase inhibitors, IBMX and cilostazol, block ex vivo Equid Herpesvirus type-1-induced platelet activation

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    Tracy Stokol


    Full Text Available Equid herpes virus type-1 (EHV-1 is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins or platelet inhibitors that impede post-receptor thrombin signaling (phosphodiesterase [PDE] antagonists would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque forming unit/cell in the presence or absence of unfractionated heparin (UFH, low-molecular-weight (LMWH heparin or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1 to 0.2 U/mL anti-Factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX or isoenzyme-3 selective (cilostazol PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

  8. Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice (United States)

    Woulfe, Donna S.; Lilliendahl, Joanne Klimas; August, Shelley; Rauova, Lubica; Kowalska, M. Anna; Åbrink, Magnus; Pejler, Gunnar; White, James G.


    Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG−/− mice. Wild-type (WT) but not SG−/− platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal α-granule structure in SG−/− platelets. However, SG−/− platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG−/− megakaryocytes showed extensive emperipolesis of neutrophils. SG−/− platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG−/− platelets. The α-granule proteins platelet factor 4, β-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG−/− platelets. Exposure of P-selectin and αIIb after thrombin treatment was similar in WT and SG−/− platelets. SG−/− mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG−/− platelet function deficiencies are related to inadequate packaging and secretion of selected α-granule proteins and reduced secretion of dense granule contents critical for platelet activation. PMID:18094327

  9. Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide. (United States)

    Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E


    Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

  10. Long-term untreated streptozotocin-diabetes leads to increased expression and elevated activity of prostaglandin H2 synthase in blood platelets. (United States)

    Siewiera, Karolina; Kassassir, Hassan; Talar, Marcin; Wieteska, Lukasz; Watala, Cezary


    In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes.

  11. Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension. (United States)

    Perros, Frédéric; Montani, David; Dorfmüller, Peter; Durand-Gasselin, Ingrid; Tcherakian, Colas; Le Pavec, Jérôme; Mazmanian, Michel; Fadel, Elie; Mussot, Sacha; Mercier, Olaf; Hervé, Philippe; Emilie, Dominique; Eddahibi, Saadia; Simonneau, Gérald; Souza, Rogério; Humbert, Marc


    Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. To analyze the expression and pathogenic role of PDGF in idiopathic PAH. PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH. Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-beta. The effects of imatinib on PDGF-B-induced proliferation and chemotaxis were tested on human PASMCs. PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. PDGF-BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. PDGF may play an important role in human PAH. Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

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

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    Shukla SD


    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

  13. Influence of HbA1c levels on platelet function profiles associated with tight glycemic control in patients presenting with hyperglycemia and an acute coronary syndrome. A subanalysis of the CHIPS Study ("Control de HIperglucemia y Actividad Plaquetaria en Pacientes con Síndrome Coronario Agudo"). (United States)

    Vivas, David; García-Rubira, Juan C; Bernardo, Esther; Angiolillo, Dominick J; Martín, Patricia; Calle-Pascual, Alfonso; Núñez-Gil, Iván; Macaya, Carlos; Fernández-Ortiz, Antonio


    Patients with hyperglycemia, an acute coronary syndrome and poor glycemic control have increased platelet reactivity and poor prognosis. However, it is unclear the influence of a tight glycemic control on platelet reactivity in these patients. This is a subanalysis of the CHIPS study. This trial randomized patients with hyperglycemia to undergo an intensive glucose control (target blood glucose 80-120 mg/dL), or conventional glucose control (target blood glucose <180 mg/dL). We analyzed platelet function at discharge on the subgroup of patients with poor glycemic control, defined with admission levels of HbA1c higher than 6.5%. The primary endpoint was maximal platelet aggregation following stimuli with 20 μM ADP. We also measured aggregation following collagen, epinephrine, and thrombin receptor-activated peptide, as well as P2Y12 reactivity index and surface expression of glycoprotein IIb/IIIa and P-selectin. A total of 67 patients presented HbA1c ≥ 6.5% (37 intensive, 30 conventional), while 42 had HbA1c < 6.5% (20 intensive, 22 conventional). There were no differences in baseline characteristics between groups. At discharge, patients with HbA1c ≥6.5% had significantly reduced MPA with intensive glucose control compared with conventional control (46.1 ± 22.3 vs. 60.4 ± 20.0%; p = 0.004). Similar findings were shown with other measures of platelet function. However, glucose control strategy did not affect platelet function parameters in patients with HbA1c < 6.5%. Intensive glucose control in patients presenting with an acute coronary syndrome and hyperglycemia results in a reduction of platelet reactivity only in the presence of elevated HbA1c levels.

  14. Effect of irradiation on gene expression of rat liver adhesion molecules. In vivo and in vitro studies

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    Moriconi, Federico; Malik, Ihtzaz; Ahmad, Ghayyor; Dudas, Joszef; Ramadori, Giuliano [Dept. of Gastroenterology and Endocrinology, Goettingen Univ. (Germany); Rave-Fraenk, Margret; Vorwerk, Hilke; Hille, Andrea; Hess, Clemens Friedrich; Christiansen, Hans [Dept. of Radiotherapy, Goettingen Univ. (Germany)


    Background and purpose: Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro. Material and methods: Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology. Results: Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), {beta}{sub 1}-integrin, {beta}{sub 2}-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, {beta}{sub 1}-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), {beta}{sub 2}-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-){alpha}, interleukin-(IL-)1{beta}, or IL-6 plus TNF-{alpha} led to an upregulation of P-selectin, ICAM-1 and VCAM-1. Conclusion: The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of

  15. Selected immunological changes in patients with Goeckerman's therapy TNF-alpha, sE-selectin, sP-selectin, sICAM-1 and IL-8

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    Borska, L.; Fiala, Z.; Krejsek, J.; Andrys, C.; Vokurkova, D.; Hamakova, K.; Kremlacek, J.; Ettler, K. [Charles University, Hradec Kralove (Czech Republic). Faculty of Medicine


    Psoriasis is one of the most frequent inflammatory skin diseases in which abnormal individual immune reactivity plays an important role. The aim of the present study was to describe selected immunological changes, concerning pro-inflammatory cytokines (TNF-alpha, IL-8) and adhesion molecules (sE-selectin, sP-selectin, sICAM-1), in 56 patients cured by Goeckerman's therapy (GT). GT includes dermal application of crude coal tar (containing polycyclic aromatic hydrocarbons) and exposure to UV radiation.

  16. RhoA signaling through platelet P2Y₁ receptor controls leukocyte recruitment in allergic mice. (United States)

    Amison, Richard T; Momi, Stefania; Morris, Abigail; Manni, Giorgia; Keir, Sandra; Gresele, Paolo; Page, Clive P; Pitchford, Simon C


    Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown. We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation. BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed. P2Y₁, but not P2Y₁₂ or P2X₁, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin-dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y₁ receptor dependent. Furthermore, platelet P2Y₁ activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y₁ stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y₁ antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y₁ receptor and subsequent activation of RhoA. RhoA signaling downstream of platelet P2Y₁, but not P2Y₁₂, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by

  17. Inhibitory Effects of Cytosolic Ca2+ Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets

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    Hyuk-Woo Kwon


    Full Text Available Intracellular Ca2+ ([Ca2+]i is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro, an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI (Ser1756 to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756 by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa, indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756 phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

  18. The human megakaryocytic cell line UT-7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor. (United States)

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Yokota, Hiroshi


    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.

  19. Gene expression profiling identifies platelet-derived growth factor as a diagnostic molecular marker for papillary thyroid carcinoma. (United States)

    Yano, Yukiko; Uematsu, Naoya; Yashiro, Tohru; Hara, Hisato; Ueno, Ei; Miwa, Masanao; Tsujimoto, Gozoh; Aiyoshi, Yuji; Uchida, Kazuhiko


    Cancer diagnostics and therapeutics are often based on clinically relevant markers that are expressed specifically in a malignant tissue at levels higher than in normal tissue. We examined potential markers for papillary thyroid carcinoma (PTC) by monitoring PTC-specific gene expression using cDNA microarray. Gene expression profiles for PTC tissue, normal thyroid tissue, and healthy peripheral blood cells were compared by use of a human 4000-gene cDNA microarray. Protein expressions of the up-regulated genes in PTC were examined in thyroid tissues by immunohistochemistry. Sixty-four genes were overexpressed in PTC tissue relative to normal thyroid tissue and healthy peripheral blood cells. The genes that were up-regulated in PTC were involved in cell cycle regulation, DNA damage response, angiogenesis, and oncogenesis. Among these genes, basic fibroblast growth factor and platelet-derived growth factor were identified by immunochemical methods as proteins that are specifically expressed at high levels in thyroid neoplasms. Basic fibroblast growth factor, which has been identified as a biomarker for PTC, was overexpressed in 54% of PTC cases, 67% of follicular thyroid carcinomas, and 36% of benign thyroid neoplasms. Platelet-derived growth factor was overexpressed in 81% of PTC cases and 100% of follicular carcinomas, but was immunonegative in normal thyroid tissues and benign thyroid neoplasms. Platelet-derived growth factor may be a potential biomarker for PTC and follicular carcinoma. Expression profile analysis using a microarray followed by immunohistochemical study can be used to facilitate the development of molecular biomarkers for cancer.

  20. Genetic determinants of on-aspirin platelet reactivity: focus on the influence of PEAR1.

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    Morten Würtz

    Full Text Available Platelet aggregation during aspirin treatment displays considerable inter-individual variability. A genetic etiology likely exists, but it remains unclear to what extent genetic polymorphisms determine platelet aggregation in aspirin-treated individuals.To identify platelet-related single nucleotide polymorphisms (SNPs influencing platelet aggregation during aspirin treatment. Furthermore, we explored to what extent changes in cyclooxygenase-1 activity and platelet activation may explain such influence.We included 985 Danish patients with stable coronary artery disease treated with aspirin 75 mg/day mono antiplatelet therapy. Patients were genotyped for 16 common SNPs in platelet-related genes using standard PCR-based methods (TaqMan. Platelet aggregation was evaluated by whole blood platelet aggregometry employing Multiplate Analyzer (agonists: arachidonic acid and collagen and VerifyNow Aspirin. Serum thromboxane B2 was measured to confirm aspirin adherence and was used as a marker of cyclooxygenase-1 activity. Soluble P-selectin was used as marker of platelet activation. Platelet aggregation, cyclooxygenase-1 activity, and platelet activation were compared across genotypes in adjusted analyses.The A-allele of the rs12041331 SNP in the platelet endothelial aggregation receptor-1 (PEAR1 gene was associated with reduced platelet aggregation and increased platelet activation, but not with cyclooxygenase-1 activity. Platelet aggregation was unaffected by the other SNPs analyzed.A common genetic variant in PEAR1 (rs12041331 reproducibly influenced platelet aggregation in aspirin-treated patients with coronary artery disease. The exact biological mechanism remains elusive, but the effect of this polymorphism may be related to changes in platelet activation. Furthermore, 14 SNPs previously suggested to influence aspirin efficacy were not associated with on-aspirin platelet NCT01383304.

  1. Controlled type II diabetes mellitus has no major influence on platelet micro-RNA expression. Results from micro-array profiling in a cohort of 60 patients. (United States)

    Stratz, Christian; Nührenberg, Thomas; Fiebich, Bernd L; Amann, Michael; Kumar, Asit; Binder, Harald; Hoffmann, Isabell; Valina, Christian; Hochholzer, Willibald; Trenk, Dietmar; Neumann, Franz-Josef


    Diabetes mellitus as a major contributor to cardiovascular disease burden induces dysfunctional platelets. Platelets contain abundant miRNAs, which are linked to inflammatory responses and, thus, may play a role in atherogenesis. While diabetes mellitus affects plasma miRNAs, no data exist on platelet miRNA profiles in this disease. Therefore, this study sought to explore the miRNA profile of platelets in patients with diabetes mellitus that is unrelated to the presence or absence of coronary artery disease (CAD). Platelet miRNA profiles were assessed in stable diabetic and non-diabetic patients (each n=30); 15 patients in each group had CAD. Platelet miRNA was isolated from leucocyte-depleted platelet-rich plasma, and miRNA profiling was performed using LNA micro-array technology (miRBase18.0, containing 1,917 human miRNAs). Effects of diabetes mellitus were explored by univariate statistical tests for each miRNA, adjusted for potential confounders, and by developing a multivariable signature; evaluated by resampling techniques. Platelets in non-diabetic patients demonstrated miRNA expression profiles comparable to previous data. The miRNA profiles of platelets in diabetics were similar. Statistical analysis unveiled three miRNAs (miR-377-5p, miR-628-3p, miR-3137) with high reselection probabilities in resampling techniques, corresponding to signatures with modest discriminatory performance. Functional annotation of predicted targets for these miRNAs pointed towards an influence of diabetes mellitus on mRNA processing. We did not find major differences in platelet miRNA profiles between diabetics and non-diabetics. Minor differences pertained to miRNAs associated with mRNA processing. Thus, described differences in plasma miRNAs between diabetic and non-diabetic patients cannot be explained by plain changes in platelet miRNA profile.

  2. Breast-cancer extracellular vesicles induce platelet activation and aggregation by tissue factor-independent and -dependent mechanisms. (United States)

    Gomes, Fausto G; Sandim, Vanessa; Almeida, Vitor H; Rondon, Araci M R; Succar, Barbara B; Hottz, Eugenio D; Leal, Ana Carolina; Verçoza, Brunno Renato F; Rodrigues, Juliany Cola F; Bozza, Patrícia T; Zingali, Russolina B; Monteiro, Robson Q


    Cancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities. EVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs. Interaction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs. Our results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Platelets and their chemokines in atherosclerosis – clinical applications

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    Philipp evon Hundelshausen


    Full Text Available The concept of platelets as important players in the process of atherogenesis has become increasingly accepted due to accumulating experimental and clinical evidence. Despite the progress in understanding the molecular details of atherosclerosis, particularly by using animal models, the inflammatory and thrombotic roles of activated platelet s especially in the human system remain difficult to dissect, as often only the complications of atherosclerosis i.e. stroke and myocardial infarction are definable but not the plaque burden.Platelet indices including platelet count and mean platelet volume and soluble mediators released by activated platelets are associated with atherosclerosis. The chemokine CXCL4 has multiple atherogenic activities e.g. altering the differentiation of T cells and macrophages by inhibiting neutrophil and monocyte apoptosis and by increasing the uptake of oxLDL and synergizing with CCL5. CCL5 is released and deposited on endothelium by activated platelets thereby triggering atherogenic monocyte recruitment, which can be attenuated by blocking the corresponding chemokine receptor CCR5. Atheroprotective and plaque stabilizing properties are attributed to CXCL12, which plays an important role in regenerative processes by attracting progenitor cells. Its release from luminal attached platelets accelerates endothelial healing after injury. Platelet surface molecules GPIIb/IIIa, GP1bα, P-selectin, JAM-A and the CD40/CD40L dyade are crucially involved in the interaction with endothelial cells, leukocytes and matrix molecules affecting atherogenesis. Beyond the effects on the arterial inflammatory infiltrate, platelets affect cholesterol metabolism by binding, modifying and endocytosing LDL particles via their scavenger receptors and contribute to the formation of lipid laden macrophages. Current medical therapies for the prevention of atherosclerotic therapies enable the elucidation of mechanisms linking platelets to inflammation

  4. Production of platelet-rich plasma gel from elderly patients under antithrombotic drugs: Perspectives in chronic wounds care. (United States)

    Velier, M; Magalon, J; Daumas, A; Cassar, M; Francois, P; Ghazouane, A; Philandrianos, C; Bertrand, B; Frere, C; Bernot, D; Villani, P; Dignat George, F; Sabatier, F


    Platelet-Rich Plasma (PRP) is an autologous biological therapy obtained by centrifuging the patient's own blood to concentrate platelets. The addition of autologous thrombin and calcium chloride to PRP allows the production of a semi-solid form called PRP gel. PRP gel is increasingly used in a variety of tissue defects and predominantly in the management of non-healing chronic wounds. The topical application of PRP gel seems promising due to the capability of platelets to store and secrete growth factors (GF), fibrin and cytokines, which are essentials for wound healing. Most patients who suffered from chronic wounds are elderly patients with co-morbidities and polypharmacy including antithrombotic drugs such as antiplatelet agents (AP) or anticoagulants (AC), which could hamper the feasibility of this autologous platelet-derived therapy. To date, no study has investigated PRP gel formation in patients with AP or AC. The aim of this study was to evaluate the influence of AP or AC drugs on the production of PRP gel formation from elderly patients. Different biological characteristics were determined to qualify the production of PRP gel from such patients (Interquartile range (IQR) = 75-92 years) compared to healthy volunteers (IQR = 23-37 years). No significant difference was observed in the volume, composition (quantity of platelets, leukocytes and red blood cells) and functionality of platelets from PRP except a higher ADP-induced P-selectin expression in healthy donors compared with elderly patients. Autologous thrombin characteristics were similar in the two groups. Gel time formation (IQR: 120-195 seconds for controls and 135-210 seconds for elderly patients) and final composition of PRP gel were not significantly modified. Concentrations of theoretical thrombin generated in the serum and in the gel were inversely correlated with the time of formation of PRP gel (r2 = 0.57, p = 0.012). Altogether these data indicate that PRP gel preparation is not

  5. Low-Level Laser Irradiation Exerts Antiaggregative Effect on Human Platelets Independently on the Nitric Oxide Metabolism and Release of Platelet Activation Markers

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    Piotr Rola


    Full Text Available Aim. The goal of the study is to develop a model allowing to investigate precisely the effect of low-level laser therapy (LLLT on platelet aggregation and to verify the hypothesis regarding the role of the nitric oxide (NO bioavailability and platelet activation markers in modulating platelet aggregation. Methods. A total of 41 healthy volunteers at the age of 21–45 years were investigated. At first, platelet aggregation in response to three agonists (TRAP, ADP, and collagen was evaluated following previous exposure to different doses of laser radiation (λ = 662 nm to assess the dose-response effect. Subsequently, plasma levels of platelet activation markers (PF4—platelet factor-4 and sP-selectin as well as the substrate for nitric oxide synthase, L-arginine, and its competitive inhibitors (ADMA—asymmetric dimethylarginine and SDMA—symmetric dimethylarginine were measured. Results. All doses of laser irradiation significantly reduced the aggregation. However, the most pronounced effect was observed for 19.7 J/cm2. No significant differences in the levels of platelet activation markers nor in the nitric-oxide-metabolic-pathway compounds between analyzed groups were noted. Conclusions. We have demonstrated in the established in vitro experimental model that the LLLT in a reproducible manner decreases the whole blood platelet aggregation regardless of the NO bioavailability or changes in the platelet activation markers.

  6. Rhesus monkey platelets

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    Harbury, C.B.


    The purpose of this abstract is to describe the adenine nucleotide metabolism of Rhesus monkey platelets. Nucleotides are labelled with /sup 14/C-adenine and extracted with EDTA-ethanol (EE) and perchlorate (P). Total platelet ATP and ADP (TATP, TADP) is measured in the Holmsen Luciferase assay, and expressed in nanomoles/10/sup 8/ platelets. TR=TATP/TADP. Human platelets release 70% of their TADP, with a ratio of released ATP/ADP of 0.7. Rhesus platelets release 82% of their TADP, with a ratio of released ATP/ADP of 0.33. Thus, monkey platelets contain more ADP than human platelets. Thin layer chromatography of EE gives a metabolic ratio of 11 in human platelets and 10.5 in monkey platelets. Perchlorate extracts metabolic and actin bound ADP. The human and monkey platelets ratios were 5, indicating they contain the same proportion of actin. Thus, the extra ADP contained in monkey platelets is located in the secretory granules.

  7. Sequential adhesion of platelets and leukocytes from flowing whole blood onto a collagen-coated surface: requirement for a GpVI-binding site in collagen. (United States)

    Butler, Lynn M; Metson-Scott, Tom; Felix, Jo; Abhyankar, Anita; Rainger, G Ed; Farndale, Richard W; Watson, Stephen P; Nash, Gerard B


    The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic responses in damaged tissue. To investigate the conditions under which platelets and leukocytes might be deposited together in vessels, we perfused fluorescently-labelled whole blood through glass capillaries coated with various collagen preparations. Video-microscopic observations of the surface showed that platelets formed numerous, individual, rolling and stationary attachments to surfaces coated with acid-soluble, monomeric collagen. However, leukocyte interactions with the deposited platelets were rare. If the blood was washed out, the adherent platelets became more activated, and many rolling adherent leukocytes were observed if a second bolus of blood was perfused over them. This suggested that platelet activation had initially been inadequate to support leukocyte capture. Next, fibrillar collagen was adsorbed to the capillaries to present an ordered array of peptide motifs to platelet receptor glycoprotein (Gp)VI and transduce an activating signal. In this case, platelets were deposited in discrete, stable aggregates and the bound platelets captured many flowing leukocytes. Alternatively, acid-soluble collagen was seeded with collagen-related peptide (CRP) known to contain a GpVI-binding motif. Again, platelet adhesion became stable, and numerous flowing leukocytes were captured. Addition of antibody against GpVI or against P-selectin greatly reduced leukocyte adhesion to the platelets. Thus, in whole blood, platelets binding to exposed collagen need to be activated through GpVI in order to expose sufficient P-selectin to allow efficient capture of flowing leukocytes to take place.

  8. Effects of high fat diet and perinatal dioxin exposure on development of body size and expression of platelet-derived growth factor receptor β in the rat brain. (United States)

    Bor, Amartuvshin; Nishijo, Muneko; Nishimaru, Hiroshi; Nakamura, Tomoya; Tran, Nghi Ngoc; Van Le, Quang; Takamura, Yusaku; Matsumoto, Jumpei; Nishino, Yoshikazu; Nishijo, Hisao


    Environmental exposure to dioxins, consumption of a high fat diet, and platelet-derived growth factor receptor β signaling in the brain affect feeding behavior, which is an important determinant of body growth. In the present study, we investigated the effects of prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and high fact diet after weaning on body growth and expression of platelet-derived growth factor receptor β in the brain in rat pups. Subjects from the control and dioxin exposure groups were assigned to 1 of 3 different diet groups: standard diet, high fat diet in the juvenile period, or high fat diet in adulthood. Body weight gain rate in the juvenile high fat diet group and the length gain rate in the adult high fat diet group were greater than the corresponding values in the standard diet group only in male offspring, although the effects of dioxin exposure on growth were not significant. Consumption of a high fat diet decreased platelet-derived growth factor receptor β levels in the amygdala and hippocampus in both sexes compared to control groups, while 2,3,7,8-tetrachlorodibenzo-p-dioxin decreased platelet-derived growth factor receptor platelet-derived growth factor receptor β levels in the amygdala and striatum only in females receiving an high fat diet. Furthermore, platelet-derived growth factor receptor β levels in the hippocampus and platelet-derived growth factor receptor β striatum were inversely correlated with increases in body length, while changes in platelet-derived growth factor receptor β in the amygdala and nucleus accumbens were significantly correlated to body weight gain or body mass index. In conclusion, these findings suggest that these 2,3,7,8-tetrachlorodibenzo-p-dioxin and high fat diet-induced changes in body growth and feeding behaviors might be partially mediated by changes in brain platelet-derived growth factor receptor β levels.

  9. Platelet-rich fibrin increases cell attachment, proliferation and collagen-related protein expression of human osteoblasts. (United States)

    Wu, C-L; Lee, S-S; Tsai, C-H; Lu, K-H; Zhao, J-H; Chang, Y-C


    Platelet-rich fibrin (PRF) prepared by Choukroun's technique is derived from an autogenous preparation of concentrated platelets without any manipulation. PRF was found to increase osteoblast growth and proliferation. However, the underlying mechanisms are not yet completely understood. This study aimed to determine the effects of PRF on cell attachment, proliferation, phosphorylated Akt, heat shock protein 47 (HSP47) and lysyl oxidase (LOX) expression on human osteoblasts. Blood collection was carried out from 10 healthy volunteers. Cell attachment and proliferation were measured by colorimetric assay with WST-1 and alamar blue in human osteoblast cell line U2OS cells, respectively. Western blot was employed to evaluate the expression of p-Akt, HSP47 and LOX. PRF alone was found to stimulate U2OS cell attachment compared with untreated controls (p proliferation during a 5-day incubation period (p proliferation and simultaneously upregulating collagen-related protein production. These actions in combination would effectively promote bone regeneration. © 2012 Australian Dental Association.

  10. Gene expression profiling reveals multiple differences in platelets from patients with stable angina or non-ST elevation acute coronary syndrome. (United States)

    Colombo, Gualtiero; Gertow, Karl; Marenzi, Giancarlo; Brambilla, Marta; De Metrio, Monica; Tremoli, Elena; Camera, Marina


    Platelets play a key role in coronary artery disease. They have the capacity of protein synthesis through translation of megakaryocyte-derived mRNAs, which may influence pathophysiological functions. The present study aimed to prove the concept that platelets from patients with non-ST elevation acute coronary syndrome (NSTE-ACS) have differential mRNA expression profiles, in the hypothesis that this may influence their thrombogenicity. Gene expression profiles were determined in RNA pools from resting platelets of patients with stable angina (SA, n = 14) or NSTE-ACS (n = 15) using a glass microarray platform. Validation was done by real-time PCR and immunoblot analyses in independent sets of individual samples (26 SA and 17 NSTE-ACS patients, in total). Parallel comparison with healthy subjects was performed to relate the relative abundance of validated genes in CAD patients to a control expression level. Microarray analysis identified 45 transcripts with a significant ≥ ± 2.0-fold difference in expression between NSTE-ACS and SA platelet pools. Thus, gene expression profiles at least partially discriminate unstable from stable CAD. Validation confirmed a significant over-expression of 3 genes in NSTE-ACS at both mRNA and protein level. In particular, the glycoprotein Ib β-polypeptide (GP1BB) was increased in NSTE-ACS also in comparison with healthy subjects. This study provides evidence that NSTE-ACS platelets are potentially preconditioned to a higher degree of reactivity on the transcriptional level. Our data suggest that a different composition of the mRNA pool might mediate an increased platelet prothrombotic potential in NSTE-ACS patients. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Comparison between in vitro properties of washed platelet concentrates suspended in M-sol and those in BRS-A, both of which were prepared with an automated cell processor. (United States)

    Iwama, Akira; Hirayama, Junichi; Nogawa, Masayuki; Shiba, Masayuki; Satake, Masahiro; Takamoto, Shigeru; Tadokoro, Kenji


    Washed platelet concentrate (WPC) is prepared manually in general, but automated preparation is desirable to minimize variation in the WPC quality and enhance WPC production. Recently, the software was improved for an automated cell processor (ACP) to control all processes of WPC preparation. M-sol and BRS-A, which are mixtures of medical solutions, are widely used for WPC preparation with a manual method in Japan. In this study, we prepared WPC suspended in M-sol (WPC-M) or BRS-A (WPC-B) with the ACP, and compared their in vitro properties during 7-day storage. PC was divided into two equal aliquots for WPC-M and WPC-B. A divided PC, medical solutions and disposable materials were set in the ACP, and it was started to prepare WPC-M or WPC-B on Day 0. Prepared WPC was stored on a flatbed shaker until Day 7. The pH of WPC-M and WPC-B was maintained above 6.8 during the 7-day storage. The differences in aggregation (%), HSR (%), P-selectin expression, GPIbα expression, and phosphatidylserine expression between WPC-M and WPC-B were minimal until Day 3. The in vitro properties of WPC-B are not markedly different from those of WPC-M until Day 3. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Comparison of VerifyNow-P2Y12 test and Flow Cytometry for monitoring individual platelet response to clopidogrel. What is the cut-off value for identifying patients who are low responders to clopidogrel therapy? (United States)

    Godino, Cosmo; Mendolicchio, Loredana; Figini, Filippo; Latib, Azeem; Sharp, Andrew Sp; Cosgrave, John; Calori, Giliola; Cera, Michela; Chieffo, Alaide; Castelli, Alfredo; Maseri, Attilio; Ruggeri, Zaverio M; Colombo, Antonio


    Dual anti-platelet therapy with aspirin and a thienopyridine (DAT) is used to prevent stent thrombosis after percutaneous coronary intervention (PCI). Low response to clopidogrel therapy (LR) occurs, but laboratory tests have a controversial role in the identification of this condition. We studied LR in patients with stable angina undergoing elective PCI, all on DAT for at least 7 days, by comparing: 1) Flow cytometry (FC) to measure platelet membrane expression of P-selectin (CD62P) and PAC-1 binding following double stimulation with ADP and collagen type I either in the presence of prostaglandin (PG) E1; 2) VerifyNow-P2Y12 test, in which results are reported as absolute P2Y12-Reaction-Units (PRU) or % of inhibition (% inhibition). Thirty controls and 52 patients were analyzed. The median percentage of platelets exhibiting CD62P expression and PAC-1 binding by FC evaluation after stimulation in the presence of PG E1 was 25.4% (IQR: 21.4-33.1%) and 3.5% (1.7-9.4%), respectively. Only 6 patients receiving DAT (11.5%) had both values above the 1st quartile of controls, and were defined as LR. Evaluation of the same patients with the VerifyNow-P2Y12 test revealed that the area under the receiver-operating-characteristic (ROC) curve was 0.94 (95% CI: 0.84-0.98, p Cut-off values of ≤ 15% inhibition or > 213 PRU gave the maximum accuracy for the detection of patients defined as having LR by FC. In conclusion our findings show that a cut-off value of ≤ 15% inhibition or > 213 PRU in the VerifyNow-P2Y12 test may provide the best accuracy for the identification of patients with LR.

  13. Comparison of VerifyNow-P2Y12 test and Flow Cytometry for monitoring individual platelet response to clopidogrel. What is the cut-off value for identifying patients who are low responders to clopidogrel therapy?

    Directory of Open Access Journals (Sweden)

    Castelli Alfredo


    Full Text Available Abstract Background Dual anti-platelet therapy with aspirin and a thienopyridine (DAT is used to prevent stent thrombosis after percutaneous coronary intervention (PCI. Low response to clopidogrel therapy (LR occurs, but laboratory tests have a controversial role in the identification of this condition. Methods We studied LR in patients with stable angina undergoing elective PCI, all on DAT for at least 7 days, by comparing: 1 Flow cytometry (FC to measure platelet membrane expression of P-selectin (CD62P and PAC-1 binding following double stimulation with ADP and collagen type I either in the presence of prostaglandin (PG E1; 2 VerifyNow-P2Y12 test, in which results are reported as absolute P2Y12-Reaction-Units (PRU or % of inhibition (% inhibition. Results Thirty controls and 52 patients were analyzed. The median percentage of platelets exhibiting CD62P expression and PAC-1 binding by FC evaluation after stimulation in the presence of PG E1 was 25.4% (IQR: 21.4–33.1% and 3.5% (1.7–9.4%, respectively. Only 6 patients receiving DAT (11.5% had both values above the 1st quartile of controls, and were defined as LR. Evaluation of the same patients with the VerifyNow-P2Y12 test revealed that the area under the receiver-operating-characteristic (ROC curve was 0.94 (95% CI: 0.84–0.98, p 213 PRU gave the maximum accuracy for the detection of patients defined as having LR by FC. Conclusion In conclusion our findings show that a cut-off value of ≤ 15% inhibition or > 213 PRU in the VerifyNow-P2Y12 test may provide the best accuracy for the identification of patients with LR.

  14. Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium changes cell migration during midline fusion and causes cerebellar ectopia

    NARCIS (Netherlands)

    Andrae, Johanna; Afink, Gijs; Zhang, Xiao-Qun; Wurst, Wolfgang; Nistér, Monica


    The platelet-derived growth factor (PDGF) and receptors are expressed in the developing central nervous system and in brain tumors. To investigate the role of PDGF during normal cerebellar development, we created transgenic mice where PDGF-B was introduced into the endogenous Engrailed1 locus (En1).

  15. The Impact of Helicobacter pylori Urease upon Platelets and Consequent Contributions to Inflammation

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    Adriele Scopel-Guerra


    Full Text Available Gastric infection by Helicobacter pylori is considered a risk factor for gastric and duodenal cancer, and extragastric diseases. Previous data have shown that, in a non-enzymatic way, H. pylori urease (HPU activates neutrophils to produce ROS and also induces platelet aggregation, requiring ADP secretion modulated by the 12-lipoxygenase pathway, a signaling cascade also triggered by the physiological agonist collagen. Here we investigated further the effects on platelets of recombinant versions of the holoenzyme HPU, and of its two subunits (HpUreA and HpUreB. Although HpUreA had no aggregating activity on platelets, it partially inhibited collagen-induced aggregation. HpUreB induced platelet aggregation in the nanomolar range, and also interfered dose-dependently on both collagen- and ADP-induced platelet aggregation. HPU-induced platelet aggregation was inhibited by antibodies against glycoprotein VI (GPVI, the main collagen receptor in platelets. Flow cytometry analysis revealed exposure of P-selectin in HPU-activated platelets. Anti-glycoprotein IIbIIIa (GPIIbIIIa antibodies increased the binding of FITC-labeled HPU to activated platelets, whereas anti-GPVI did not. Evaluation of post-transcriptional events in HPU-activated platelets revealed modifications in the pre-mRNA processing of pro-inflammatory proteins, with increased levels of mRNAs encoding IL-1β and CD14. We concluded that HPU activates platelets probably through its HpUreB subunit. Activation of platelets by HPU turns these cells into a pro-inflammatory phenotype. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked, role in gastric inflammation mediated by urease-activated neutrophils and platelets.

  16. The Impact of Helicobacter pylori Urease upon Platelets and Consequent Contributions to Inflammation. (United States)

    Scopel-Guerra, Adriele; Olivera-Severo, Deiber; Staniscuaski, Fernanda; Uberti, Augusto F; Callai-Silva, Natália; Jaeger, Natália; Porto, Bárbara N; Carlini, Celia R


    Gastric infection by Helicobacter pylori is considered a risk factor for gastric and duodenal cancer, and extragastric diseases. Previous data have shown that, in a non-enzymatic way, H. pylori urease (HPU) activates neutrophils to produce ROS and also induces platelet aggregation, requiring ADP secretion modulated by the 12-lipoxygenase pathway, a signaling cascade also triggered by the physiological agonist collagen. Here we investigated further the effects on platelets of recombinant versions of the holoenzyme HPU, and of its two subunits (HpUreA and HpUreB). Although HpUreA had no aggregating activity on platelets, it partially inhibited collagen-induced aggregation. HpUreB induced platelet aggregation in the nanomolar range, and also interfered dose-dependently on both collagen- and ADP-induced platelet aggregation. HPU-induced platelet aggregation was inhibited by antibodies against glycoprotein VI (GPVI), the main collagen receptor in platelets. Flow cytometry analysis revealed exposure of P-selectin in HPU-activated platelets. Anti-glycoprotein IIbIIIa (GPIIbIIIa) antibodies increased the binding of FITC-labeled HPU to activated platelets, whereas anti-GPVI did not. Evaluation of post-transcriptional events in HPU-activated platelets revealed modifications in the pre-mRNA processing of pro-inflammatory proteins, with increased levels of mRNAs encoding IL-1β and CD14. We concluded that HPU activates platelets probably through its HpUreB subunit. Activation of platelets by HPU turns these cells into a pro-inflammatory phenotype. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked, role in gastric inflammation mediated by urease-activated neutrophils and platelets.

  17. Comprehensive metabolomic study of platelets reveals the expression of discrete metabolic phenotypes during storage

    DEFF Research Database (Denmark)

    Paglia, Giuseppe; Sigurjónsson, Ólafur E; Rolfsson, Óttar


    BACKGROUND: Platelet (PLT) concentrates are routinely stored for 5 to 7 days. During storage they exhibit what has been termed PLT storage lesion (PSL), which is evident by a loss of hemostatic function when transfused into patients. The overall goal of this study was to obtain a comprehensive data...... set describing PLT metabolism during storage. STUDY DESIGN AND METHODS: The experimental approach adopted to achieve this goal combined a series of standard assays to monitor the quality of stored PLTs and a deep-coverage metabolomics study using liquid chromatography coupled with mass spectrometry...... not undergo a monotonic decay, but experienced systematic changes in metabolism reflected in three discrete metabolic phenotypes: The first (Days 0-3) was associated with active glycolysis, pentose phosphate pathway, and glutathione metabolism and down regulation of tricarboxylic acid (TCA) cycle. The second...

  18. Effect of Follicular Fluid and Platelet-Activating Factor on Lactate Dehydrogenase C Expression in Human Asthenozoospermic Samples

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    Tahereh Esmaeilpour


    Full Text Available Background: Application of follicular fluid (FF and platelet-activating factor (PAF in artificial insemination improves sperm motility. Lactate dehydrogenase C (LDH-C is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples. Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction (q-RT PCR and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples. Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms. Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples (P=0.0001, although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients.

  19. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation. (United States)

    Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D


    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

  20. Expression of platelet-bound stromal cell-derived factor-1 in patients with non-valvular atrial fibrillation and ischemic heart disease. (United States)

    Stellos, Konstantinos; Rahmann, A; Kilias, A; Ruf, M; Sopova, K; Stamatelopoulos, K; Jorbenadze, R; Weretka, S; Geisler, T; Gawaz, M; Weig, H-J; Bigalke, B


    Blood cell infiltration and inflammation are involved in atrial remodelling during atrial fibrillation (AF) although the exact mechanisms of inflammatory cell recruitment remain poorly understood. Platelet-bound stromal cell-derived factor-1 (SDF-1) is increased in cases of ischemic myocardium and regulates recruitment of CXCR4(+) cells on the vascular wall. Whether platelet-bound SDF-1 expression is differentially influenced by non-valvular paroxysmal or permanent atrial fibrillation (AF) in patients with stable angina pectoris (SAP) or acute coronary syndrome (ACS) has not been reported so far. A total of 1291 consecutive patients with coronary artery disease (CAD) undergoing coronary angiography were recruited. Among the patients with SAP, platelet-bound-SDF-1 is increased in patients with paroxysmal AF compared with SR or to persistent/permanent AF (P disease. Further in vivo studies are required to elucidate the role of SDF-1 in atrial remodeling and the atrial fibrillation course.

  1. Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. (United States)

    Pinzani, M; Milani, S; Herbst, H; DeFranco, R; Grappone, C; Gentilini, A; Caligiuri, A; Pellegrini, G; Ngo, D V; Romanelli, R G; Gentilini, P


    Expression of platelet-derived growth factor (PDGF) and its receptor (R) subunits was evaluated in normal human liver and in cirrhotic liver tissue by in situ hybridization and immunohistochemistry. In normal liver, PDGF and PDGF-R subunit expression was limited to a few mesenchymal cells of the portal tract stroma and vessels. In cirrhotic liver, PDGF-A and -B chain mRNA expression was markedly increased and was co-distributed with immunoreactivity for PDGF-AA and -BB in infiltrating inflammatory cells and along vascular structures within fibrous septa. These aspects were paralleled by a marked overexpression of PDGF-R alpha- and beta-subunit mRNAs and of the relative immunoreactivities in a wide range of mesenchymal cells in fibrous septa and in perisinusoidal alpha-smooth-muscle-actin-positive cells. In general expression and distribution of PDGF-R subunits appeared to be related to the activation of different mesenchymal cell types involved in the fibroproliferative process. Therefore, we evaluated the expression of PDGF-R subunits in liver tissue specimens with increasing degrees of necroinflammatory activity. The results of this additional study confirmed that expression of PDGF-R subunits is highly correlated with the severity of histological lesions and collagen deposition. Our results, providing evidence for a functional involvement of PDGF/PDGF-R in liver fibrogenesis, greatly support the results of previous in vitro studies and direct attention toward pharmacological strategies able to affect the series of signaling events arising from the autophosphorylation of PDGF-R subunits.

  2. Expression of platelet derived growth factor family members and the potential role of imatinib mesylate for cervical cancer

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    Robles Elizabeth


    Full Text Available Abstract Background Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease; hence newer therapeutical modalities are needed. Preliminary investigations suggest that platelet-derived growth factor (PDGF might have a role in the development of cervical cancer, therefore it is important to determine whether this growth factor pathway is functional and its targeting with imatinib mesylate leads to growth inhibition of cervical cancer cells. Results PDGF receptors (PDGFR and their ligands are frequently expressed in cervical cancer and the majority exhibited a combination of family members co-expression. A number of intronic and exonic variations but no known mutations in the coding sequence of the PDGFRα gene were found in cancer cell lines and primary tumors. Growth assays demonstrated that PDGFBB induces growth stimulation that can be blocked by imatinib and that this tyrosine kinase inhibitor on its own inhibits cell growth. These effects were associated with the phosphorylation status of the receptor. Conclusion The PDGFR system may have a role in the pathogenesis of cervical cancer as their members are frequently expressed in this tumor and cervical cancer lines are growth inhibited by the PDGFR antagonist imatinib.

  3. Immunologic changes in TNF-alpha, sE-selectin, sP-selectin, sICAM-1, and IL-8 in pediatric patients treated for psoriasis with the Goeckerman regimen

    Energy Technology Data Exchange (ETDEWEB)

    Borska, L.; Fiala, Z.; Krejsek, J.; Andrys, C.; Vokurkova, D.; Hamakova, K.; Kremlacek, J.; Ettler, K. [Charles University of Prague, Hradec Kralove (Czech Republic). Faculty of Medicine


    Psoriasis is a chronic inflammatory skin disease which is often manifested during childhood. The present study investigated changes in the serum levels of proinflammatory cytokines and soluble forms of adhesion molecules in children with psoriasis. The observed patient group of 26 children was treated with the Goeckerman regimen. This therapy combines dermal application of crude coal tar with ultraviolet radiation. The Psoriasis Area Severity Index decreased significantly after treatment by with the Goeckerman regimen (p < 0.001). Serum levels of the proinflammatory cytokine TNF-alpha and adhesion molecules sICAM-1, sP-selectin and sE-selectin decreased after the Goeckerman regimen. The TNF-alpha and sICAM-1 decreased significantly (p < 0.05). Our findings support the complex role of these immune parameters in the immunopathogenesis of psoriasis in children. The serum level of IL-8 increased after the Goeckerman regimen. This fact indicates that the chemokine pathway of IL-8 activity could be modulated by this treatment, most likely by polycyclic aromatic hydrocarbons.

  4. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil


    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Platelet mimicry

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Hunter, Alan Christy; Peer, Dan


    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...

  6. Morphine induces expression of platelet-derived growth factor in human brain microvascular endothelial cells: implication for vascular permeability.

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

    Full Text Available Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling and loss-of-function (Egr-1 approaches demonstrated the role of mitogen-activated protein kinases (MAPKs, PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

  7. Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis (United States)

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


    AIM: To determine the platelet-activating factor (PAF) synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis. 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. PMID:18205269

  8. Over-Expression of Platelet-Derived Growth Factor-D Promotes Tumor Growth and Invasion in Endometrial Cancer

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    Yuan Wang


    Full Text Available The platelet-derived growth factor-D (PDGF-D was demonstrated to be able to promote tumor growth and invasion in human malignancies. However, little is known about its roles in endometrial cancer. In the present study, we investigated the expression and functions of PDGF-D in human endometrial cancer. Alterations of PDGF-D mRNA and protein were determined by real time PCR, western blot and immunohistochemical staining. Up-regulation of PDGF-D was achieved by stably transfecting the pcDNA3-PDGF-D plasmids into ECC-1 cells; and knockdown of PDGF-D was achieved by transient transfection with siRNA-PDGF-D into Ishikawa cells. The MTT assay, colony formation assay and Transwell assay were used to detect the effects of PDGF-D on cellular proliferation and invasion. The xenograft assay was used to investigate the functions of PDGF-D in vivo. Compared to normal endometrium, more than 50% cancer samples showed over-expression of PDGF-D (p < 0.001, and high level of PDGF-D was correlated with late stage (p = 0.003, deep myometrium invasion (p < 0.001 and lympha vascular space invasion (p = 0.006. In vitro, over-expressing PDGF-D in ECC-1 cells significantly accelerated tumor growth and promoted cellular invasion by increasing the level of MMP2 and MMP9; while silencing PDGF-D in Ishikawa cells impaired cell proliferation and inhibited the invasion, through suppressing the expression of MMP2 and MMP9. Moreover, we also demonstrated that over-expressed PDGF-D could induce EMT and knockdown of PDGF-D blocked the EMT transition. Consistently, in xenografts assay, PDGF-D over-expression significantly promoted tumor growth and tumor weights. We demonstrated that PDGF-D was commonly over-expressed in endometrial cancer, which was associated with late stage deep myometrium invasion and lympha vascular space invasion. Both in vitro and in vivo experiments showed PDGF-D could promote tumor growth and invasion through up-regulating MMP2/9 and inducing EMT. Thus, we

  9. Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions. (United States)

    Biasetti, Jacopo; Sampath, Kaushik; Cortez, Angel; Azhir, Alaleh; Gilad, Assaf A; Kickler, Thomas S; Obser, Tobias; Ruggeri, Zaverio M; Katz, Joseph


    Tracking cells and proteins' phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique's ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.

  10. Differential expression of neuronal dopamine and serotonin transporters DAT and SERT in megakaryocytes and platelets generated from human MEG-01 megakaryoblasts. (United States)

    Hohmann, Sarah; Schweinfurth, Nina; Lau, Thorsten; Deuschle, Michael; Lederbogen, Florian; Banaschewski, Tobias; Schloss, Patrick


    In the central nervous system, serotonergic and dopaminergic signaling is terminated by the activity of specialized transporter proteins for serotonin (SERT) and dopamine (DAT). These transporter proteins are found both on the cell surface and in intracellular transport vesicles. Trafficking between these subcellular domains regulates the efficiency of removal of extracellular neurotransmitters and hence the efficacy of neuronal signaling. Therefore, it is of high interest to gain more insight into the regulatory mechanisms of the human DAT and SERT cell surface expression in their natural surroundings, i.e., in human cells. Because it is not possible to cultivate human neuronal cells expressing these transporter proteins, there is a need to find other human cells expressing these neuronal proteins. Here, we have investigated the expression of human SERT and DAT on developing megakaryocytes and platelet-like particles derived from the megakaryocyte progenitor cell line MEG-01 upon differentiation by valproic acid (VPA) and all-trans retinoic acid (ATRA). Our results show that MEG-01 cells express SERT and DAT and that VPA and ATRA induce a significant increase of transporter expression on developing megakaryocytes and platelets. As compared to ATRA, VPA more efficiently induced SERT expression but not DAT expression. Comparable to naïve platelets and neurons, SERT was localized to both the cell surface and intracellular compartments. Hence, VPA and ATRA-treated MEG-01 cells provide a model well-suited to studying neuronal monoamine transporter expression, not only during transcription and translation but also with respect to protein trafficking to and from the cell surface.

  11. Increased expression of platelet-derived growth factor A and collagenous matrix proteins in congenital multicystic renal dysplasia. (United States)

    Liapis, H; Yu, H; Flath, A; Steinhardt, G F


    The expression of platelet-derived growth factor A (PDGF-A), and its spatial and temporal relationship to interstitial collagens in kidneys with congenital multicystic dysplasia using in situ hybridization, have been examined. Seventeen dysplastic kidneys (16 weeks to 7 months) and 20 normal age-matched controls were used in the study. Increased PDGF-A mRNA was detected in dysplastic compared to normal kidneys in all age groups including extensively fibrotic postnatal kidneys. An abundant PDGF-A mRNA signal was seen within the epithelial cells of cystically dilated or dysplastic tubules and within interstitial fibroblasts and disorganized primitive mesenchyme. A comparable amount of PDGF-A protein was detected by Western blotting. Procollagen I and III mRNA were increased in fibroblasts surrounding cystic and dysplastic tubules. We conclude that tubular epithelial production of PDGF-A may induce collagenous matrix production by adjacent fibroblasts, while marked up-regulation of PDGF-A by interstitial cells may be responsible for sustainable fibrogenic effects in the fetal kidney contributing to renal maldevelopment.

  12. Immunohistochemical Expression of Collagens in the Skin of Horses Treated with Leukocyte-Poor Platelet-Rich Plasma (United States)

    Silva, Mariana Brettas; Pinto, José de Oliveira; Lima, Marianna Barros de Souza; Crepaldi, Júlio; Lopes, Gabriela Francine Martins; dos Santos, Hélio Batista; Ribeiro, Rosy Iara Maciel de Azambuja; Thomé, Ralph Gruppi


    This study evaluated the immunohistochemical expression of type I (COL I) and III (COL III) collagens during the healing process of skin treated with leukocyte-poor platelet-rich plasma (LP-PRP). Seven healthy gelding crossbred horses aged 16 to 17 years were used. Two rectangle-shaped wounds were created surgically in the right and left gluteal regions. Twelve hours after wound induction, 0.5 mL of the LP-PRP was administered in each edge of the wounds of one of the gluteal regions. The contralateral region was used as control (CG). Three samples were obtained: after wound induction (T0), 14 days (T1) of healing process, and after complete closure of the skin (T2). The normal skin (T0) showed strong staining for type III and I collagen in papillary and reticular dermis, respectively. In the scar of the treated group, COL III showed important (p < 0.05) increase in immunoreaction in T2 compared with T1. The administration of a single dose of LP-PRP 12 h after induction of wound in horses does not influence formation of collagens I and III. However, the intense labeling for COL III suggests that the tissue was still weak during the macroscopic closure of the wound, demonstrating that healing was not completely finished. PMID:26236743

  13. CD24 expression causes the acquisition of multiple cellular properties associated with tumor growth and metastasis

    NARCIS (Netherlands)

    Baumann, P; Cremers, N; Kroese, F; Orend, G; Chiquet-Ehrismann, R; Uede, T; Yagita, H; Sleeman, JP


    The glycosylphosphatidylinositol-anchored membrane protein CD24 functions as an adhesion molecule for P-selectin and L1 and plays a role in B-cell development and neurogenesis. Over the last few years, a large body of literature has also implicated CD24 expression in tumorigenesis and progression.

  14. 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.

  15. Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression.

    Directory of Open Access Journals (Sweden)

    Yusuke Ando

    Full Text Available We previously reported that mouse bone marrow-derived macrophages (BMDMs that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS and produced lower levels of nitric oxide (NO and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup and found that BMDMs cultured with PLT-sup (PLT-BMDMs expressed a lower level of inducible NO synthase (iNOS and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.

  16. 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.

  17. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink. (United States)

    Babavalian, H; Latifi, A M; Shokrgozar, M A; Bonakdar, S; Tebyanian, H; Shakeri, F


    The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

  18. Impact of exercise training on inflammation and platelet activation in patients with intermittent claudication. (United States)

    Schlager, Oliver; Hammer, Alexandra; Giurgea, Aura; Schuhfried, Othmar; Fialka-Moser, Veronika; Gschwandtner, Michael; Koppensteiner, Renate; Steiner, Sabine


    Serum markers of inflammation and platelet activation are related to cardiovascular risk. Cardiovascular risk reduction is a major treatment goal in patients with peripheral arterial disease (PAD). Although current guidelines recommend supervised exercise training (SET) for PAD patients with intermittent claudication, its contribution to risk reduction remains unclear. Aim of the present study was to assess the impact of SET on inflammation and platelet activation as surrogates for cardiovascular risk. Fifty-three patients with intermittent claudication were randomly assigned to SET on top of best medical treatment (BMT) for 6 months (SET-group) or to BMT only (BMT-group). High sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and fibrinogen as well as soluble P-selectin (sP-sel), prothrombin fragment 1+2 (F1.2) and monocyte-platelet aggregates (MPA) were determined at study entry, after 3, 6 and 12 months. While clinical improvement, reflected by an increase of walking capacity, was observed upon SET, no lasting changes of markers of inflammation and platelet activation were found within the SET-group during the training period. Compared to the BMT-group no improvements of these markers were observed in response to training at any time point (all p >0.05). Regular SET added no further anti-inflammatory effect and had no effect on platelet activation when provided on top of BMT in PAD patients with intermittent claudication.

  19. Factors associated with early platelet activation in obese children. (United States)

    Gómez García, Anel; Núñez, Guillermina García; Sandoval, Martha Eva Viveros; Castellanos, Sergio Gutierrez; Alvarez Aguilar, Cleto


    To investigate the factors associated with platelet activation in obese children. Cross-sectional study. Department of Pediatrics of Regional Hospital N∘ 1 of Mexican Institute of Social Security in Morelia, Michoacán, Mexico. 79 obese and 64 non-obese children between the ages of 5 and 10 years. Obese children (body mass index [BMI] >85 in growth curves for Centers for Disease Control/National Center for Health Statistics), and the control group of 64 non-obese children (percentile Obese children displayed higher plasma sP-selectin, leptin, PAI-1, and vWF than non-obese children. In the univariate logistic regression analysis, leptin, vWF, UA, and high density lipoprotein (HDL), but not with PAI-1, were factors associated with platelet activation. By stepwise linear regression analysis adjusted by sex and age, the best predictor variables for platelet activation were leptin (β:0.381; t:4.665; P=0.0001), vWF (β:0.211; t:2.926; P=0.004), UA (β:0.166; t:2.146; P=0.034), and HDL (β:-0.215; t:-2.819; P=0.006). Obese children have a higher risk of developing early platelet activation. Factors associated with platelet activation were Leptin, vWF, UA, and HDL. Further studies involving larger numbers of patients over a longer duration are needed to understand the possible molecular mechanism underlying the association between leptin, vWF, and UA and endothelial activation and/or endothelial damage/dysfunction in obese children and its influence in cardiovascular disease in adults. © 2014 Marshfield Clinic.

  20. Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders. (United States)

    Dovlatova, N; Lordkipanidzé, M; Lowe, G C; Dawood, B; May, J; Heptinstall, S; Watson, S P; Fox, S C


    Mild platelet function disorders (PFDs) are complex and difficult to diagnose. The current gold standard test, light transmission aggregometry (LTA), including lumi-aggregometry, is time and labour intensive and blood samples must be processed within a limited time after venepuncture. Furthermore, many subjects with suspected PFDs do not show a platelet abnormality on LTA. To assess the diagnostic potential of an easy-to-use remote platelet function test (RPFT) as a diagnostic pre-test for suspected PFDs. A remote platelet function test was compared with lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets Study (GAPP, ISRCTN 77951167). For the RPFT, whole blood was stimulated with platelet agonists, stabilized with PAMFix and returned to the central laboratory for analysis of P-selectin and CD63 by flow cytometry. For the 61 study participants (42 index cases and 19 relatives) there was a good agreement between lumi-aggregometry and the RPFT, with diagnosis being concordant in 84% of cases (κ = 0.668, P tests, 29 participants were identified to have a deficiency in platelet function and 22 participants appeared normal. There were four participants where lumi-aggregometry revealed a defect but the RPFT did not, and six participants where the RPFT detected an abnormal platelet response that was not identified by lumi-aggregometry. This study suggests that the RPFT could be an easy-to-use pre-test to select which participants with bleeding disorders would benefit from extensive platelet phenotyping. Further development and evaluation of the test are warranted in a wider population of patients with excessive bleeding and could provide informative screening tests for PFDs. © 2014 International Society on Thrombosis and Haemostasis.

  1. cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects. (United States)

    Suntravat, Montamas; Jia, Ying; Lucena, Sara E; Sánchez, Elda E; Pérez, John C


    A 5' truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5'-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, Crotalus atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC(50) of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC(50)s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders. Published by Elsevier Ltd.

  2. Platelet Donation (United States)

    ... body. I imagined the faces of many different strangers, taking time out of their day… their jobs… ... thing to do for another human being. A stranger. Someone’s platelets made their way to Phil that ...

  3. Characterization of platelet-derived growth factor-A expression in mouse tissues using a lacZ knock-in approach.

    Directory of Open Access Journals (Sweden)

    Johanna Andrae

    Full Text Available Expression of the platelet-derived growth factor A-chain gene (Pdgfa occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology.

  4. Platelet Function Tests (United States)

    ... Patient Resources For Health Professionals Subscribe Search Platelet Function Tests Send Us Your Feedback Choose Topic At ... Also Known As Platelet Aggregation Studies PFT Platelet Function Assay PFA Formal Name Platelet Function Tests This ...

  5. Platelet antibodies blood test (United States)

    This blood test shows if you have antibodies against platelets in your blood. Platelets are a part of the blood ... Chernecky CC, Berger BJ. Platelet antibody - blood. In: Chernecky ... caused by platelet destruction, hypersplenism, or hemodilution. ...

  6. Antiplatelet Effects of Qishen Yiqi Dropping Pill in Platelets Aggregation in Hyperlipidemic Rabbits

    Directory of Open Access Journals (Sweden)

    Yi Wang


    Full Text Available We investigated the effects of Qishen Yiqi Dropping Pill (QSYQ on platelets aggregation and its possible mechanisms. Hyperlipidemic model in rabbits was produced by a high fat/cholesterol diet for 6 weeks, the therapeutic effect of QSYQ with 2.0 g/kg, 1.0 g/kg, and 0.5 g/kg was observed. Fourteen days after drug treatment, platelet aggregation induced by adenosine diphosphate (ADP, arachidonic acid (AA, and collagen (COLL was significantly reduced in rabbits of model group. Moreover, β-thromboglobulin (β-TG level decreased obviously but no significant change in P-selectin and platelet factor 4 (PF4 level, while QSYQ significantly decreased the ratio of thromboxane B2 (TXB2 to 6-keto-prostaglandin F1α (6-Keto-PGF1α and increased cyclic adenosine monophosphate (cAMP level in rabbits. In summary, QSYQ can improve platelets aggregation and inhibit the over-release of β-TG in hyperlipidemic rabbits; and the increased cAMP level may be involved in this process. These results suggest that the antiplatelet aggregation effect of QSYQ may be due to its ability to increase cAMP level for improving cAMP metabolism.

  7. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia. (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A


    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  8. Magnetic Nanoparticle Labeling of Human Platelets from Platelet Concentrates for Recovery and Survival Studies. (United States)

    Aurich, Konstanze; Wesche, Jan; Palankar, Raghavendra; Schlüter, Rabea; Bakchoul, Tamam; Greinacher, Andreas


    Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation.

  9. Letter to the Editor

    African Journals Online (AJOL)

    I read the article documented by Xianghong et al. with great interest1. They assessed the mean platelet volume. (MPV), platelet count and P selectin (CD62 P) expression in liver cirrhosis. The values of CD62P and MPV in patients with cirrhosis were significantly higher than those of the control group, while the platelet count ...

  10. The diversity of platelet microparticles. (United States)

    Boilard, Eric; Duchez, Anne-Claire; Brisson, Alain


    Platelet microparticles are small extracellular vesicles abundant in blood. The present review will introduce the mechanisms underlying the generation of microparticles, and will describe the diverse microparticle subtypes identified to date. The most appropriate methodologies used to distinguish microparticle subtypes will be also presented. Both the megakaryocytes and platelets can generate microparticles. Circulating microparticles originating from megakaryocytes are distinguished from those derived from activated platelets by the presence of CD62P, LAMP-1, and immunoreceptor-based activation motif receptors. Close examination of platelet activation has shed light on a novel mechanism leading to microparticle production. Under physiologic flow, microparticles bud off from long membrane strands formed by activated platelets. Furthermore, mounting evidence supports the notion of microparticle heterogeneity. Platelet microparticles are commonly characterized by the expression of surface platelet antigens and phosphatidylserine. In fact, only a fraction of platelet microparticles harbor phosphatidylserine, and a distinct subset contains respiratory-competent mitochondria. During disease, the microparticle surface may undergo posttranslational modifications such as citrullination, further supporting the concept of microparticle diversity. An appreciation of the microparticle heterogeneity will support their development as potential biomarkers and may reveal functions unique to each microparticle subtype in health and disease.

  11. The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors.

    Directory of Open Access Journals (Sweden)

    Frank A J L Scheer

    Full Text Available BACKGROUND: Platelets are involved in the thromboses that are central to myocardial infarctions and ischemic strokes. Such adverse cardiovascular events have day/night patterns with peaks in the morning (~9 AM, potentially related to endogenous circadian clock control of platelet activation. The objective was to test if the human endogenous circadian system influences (1 platelet function and (2 platelet response to standardized behavioral stressors. We also aimed to compare the magnitude of any effects on platelet function caused by the circadian system with that caused by varied standardized behavioral stressors, including mental arithmetic, passive postural tilt and mild cycling exercise. METHODOLOGY/PRINCIPAL FINDINGS: We studied 12 healthy adults (6 female who lived in individual laboratory suites in dim light for 240 h, with all behaviors scheduled on a 20-h recurring cycle to permit assessment of endogenous circadian function independent from environmental and behavioral effects including the sleep/wake cycle. Circadian phase was assessed from core body temperature. There were highly significant endogenous circadian rhythms in platelet surface activated glycoprotein (GP IIb-IIIa, GPIb and P-selectin (6-17% peak-trough amplitudes; p ≤ 0.01. These circadian peaks occurred at a circadian phase corresponding to 8-9 AM. Platelet count, ATP release, aggregability, and plasma epinephrine also had significant circadian rhythms but with later peaks (corresponding to 3-8 PM. The circadian effects on the platelet activation markers were always larger than that of any of the three behavioral stressors. CONCLUSIONS/SIGNIFICANCE: These data demonstrate robust effects of the endogenous circadian system on platelet activation in humans--independent of the sleep/wake cycle, other behavioral influences and the environment. The 9 AM timing of the circadian peaks of the three platelet surface markers, including platelet surface activated GPIIb-IIIa, the

  12. Increased megakaryocytic proliferation, pro-platelet deposition and expression of fibrosis-associated factors in children with chronic myeloid leukaemia with bone marrow fibrosis. (United States)

    Hussein, K; Stucki-Koch, A; Göhring, G; Kreipe, H; Suttorp, M


    Paediatric chronic myeloid leukaemia (ped-CML) is rare and ped-CML with fibre accumulation in the bone marrow (MF) is thought to be even rarer. In adults (ad-CML), fibrosis represents an adverse prognostic factor. So far, the pro-fibrotic changes in the bone marrow microenvironment have not been investigated in detail in ped-CML. From a total of 66 ped-CML in chronic phase, biopsies were analysable and 10 had MF1/2 (MF1, n=8/10; MF2, n=2/10). We randomly selected 16 ped-CML and 16 ad-CML cases with and without fibrosis (each n=8) as well as 18 non-neoplastic controls. Bone marrow samples were analysed with a real-time PCR-based assay (including 127 genes for paediatric cases) and by immunohistochemistry. We found increased expression of megakaryocytic genes in ped-CML. The number of megakaryocytes and pro-platelets are increased in CML patients, but the most significant increase was noted for ped-CML-MF1/2. Anti-fibrotic MMP9 expression was lower in children than in adults. Cell mobilisation-related CXCL12 was decreased in young and adult patients with CML but not the corresponding receptor CXCR4. In summary, fibre accumulation in ped-CML-MF1/2 is associated with increased megakaryocytic proliferation and increased interstitial pro-platelet deposition. Deregulated expression of matrix-modulating factors shifts the bone marrow microenvironment towards fibrosis.

  13. Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels. (United States)

    Yuasa, Tomoyuki; Kakuhata, Rei; Kishi, Kazuhiro; Obata, Toshiyuki; Shinohara, Yasuo; Bando, Yoshimi; Izumi, Keisuke; Kajiura, Fumiko; Matsumoto, Mitsuru; Ebina, Yousuke


    Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.

  14. Platelet function investigation by flow cytometry: Sample volume, needle size, and reference intervals. (United States)

    Pedersen, Oliver Heidmann; Nissen, Peter H; Hvas, Anne-Mette


    Flow cytometry is an increasingly used method for platelet function analysis because it has some important advantages compared with other platelet function tests. Flow cytometric platelet function analyses only require a small sample volume (3.5 mL); however, to expand the field of applications, e.g., for platelet function analysis in children, even smaller volumes are needed. Platelets are easily activated, and the size of the needle for blood sampling might be of importance for the pre-activation of the platelets. Moreover, to use flow cytometry for investigation of platelet function in clinical practice, a reference interval is warranted. The aims of this work were 1) to determine if small volumes of whole blood can be used without influencing the results, 2) to examine the pre-activation of platelets with respect to needle size, and 3) to establish reference intervals for flow cytometric platelet function assays. To examine the influence of sample volume, blood was collected from 20 healthy individuals in 1.0 mL, 1.8 mL, and 3.5 mL tubes. To examine the influence of the needle size on pre-activation, blood was drawn from another 13 healthy individuals with both a 19- and 21-gauge needle. For the reference interval study, 78 healthy adults were included. The flow cytometric analyses were performed on a NAVIOS flow cytometer (Beckman Coulter, Miami, Florida) investigating the following activation-dependent markers on the platelet surface; bound-fibrinogen, CD63, and P-selectin (CD62p) after activation with arachidonic acid, ristocetin, adenosine diphosphate, thrombin-receptor-activating-peptide, and collagen. The study showed that a blood volume as low as 1.0 mL can be used for platelet function analysis by flow cytometry and that both a 19- and 21-gauge needle can be used for blood sampling. In addition, reference intervals for platelet function analyses by flow cytometry were established.

  15. Prostate field cancerization: deregulated expression of macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) in tumor adjacent tissue. (United States)

    Jones, Anna C; Antillon, Kresta S; Jenkins, Shannon M; Janos, Sara N; Overton, Heidi N; Shoshan, Dor S; Fischer, Edgar G; Trujillo, Kristina A; Bisoffi, Marco


    Prostate field cancerization denotes molecular alterations in histologically normal tissues adjacent to tumors. Such alterations include deregulated protein expression, as we have previously shown for the key transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS). Here we add the two secreted factors macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) to the growing list of protein markers of prostate field cancerization. Expression of MIC-1 and PDGF-A was measured quantitatively by immunofluorescence and comprehensively analyzed using two methods of signal capture and several groupings of data generated in human cancerous (n = 25), histologically normal adjacent (n = 22), and disease-free (n = 6) prostate tissues. A total of 208 digitized images were analyzed. MIC-1 and PDGF-A expression in tumor tissues were elevated 7.1x to 23.4x and 1.7x to 3.7x compared to disease-free tissues, respectively (pcancerization, MIC-1 and PDGF-A expression in adjacent tissues were elevated 7.4x to 38.4x and 1.4x to 2.7x, respectively (pcancerization. These secreted factors could promote tumorigenesis in histologically normal tissues and lead to tumor multifocality. Among several clinical applications, they could also be exploited as indicators of disease in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

  16. 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


    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.

  17. Aspirin 'resistance': impact on no-reflow, platelet and inflammatory biomarkers in diabetics after ST-segment elevation myocardial infarction. (United States)

    Kuliczkowski, Wiktor; Gasior, Mariusz; Pres, Damian; Kaczmarski, Jacek; Laszowska, Anna; Szewczyk, Marta; Hawranek, Michal; Tajstra, Mateusz; Zeglen, Slawomir; Polonski, Lech; Serebruany, Victor L


    The no-reflow (NR) phenomenon exists despite percutaneous coronary intervention (PCI), and is especially prevalent in diabetics. The causes(s) of NR are not fully elucidated, but may be associated with impaired residual platelet and inflammatory reactivity during dual-antiplatelet therapy. To assess the relationship between dual-antiplatelet therapy, NR and conventional biomarkers suggestive of platelet and inflammatory response in diabetics following ST-segment elevation myocardial infarction (STEMI) treated with PCI. Sixty diabetics with (n = 27) and without NR (n = 33) were prospectively enrolled. All patients were treated with clopidogrel and aspirin. Platelet and inflammatory biomarkers were assessed serially in the peripheral blood and right atrium before and after PCI and then at 24 h, 7 days and 30 days. Arachidonic acid (AA)-induced platelet aggregation and the serum thromboxane B2 level before and after PCI (in the peripheral and right atrium blood) were significantly higher in the NR patients than in those with no NR. AA-induced aggregation >100 (AUC*min) before PCI predicted NR in diabetic patients with 96.2% sensitivity and 38.5% specificity (AUC 0.66; 95% CI 0.52-0.71; p = 0.029). There were no other correlations between NR and platelet reactivity (collagen, adenosine diphosphate, thrombin receptor agonist peptide-induced aggregation, vasodilator-stimulated phosphoprotein platelet reactivity index, soluble P-selectin, soluble CD40 ligand, platelet-derived growth factor AB and the level of platelet-monocyte aggregates) or between NR and inflammatory indices (i.e. high-sensitivity C-reactive protein, interleukin 6 and interleukin 10). An inadequate response to aspirin, but not to clopidogrel, may be associated with the occurrence of the NR phenomenon in diabetics with STEMI who have been treated with primary PCI. © 2015 S. Karger AG, Basel.

  18. Global gene expression analysis in the mouse brainstem after hyperalgesia induced by facial carrageenan injection--evidence for a form of neurovascular coupling? (United States)

    Poh, Kay-Wee; Lutfun, Nahar; Manikandan, Jayapal; Ong, Wei-Yi; Yeo, Jin-Fei


    The present study was carried out to examine global gene expression in the brainstem, in a mouse facial carrageenan injection model of orofacial pain. Mice that received facial carrageenan injection showed increased mechanical allodynia, demonstrated by increased responses to von Frey hair stimulation of the face. The brainstem was harvested at 3 days post-injection, corresponding to the time of peak responses, and analyzed by Affymetrix Mouse Genome 430 2.0 microarrays. We sought to identify common genes that are changed in the respective sides of the brainstem after either right- or left-sided facial carrageenan injection. The result is a relatively small list of genes (22 genes), which were then classified using DAVID software. Many of them fell into the categories of "response to stress", "defence response", "response to biotic stimulus", "cell adhesion" and "leukocyte adhesion". Of these, increased expression of P-selectin, ICAM-1 and CCL12 after carrageenan injection could be verified by real-time RT-PCR on both the right and left sides, and increased in P-selectin and ICAM-1 further verified by Western blot analysis. P-selectin and ICAM-1 were immunolocalized to endothelial cells, and were double labelled with von Willebrand factor. Intraperitoneal injection of the P-selectin inhibitor KF38789 significantly reduced mechanical allodynia in the facial carrageenan-injected mice. P-selectin mediates the capturing of leukocytes from the bloodstream and rolling of leukocytes along the endothelial surface. We hypothesize that increased nociceptive input to the brainstem could attract circulating macrophages into the brain, resulting in neuroinflammation and pain.

  19. Increased platelet aggregation and serum thromboxane levels in aspirin-treated patients with prior myocardial infarction. (United States)

    Larsen, Sanne Bøjet; Neergaard-Petersen, Søs; Grove, Erik Lerkevang; Kristensen, Steen Dalby; Hvas, Anne-Mette


    The antiplatelet effect of aspirin displays considerable inter-individual variability. We investigated the antiplatelet effect of aspirin in patients with coronary artery disease on aspirin mono-therapy with and without prior myocardial infarction (MI). Further, we investigated whether the effect of aspirin differed between patients with and without aspirin use at the time of MI onset. We performed a study on 231 patients, including 171 with prior MI. Among patients with only one prior MI (116 patients), 59 patients were on aspirin at the time of MI onset. All patients received 75 mg aspirin as mono-therapy. Platelet aggregation was assessed by multiple electrode aggregometry (Multiplate) and VerifyNow, and platelet activation was evaluated by soluble P-selectin. Furthermore, we measured serum thromboxane B2. MI patients had higher median platelet aggregation levels than patients without prior MI when evaluated by Multiplate (parachidonic acidaspirin before MI onset had significantly higher median aggregation levels compared with MI patients not on aspirin when evaluated by Multiplate (pcollagen=0.02) and VerifyNow (paspirin dose and optimal compliance. Serum thromboxane B2 levels were higher in MI patients than in patients without prior MI. Finally, patients on aspirin before MI onset had higher aggregation levels compared with patients not on aspirin.

  20. Platelet signaling-a primer. (United States)

    Goggs, Robert; Poole, Alastair W


    To review the receptors and signal transduction pathways involved in platelet plug formation and to highlight links between platelets, leukocytes, endothelium, and the coagulation system. Original studies, review articles, and book chapters in the human and veterinary medical fields. Platelets express numerous surface receptors. Critical among these are glycoprotein VI, the glycoprotein Ib-IX-V complex, integrin α(IIb) β(3) , and the G-protein-coupled receptors for thrombin, ADP, and thromboxane. Activation of these receptors leads to various important functional events, in particular activation of the principal adhesion receptor α(IIb) β(3) . Integrin activation allows binding of ligands such as fibrinogen, mediating platelet-platelet interaction in the process of aggregation. Signals activated by these receptors also couple to 3 other important functional events, secretion of granule contents, change in cell shape through cytoskeletal rearrangement, and procoagulant membrane expression. These processes generate a stable thrombus to limit blood loss and promote restoration of endothelial integrity. Improvements in our understanding of how platelets operate through their signaling networks are critical for diagnosis of unusual primary hemostatic disorders and for rational antithrombotic drug design. © Veterinary Emergency and Critical Care Society 2012.

  1. Platelet transfusion. (United States)


    The statement printed below was agreed at a consensus conference on platelet transfusion organised by the Royal College of Physicians of Edinburgh and held in Edinburgh in November 1997. We publish this statement at the request of the organising committee to bring it to the attention of physicians who do not read the haematological literature. The statement will also appear in the British Journal of Haematology in 1998 with the scientific evidence upon which it is based.

  2. The Effect of Platelet-rich Plasma on Wounds of OLETF Rats Using Expression of Matrix Metalloproteinase-2 and -9 mRNA (United States)

    Oh, Hwa Young


    Background Complicated diabetic patients show impaired, delayed wound healing caused by multiple factors. A study on wound healing showed that platelet-rich plasma (PRP) was effective in normal tissue regeneration. Nonetheless, there is no evidence that when plateletrich plasma is applied to diabetic wounds, it normalizes the diabetic wound healing process. In this study, we have analyzed matrix metalloproteinase (MMP)-2, MMP-9 expression to investigate the effect of PRP on diabetic wounds. Methods Twenty-four-week-old male Otsuka Long-Evans Tokushima Fatty rats were provided by the Tokushima Research Institute. At 50 weeks, wounds were arranged in two sites on the lateral paraspinal areas. Each wound was treated with PRP gel and physiologic saline gauze. To determine the expression of MMP-2, MMP-9, which was chosen as a marker of wound healing, reverse transcription polymerase chain reaction (RT-PCR) was performed and local distribution and expression of MMP-2, MMP-9 was also observed throughout the immunohistochemical staining. Results RT-PCR and the immunohistochemical study showed that the levels of MMP-2, MMP-9 mRNA expression in PRP applied tissues were higher than MMP-2, MMP-9 mRNA expression in saline-applied tissues. MMP-9 mRNA expression in wounds of diabetic rats decreased after healing began to occur. But no statistical differences were detected on the basis of body weight or fasting blood glucose levels. Conclusions This study could indicate the extracellular matrix-regulating effect observed with PRP. Our results of the acceleration of wound healing events by PRP under hyperglycemic conditions might be a useful clue for future clinical treatment for diabetic wounds. PMID:22783508

  3. Differences in Dural Penetration of Clival Chordomas Are Associated with Different Prognosis and Expression of Platelet-Derived Growth Factor Receptor-β. (United States)

    Zhai, Yixuan; Bai, Jiwei; Wang, Shuai; Du, Jiang; Wang, Jichao; Li, Chuzhong; Gui, Songbai; Zhang, Yazhuo


    We sought to compare the prognosis of clival chordomas with different dural penetration and establish the relationship between dural penetration and platelet-derived growth factor receptor (PDGFR)-β signaling pathway. Tumors in Type I (33 cases) showed limited dural penetration, while those in Type II (34 cases) had more serious dural penetration. Cox multivariate regression analysis was used to analyze risk factors affecting survival. Kaplan-Meier analysis measured overall survival (OS) and progression-free survival (PFS). To determine the relationship between dural penetration and PDGFR-β signaling, expression of PDGFR-β, Akt, mammalian target of rapamycin (mTOR), and phosphatase and tensin homolog (PTEN) expression was compared using immunohistochemistry, quantitative reverse transcription polymerase chain reaction, and Western blotting. Total resection was achieved in 9 cases in Type I and 11 in Type II. There were significant correlations between OS and dural penetration (P = 0.032) and age (P = 0.034). PFS correlated significantly with dural penetration (P = 0.022), gender (P = 0.001), and degree of resection (P = 0.001). Mean OS in Type I was significantly longer than in Type II (P = 0.046). Patients aged penetration. Patients with chordomas with serious dural penetration have poorer prognosis. Higher expression of PDGFR-β is related to more serious dural penetration of clival chordomas. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC). (United States)

    Basabe-Desmonts, L; Ramstrom, S; Meade, G; O'Neill, S; Riaz, A; Lee, L P; Ricco, A J; Kenny, D


    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 (

  5. Prevotella intermedia induces severe bacteremic pneumococcal pneumonia in mice with upregulated platelet-activating factor receptor expression. (United States)

    Nagaoka, Kentaro; Yanagihara, Katsunori; Morinaga, Yoshitomo; Nakamura, Shigeki; Harada, Tatsuhiko; Hasegawa, Hiroo; Izumikawa, Koichi; Ishimatsu, Yuji; Kakeya, Hiroshi; Nishimura, Masaharu; Kohno, Shigeru


    Streptococcus pneumoniae is the leading cause of respiratory infection worldwide. Although oral hygiene has been considered a risk factor for developing pneumonia, the relationship between oral bacteria and pneumococcal infection is unknown. In this study, we examined the synergic effects of Prevotella intermedia, a major periodontopathic bacterium, on pneumococcal pneumonia. The synergic effects of the supernatant of P. intermedia (PiSup) on pneumococcal pneumonia were investigated in mice, and the stimulation of pneumococcal adhesion to human alveolar (A549) cells by PiSup was assessed. The effects of PiSup on platelet-activating factor receptor (PAFR) transcript levels in vitro and in vivo were analyzed by quantitative real-time PCR, and the differences between the effects of pneumococcal infection induced by various periodontopathic bacterial species were verified in mice. Mice inoculated with S. pneumoniae plus PiSup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, spleen, and blood, and higher inflammatory cytokine levels in the bronchoalveolar lavage fluid (macrophage inflammatory protein 2 and tumor necrosis factor alpha) than those infected without PiSup. In A549 cells, PiSup increased pneumococcal adhesion and PAFR transcript levels. PiSup also increased lung PAFR transcript levels in mice. Similar effects were not observed in the supernatants of Porphyromonas gingivalis or Fusobacterium nucleatum. Thus, P. intermedia has the potential to induce severe bacteremic pneumococcal pneumonia with enhanced pneumococcal adhesion to lower airway cells.

  6. Transcriptional regulator PerA influences biofilm-associated, platelet binding, and metabolic gene expression in Enterococcus faecalis.

    Directory of Open Access Journals (Sweden)

    Scott M Maddox

    Full Text Available Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections, traits facilitated by the ability to quickly acquire and transfer virulence determinants. A 150 kb pathogenicity island (PAI comprised of genes contributing to virulence is found in many enterococcal isolates and is known to undergo horizontal transfer. We have shown that the PAI-encoded transcriptional regulator PerA contributes to pathogenicity in the mouse peritonitis infection model. In this study, we used whole-genome microarrays to determine the PerA regulon. The PerA regulon is extensive, as transcriptional analysis showed 151 differentially regulated genes. Our findings reveal that PerA coordinately regulates genes important for metabolism, amino acid degradation, and pathogenicity. Further transcriptional analysis revealed that PerA is influenced by bicarbonate. Additionally, PerA influences the ability of E. faecalis to bind to human platelets. Our results suggest that PerA is a global transcriptional regulator that coordinately regulates genes responsible for enterococcal pathogenicity.

  7. Platelet-Rich Plasma Attenuates 30-kDa Fibronectin Fragment-Induced Chemokine and Matrix Metalloproteinase Expression by Meniscocytes and Articular Chondrocytes. (United States)

    Wang, Chih-Chien; Lee, Chian-Her; Peng, Yi-Jen; Salter, Donald M; Lee, Herng-Sheng


    Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. Controlled laboratory study. Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase (MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin (IL)-6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. PRP treatment attenuates the 30-kDa FN-f-induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries. © 2015 The Author(s).

  8. Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells. (United States)

    Ulsemer, P; Lanza, F; Baas, M J; Schwartz, A; Ravanat, C; Briquel, M E; Cranmer, S; Jackson, S; Cazenave, J P; de la Salle, C


    The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and

  9. Endothelial cell-borne platelet bridges selectively recruit monocytes in human and mouse models of vascular inflammation. (United States)

    Kuckleburg, Christopher J; Yates, Clara M; Kalia, Neena; Zhao, Yan; Nash, Gerard B; Watson, Steve P; Rainger, George Ed


    Cells of the monocyte lineage are the most abundant inflammatory cells found in atherosclerotic lesions. Dominance of the inflammatory infiltrate by monocytes indicates that there is a disease-driven mechanism supporting their selective recruitment. Previous studies have demonstrated that interactions between endothelial cells (ECs) and platelets may promote monocyte recruitment. In this study, we sought to expand on this knowledge using a complex coculture model of the diseased vessel wall. Using primary human cells in an in vitro flow-based adhesion assay, we found that secretory arterial smooth muscle cells (SMCs), cocultured with ECs, promote preferential recruitment of monocytes from blood in a TGF-β1-dependent manner. Approximately 85% of leucocytes recruited to the endothelium were CD14(+). Formation of adhesive platelet bridges on ECs was essential for monocyte recruitment as platelet removal or inhibition of adhesion to the ECs abolished monocyte recruitment. Monocytes were recruited from flow by platelet P-selectin and activated by EC-derived CC chemokine ligand 2 (CCL2), although the presentation of CCL2 to adherent monocytes was dependent upon platelet activation and release of CXC chemokine ligand 4 (CXCL4). In an intravital model of TGF-β1-driven vascular inflammation in mice, platelets were also necessary for efficient leucocyte recruitment to vessels of the microcirculation in the cremaster muscle. In this study, we have demonstrated that stromal cells found within the diseased artery wall may promote the preferential recruitment of monocytes and this is achieved by establishing a cascade of interactions between SMCs, ECs, platelets, and monocytes.

  10. Lack in efficacy for imatinib mesylate as second-line treatment of recurrent or metastatic cervical cancer expressing platelet-derived growth factor receptor alpha. (United States)

    Candelaria, Myrna; Arias-Bonfill, Daymi; Chávez-Blanco, Alma; Chanona, José; Cantú, David; Pérez, Cetina; Dueñas-González, Alfonso


    Imatinib mesylate inhibits platelet-derived growth factor receptor (PDGFR), and there are evidences that the PDGFR participates in development and progression of cervical cancer. This pilot study was set to evaluate the efficacy in response rate and progression-free survival of imatinib. A secondary end point was to evaluate its safety as second-line treatment of recurrent or metastatic cervical cancer expressing PDGFRalpha. Imatinib mesylate was administered in daily dosages of 600 mg. Response was evaluated by positron emission tomography/computed tomography every two 28-day courses, and toxicity was evaluated weekly and thereafter. Twelve patients were included in the study. The median age was 49.8 years; all but 1 tumor were squamous cell carcinomas. First-line palliative chemotherapy with carboplatin-paclitaxel was the most frequently used scheme (75.0%). Ten (83.3%) had pelvic and systemic disease, whereas only 2 had systemic disease alone. All patients expressed the PDGFRalpha in more than 10% of malignant cells, whereas only 4 coexpressed the PDGFRbeta. No patient showed response. A single patient having metastatic disease in the lung showed stabilization for 6 months to then progressing in bone. No severe toxicities were seen except for the patient with worsening of bleeding from proctitis. Grades 1 and 2 gastrointestinal toxicities were common. Despite lack of activity of single-agent imatinib, further studies in cervical cancer are deserved to better define the status of imatinib targets in this tumor and to investigate its activity in combination with cytotoxic drugs.

  11. 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. (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


    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. © 2012 Blackwell Verlag GmbH.

  12. The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Wei [Jiangsu Diabetes Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China); Department of cardiology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150081 (China); Guo, Ting; Zhang, Yan; Jiang, Xiaohong; Zhang, Yongxian [Jiangsu Diabetes Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China); Zen, Ke, E-mail: [Jiangsu Diabetes Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China); Yu, Bo, E-mail: [Department of cardiology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150081 (China); Zhang, Chen-Yu, E-mail: [Jiangsu Diabetes Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China)


    Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.

  13. Role of sialic acid for platelet life span: exposure of beta-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor-expressing liver macrophages and hepatocytes

    DEFF Research Database (Denmark)

    Sørensen, Anne Louise; Rumjantseva, Viktoria; Nayeb-Hashemi, Sara


    Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet circulation lifetime is not fully clarified. We show that thrombocytopenia in mice deficient in the St3gal4 sialyltransferase gene (St3Gal-IV(-/-) mice)...

  14. The relation between oxidative stress and adhesion molecules in ...

    African Journals Online (AJOL)


    P-selectin is found in the storage granules of resting platelets as well as in the weibel–palade bodies of endothelial cells. After endothelial or platelet activation ..... in the regulation of actin binding proteins and adhesion molecules expressed on the endothelial cell membrane. In the present study MDA level was positively.

  15. Immunohistochemical expression of platelet-derived growth factor receptors in ovarian cancer patients with long-term follow-up

    DEFF Research Database (Denmark)

    Madsen, Christine Vestergaard; Dahl Steffensen, Karina; Waldstrøm, Marianne


    relation to histopathological parameters and long-term overall survival. Methods. The immunohistochemical expression of PDGFR-α and PDGFR-β was investigated in tumor and stromal cells in 170 patients with histologically verified epithelial ovarian cancer. Results. Almost half of the tumor specimens showed......Introduction. The well-documented role of the PDGF system in tumor growth and angiogenesis has prompted the development of new biological agents targeting the PDGF system. The aim of the present study was to analyze the expression of the PDGF-receptors in ovarian cancer and to investigate its...

  16. Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Passacquale

    Full Text Available BACKGROUND: Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA induces a pro-inflammatory phenotype in circulating monocytes. METHODOLOGY/PRINCIPAL FINDINGS: CD62P(+ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14(+CD16(-, CD14(highCD16(+and CD14(lowCD16(+. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01, along with enhancement of circulating CD14(highCD16(+ cells (4.7±3.6 vs 10.4±4.8; p = 0.003, their percentage being linearly related to levels of CD62P(+-platelets (r(2 = 0.4347; p = 0.0008. In separate in vitro experiments, co-incubation of CD14(+CD16(- cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14(+CD16(+ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001. This effect correlated directly with degree of MPA formation (r(2 = 0.7731; p<0.0001 and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1 blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809. CONCLUSIONS/SIGNIFICANCE: These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14(highCD16(+ monocytes in a

  17. Differences between Platelets Derived from Neonatal Cord Blood and Adult Peripheral Blood Assessed by Mass Spectrometry

    NARCIS (Netherlands)

    Stokhuijzen, Eva; Koornneef, Johanna M; Nota, Benjamin; van den Eshof, Bart Laurens; van Alphen, Floris Pieter Joachim; van den Biggelaar, Maartje; Van Der Zwaan, Carmen; Kuijk, Carlijn; Mertens, Koen|info:eu-repo/dai/nl/070940258; Fijnvandraat, Karin; Meijer, Alexander Benjamin|info:eu-repo/dai/nl/229834221


    It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free

  18. Sestrin 2 protein regulates platelet-derived growth factor receptor β (Pdgfrβ) expression by modulating proteasomal and Nrf2 transcription factor functions. (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


    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). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals. (United States)

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


    Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10(9)/L) than healthy individuals (240 × 10(9)/L, p healthy individuals using whole blood (pCOX-1 healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values healthy individuals (all p-values healthy individuals (all p-values >0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.

  20. Role of Siglec-7 in apoptosis in human platelets.

    Directory of Open Access Journals (Sweden)

    Kim Anh Nguyen

    Full Text Available Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs are well-characterized I-type lectins, which control apoptosis.We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i mitochondrial inner transmembrane potential (ΔΨm depolarization; (ii elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii phosphatidylserine exposure and (iv, microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis.The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions, Siglec-7 might be a molecular target for therapeutic intervention/prevention.

  1. Endothelial dysfunction, platelet activation, thrombogenesis and fibrinolysis in patients with hypertensive crisis. (United States)

    van den Born, Bert-Jan H; Löwenberg, Ester C; van der Hoeven, Niels V; de Laat, Bas; Meijers, Joost C M; Levi, Marcel; van Montfrans, Gert A


    Hypertensive crisis is an extreme phenotype of hypertension and hypertension-related thrombotic complications. This is most evident in patients with hypertensive crisis having advanced retinopathy and thrombotic microangiopathy (TMA). We examined whether hypertensive crisis complicated by advanced retinopathy is associated with endothelial dysfunction, platelet activation, thrombin generation and decreased fibrinolytic activity. In addition, we tested the association between these procoagulant changes and the development of TMA and end-organ dysfunction. Several key mediators of coagulation were assessed in 40 patients with hypertensive crisis with and without retinopathy and compared with 20 age, sex and ethnicity-matched normotensive controls. In patients with hypertensive crisis, associations with markers of TMA and renal dysfunction were assessed by regression analysis. Soluble P-selectin levels were higher in patients with hypertensive crisis compared with controls regardless of the presence or absence of retinopathy (Phypertensive crisis with retinopathy compared with normotensive controls (P-valuesHypertensive crisis with retinopathy confers a prothrombotic state characterized by endothelial dysfunction, platelet activation and increased thrombin generation, whereas fibrinolytic activity is enhanced. The observed changes in prothrombotic and antithrombotic pathways may contribute to the increased risk of ischaemic and haemorrhagic complications in this extreme hypertension phenotype. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

  2. Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells. (United States)

    Lange, Claudia; Brunswig-Spickenheier, Bärbel; Eissing, Leah; Scheja, Ludger


    Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Human blood platelets lack nitric oxide synthase activity. (United States)

    Böhmer, Anke; Gambaryan, Stepan; Tsikas, Dimitrios


    Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography-mass spectrometry (GC-MS) method to detect NOS activity in human platelets. The method measures simultaneously [(15)N]nitrite and [(15)N]nitrate formed from oxidized (15)N-labeled nitric oxide ((15)NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-(15)N2]-arginine. Using this GC-MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.

  4. Chronic exposure to endosulfan induces inflammation in murine colon via β-catenin expression and IL-6 production. (United States)

    Téllez-Bañuelos, Martha Cecilia; Haramati, Jesse; Franco-Topete, Karina; Peregrina-Sandoval, Jorge; Franco-Topete, Ramon; Zaitseva, Galina P


    Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of β-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant β-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose < LD50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2 mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20 mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30-42 weeks. Immunohistochemical analysis of the colon also showed that expression of β-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of β-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.

  5. Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis (United States)

    Lievens, Dirk; Zernecke, Alma; Seijkens, Tom; Soehnlein, Oliver; Beckers, Linda; Munnix, Imke C. A.; Wijnands, Erwin; Goossens, Pieter; van Kruchten, Roger; Thevissen, Larissa; Boon, Louis; Flavell, Richard A.; Noelle, Randolph J.; Gerdes, Norbert; Biessen, Erik A.; Daemen, Mat J. A. P.; Heemskerk, Johan W. M.; Weber, Christian


    CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l−/−) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l−/− platelets into Apoe−/− mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l−/− platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l−/− platelets in contrast to Cd40l+/+ platelets did not promote lesion formation. Remarkably, injection of Cd40l+/+, but not Cd40l−/−, platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l−/− platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions. PMID:20705757

  6. Effects of acute exercise on hemorheological, endothelial, and platelet markers in patients with chronic heart failure in sinus rhythm. (United States)

    Gibbs, C R; Blann, A D; Edmunds, E; Watson, R D; Lip, G Y


    Chronic heart failure (CHF) is associated with an increased risk of thrombosis and thromboembolic events, including stroke and venous thromboembolism. which may be related to a prothrombotic or hypercoagulable state. Acute vigorous exercise has been associated with activation of hemostasis, and this risk may well be particularly increased in patients with CHF. The study was undertaken to determine whether acute exercise would adversely affect abnormalities of hemorheological (fibrinogen, plasma viscosity, hematocrit), endothelial (von Willebrand factor), and platelet markers (soluble P selectin) in patients with CHF. We studied 22 ambulant outpatients (17 men; mean age 65+/-9 years) with stable CHF (New York Heart Association class II-III and a left ventricular ejection fraction of exercised to exhaustion on a treadmill. Results were compared with 20 hospital controls (patients with vascular disease, but free of CHF) and 20 healthy controls. Baseline von Willebrand factor (p = 0.01) and soluble P-selectin (p = 0.006) levels were significantly elevated in patients with CHF when compared with controls. In the patients with CHF who were exercised, plasma viscosity, fibrinogen, and hematocrit levels increased significantly, both immediately post exercise and at 20 min into the recovery period (repeated measures analysis of variance, all pexercise workload and the maximal changes in plasma viscosity in the patients with CHF (Spearman r = 0.5, p = 0.02). Plasma viscosity levels increased with exercise in the hospital control group, although no other exercise-induced changes were noted in this group. The present study indicates that the hemorheological indices. fibrinogen, and hematocrit specifically increase during acute exercise in patients with CHF. Although moderate exercise should be encouraged in patients with CHF, vigorous exercise should probably be avoided in view of its potential prothrombotic effects in this high-risk group of patients.

  7. 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.

  8. Platelets: developmental biology, physiology, and translatable platforms for preclinical investigation and drug development. (United States)

    Kleiman, Neal S; Freedman, Jane E; Tracy, Paula B; Furie, Barbara C; Bray, Paul F; Rao, Sunil V; Phillips, David R; Storey, Robert F; Rusconi, Christopher P; French, Patricia A; Steinhubl, Steven R; Becker, Richard C


    This paper, developed from the proceedings of the 2007 Platelet Colloquium, considers emerging constructs in platelet biology, preclinical models of thrombosis, and their potential application to the development of platelet-directed pharmacotherapies. Discussed first is the developmental biology of platelets, including megakaryocyte maturation, and the role of apoptotic and growth factors and other proteins in thrombopoiesis. A brief overview of current methods and observations from platelet proteomic analyses is also presented, illustrating the complex interplay of genes, gene expression, protein expression, and protein modification in various atherothrombotic phenotypes. The factor Xa-platelet interface is used as a working model for discussion of anticoagulants as platelet antagonists, highlighting the importance of receptor expression, substrate binding kinetics, platelet subpopulations, and cofactors in thrombosis. Finally, we discuss the use of emerging technologies--such as intravital microscopy and ex vivo perfusion chambers--as translatable platforms for investigating the role of platelets and their pharmacologic inhibition in human health and disease.

  9. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. (United States)

    Heijnen, H F; Schiel, A E; Fijnheer, R; Geuze, H J; Sixma, J J


    Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

  10. Platelet Count and Plateletcrit

    African Journals Online (AJOL)

    Aim: To determine whether platelet count, plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width. (PDW) and their ratios can predict mortality in hospitalised children. Methods: Children who died during hospital stay were the cases. Controls were age matched children admitted contempora- neously.

  11. Interactions of periodontal pathogens with megakaryocytic cells and platelets (United States)

    Andrews, A.M.; Haywood-Small, S.; Smith, T.; Stafford, P.


    ABSTRACT Introduction: Cardiovascular disease (CVD) is a leading cause of morbidity, accounting for around 17.3 million deaths worldwide. Recent studies have linked periodontitis to CVD with the periodonto-pathogens Porphyromonas gingivalis and Tannerella forsythia thought to contribute and exacerbate atherosclerosis through interactions with platelets. To date, while platelet activation following challenge with periodonto-pathogens has been reported, the underlying mechanisms of these interactions are yet to be elucidated. The aim of this study is to determine how periodonto-pathogens interact with platelets using both megakaryocytic cells and isolated platelets. Methods: To characterise expression levels of surface markers including ubiquitously expressed platelet-specific markers (CD41, CD42b) and platelet activation markers (CD62P, PAC-1), a multi-colour flow cytometry panel was developed using undifferentiated megakaryocytic cells CHRF-288-11 before validation using platelets isolated from healthy donors. Changes in levels of surface markers following bacterial challenge both with megakaryocytic cells and isolated platelets were determined using flow cytometry. Interaction with pathogens was visualised by platelet aggregometry and fluorescence microscopy using pathogen-specific antibodies. Results and conclusions: Both pathogens invaded megakaryocytic cells as visualised by immunofluorescence microscopy. The pathogens also bound platelets causing increased levels of aggregation and upregulated expression of activation markers including in CD62P in flow cytometric assays.

  12. 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


    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...

  13. Comprehensive comparison of neonate and adult human platelet transcriptomes.

    Directory of Open Access Journals (Sweden)

    Eva Caparrós-Pérez

    Full Text Available Understanding the underlying mechanisms of the well-substantiated platelet hyporeactivity in neonates is of interest given their implications for the clinical management of newborns, a population at higher bleeding risk than adults (especially sick and preterm infants, as well as for gaining insight into the regulatory mechanisms of platelet biology. Transcriptome analysis is useful in identifying mRNA signatures affecting platelet function. However, human fetal/neonatal platelet transcriptome analysis has never before been reported. We have used mRNA expression array for the first time to compare platelet transcriptome changes during development. Microarray analysis was performed in pure platelet RNA obtained from adult and cord blood, using the same platform in two independent laboratories. A high correlation was obtained between array results for both adult and neonate platelet samples. There was also good agreement between results in our adult samples and outcomes previously reported in three different studies. Gene enrichment analysis showed that immunity- and platelet function-related genes are highly expressed at both developmental stages. Remarkably, 201 genes were found to be differentially expressed throughout development. In particular, neonatal platelets contain higher levels of mRNA that are associated with protein synthesis and processing, while carrying significantly lower levels of genes involved in calcium transport/metabolism and cell signaling (including GNAZ. Overall, our results point to variations in platelet transcriptome as possibly underlining the hypo-functional phenotype of neonatal platelets and provide further support for the role of platelets in cellular immune response. Better characterization of the platelet transcriptome throughout development can contribute to elucidate how transcriptome changes impact different pathological conditions.

  14. Genetic engineering of platelets to neutralize circulating tumor cells. (United States)

    Li, Jiahe; Sharkey, Charles C; Wun, Brittany; Liesveld, Jane L; King, Michael R


    Mounting experimental evidence demonstrates that platelets support cancer metastasis. Within the circulatory system, platelets guard circulating tumor cells (CTCs) from immune elimination and promote their arrest at the endothelium, supporting CTC extravasation into secondary sites. Neutralization of CTCs in blood circulation can potentially attenuate metastases to distant organs. Therefore, extensive studies have explored the blockade of platelet-CTC interactions as an anti-metastatic strategy. Such an intervention approach, however, may cause bleeding disorders since the platelet-CTC interactions inherently rely on the blood coagulation cascade including platelet activation. On the other hand, platelets have been genetically engineered to correct inherited bleeding disorders in both animal models and human clinical trials. In this study, inspired by the physical association between platelets and CTCs, platelets were genetically modified to express surface-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine known to induce apoptosis specifically in tumor cells. The TRAIL-expressing platelets were demonstrated to kill cancer cells in vitro and significantly reduce metastases in a mouse model of prostate cancer metastasis. Our results suggest that using platelets to produce and deliver cancer-specific therapeutics can provide a Trojan-horse strategy of neutralizing CTCs to attenuate metastasis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment. (United States)

    Pignatelli, P; Di Santo, S; Buchetti, B; Sanguigni, V; Brunelli, A; Violi, F


    Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L-NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration-dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down-regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC-dependent NADPH oxidase activation. This effect, resulting in NO-mediated platelet glycoprotein GpIIb/IIIa down-regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.

  16. On the origin of matrix metalloproteinase-2 and -9 in blood platelets. (United States)

    Wrzyszcz, Aneta; Wozniak, Mieczyslaw


    To date, several matrix metalloproteinases (MMPs) have been identified in human platelets. In most research studies, the platelets are obtained using the isolation method from plasma by centrifugation and washing. The metalloproteinase content in the platelets can be affected by the isolation technique and the leukocyte contamination. In this work, we studied the influence of the isolation method on the detection of platelet MMPs and explore the expression of these enzymes in megakaryoblastic MEG-01 cells. We investigated the expression of mRNAs encoding for MMP-2 and -9 in platelets and MEG-01 cells. Using gelatin zymography and western blotting, we examined the expression and release of MMP-2 and 9 by platelets and MEG-01 cells and checked whether the amount of the released MMPs depends on the volume of tested platelet and leukocyte contamination. To investigate the MMP-2 expression profile, we used zymography and flow cytometry. Platelets, in contrast to the MEG-01 cells, neither contain mRNA for MMP-2 nor -9. The platelets contain pro-MMP-2 and release it during the activation. The population of uncontaminated (leukocytesplatelets contained no MMP-9 or the active form of MMP-2. We have observed that the activity of MMP-2 in platelet lysate is proportional to their mean volume and that the MMP-2 activity may not be detected if very small platelets are examined. We conclude that the detection of gelatinases in platelets depends on platelet isolation techniques and the degree of leukocyte contamination.

  17. Platelet alloimmunization after transfusion

    DEFF Research Database (Denmark)

    Taaning, E; Simonsen, A C; Hjelms, E


    BACKGROUND AND OBJECTIVES: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. MATERIALS AND METHODS: We studied the frequency of alloimmunization against platelet antigens in 117 patients...... who received a single series of blood transfusions. They received mostly saline-adenine-glucose+mannitol red blood cell components (poor in leukocytes and platelets) in connection with cardiac surgery. Platelet-specific antibodies were detected with the platelet ELISA and the monoclonal...... immunization. CONCLUSION: There was a low incidence of platelet-specific antibodies after one series of blood transfusions in this group of patients. This is similar to the results of some previous studies in multiply transfused patients, but not with those of others who found a higher incidence....

  18. Mean platelet volume and mean platelet volume/platelet count ratio ...

    African Journals Online (AJOL)

    Mean platelet volume and mean platelet volume/platelet count ratio as a risk stratification tool in the assessment of severity of acute ischemic stroke. ... The mean platelet volume (MPV) is a laboratory marker associated with platelet function and activity. Increased MPV in thromboembolic disease is reflected as an important ...

  19. A proteomics study reveals a predominant change in MaoB expression in platelets of healthy volunteers after high protein meat diet

    DEFF Research Database (Denmark)

    Zellner, Maria; Babeluk, Rita; Jakobsen, Lene Holm


    Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP)...... showed improved cognitive function and counteract well-known dementia biomarkers such as platelet MaoB and components of the methylation cycle such as vitamin B(12) and homocysteine....

  20. The effect of green laser light irradiation on whole blood platelets. (United States)

    Gresner, P; Watała, C; Sikurová, L


    Laser light irradiation is assumed to have biostimulating effect in various cell types. However, there is still a lack of information concerning response of blood platelets to laser light irradiation. In our study we used flow cytometry to monitor the effect of a green Nd-YAG laser (532 nm, 30 mW) irradiation on platelet activation and the expression of activated GPIIbIIIa glycoprotein complex (fibrinogen receptor) of whole blood platelets stained with fluorolabelled monoclonal antibody PAC-1. Also the formation of platelet microparticles and aggregates in a population of whole blood platelets following such irradiation was evaluated. Effects of laser light on platelet activation and reactivity were significant over a wide range of applied energies (plaser light energies (18 and 54 J) increased platelet activation, the irradiation with a high-energy laser light (108 J) resulted in depressed platelet reactivity and attenuated platelet response to activators. In addition, laser light irradiation had significant influence on the formation of platelet microparticles in either resting (plaser light irradiation significantly increased the formation of platelet aggregates both in resting (plaser light irradiation of blood platelets can trigger signal transduction, leading to platelet activation, as well as the gradual loss of natural platelet reactivity and platelets' ability to respond to activating agents.

  1. Potential role of platelet-leukocyte aggregation in trauma-induced coagulopathy: Ex vivo findings. (United States)

    Zipperle, Johannes; Altenburger, Katrin; Ponschab, Martin; Schlimp, Christoph J; Spittler, Andreas; Bahrami, Soheyl; Redl, Heinz; Schöchl, Herbert


    Platelet dysfunction has been identified as an important contributor of trauma-induced coagulopathy, but the underlying mechanism still remains to be elucidated. Trauma-associated proinflammatory stimuli strongly activate leukocytes, which in turn bind activated platelets. Therefore, we investigated the role of platelet-leukocyte aggregation (PLA) as a potential feature of trauma-induced platelet dysfunction. Whole blood from 10 healthy donors was exposed to selective and collective platelet and leukocyte agonists in order to simulate differential states of activation. PLA formation and CD11b expression as a measure of leukocyte activation were determined by flow cytometry. Platelet-mediated hemostatic function was measured by thromboelastometry (ROTEM) and impedance aggregometry (Multiplate). Activation of platelets and leukocytes was associated with diminished platelet-mediated hemostatic potential. Aggregation of platelets with monocytes rather than granulocytes resulted in a reduction of hemostatic function, as indicated by an impaired responsiveness in platelet aggregometry and a reduction of thromboelastometric maximum clot firmness. This finding was irrespective of CD11b expression and was not paralleled by a reduction of measurable platelet counts. PLA formation occurs primarily between monocytes and activated platelets and is associated with impaired platelet-mediated hemostatic function. PLA formation was not paralleled by a reduction in platelet complete blood counts.

  2. Measurement of platelet aggregation, independently of patient platelet count

    DEFF Research Database (Denmark)

    Vinholt, P J; Frederiksen, H; Hvas, A-M


    Essentials •Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. •A flow cytometric platelet aggregation test independent of the patient platelet count was made. •Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer....... •High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count...

  3. A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex. (United States)

    Lanza, François; De La Salle, Corinne; Baas, Marie-Jeanne; Schwartz, Agnès; Boval, Bernadette; Cazenave, Jean-Pierre; Caen, Jacques P


    This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.

  4. Surfactants reduce platelet-bubble and platelet-platelet binding induced by in vitro air embolism. (United States)

    Eckmann, David M; Armstead, Stephen C; Mardini, Feras


    The effect of gas bubbles on platelet behavior is poorly characterized. The authors assessed platelet-bubble and platelet-platelet binding in platelet-rich plasma in the presence and absence of bubbles and three surface-active compounds. Platelet-rich plasma was prepared from blood drawn from 16 volunteers. Experimental groups were surfactant alone, sparging (microbubble embolization) alone, sparging with surfactant, and neither sparging nor surfactant. The surfactants were Pluronic F-127 (Molecular Probes, Eugene, OR), Perftoran (OJSC SPC Perftoran, Moscow, Russia), and Dow Corning Antifoam 1510US (Dow Corning, Midland, MI). Videomicroscopy images of specimens drawn through rectangular glass microcapillaries on an inverted microscope and Coulter counter measurements were used to assess platelet-bubble and platelet-platelet binding, respectively, in calcium-free and recalcified samples. Histamine-induced and adenosine diphosphate-induced platelet-platelet binding were measured in unsparged samples. Differences between groups were considered significant for P bubbles in sparged, surfactant-free samples. With sparging and surfactant, few platelets adhered to bubbles. Numbers of platelet singlets and multimers not adherent to bubbles were different (P surfactant. No significant platelet-platelet binding occurred in uncalcified, sparged samples, although 20-30 platelets adhered to bubbles. Without sparging, histamine and adenosine diphosphate provoked platelet-platelet binding with and without surfactants present. Sparging causes platelets to bind to air bubbles and each other. Surfactants added before sparging attenuate platelet-bubble and platelet-platelet binding. Surfactants may have a clinical role in attenuating gas embolism-induced platelet-bubble and platelet-platelet binding.

  5. Progress in bio-manufacture of platelets for transfusion. (United States)

    Heazlewood, Shen Y; Nilsson, Susan K; Cartledge, Kellie; Be, Cheang Ly; Vinson, Andrew; Gel, Murat; Haylock, David N


    Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

  6. Platelets: much more than bricks in a breached wall. (United States)

    Mancuso, Maria Elisa; Santagostino, Elena


    Platelets have various roles in vascular biology and homeostasis. They are the first actor in primary haemostasis and play important roles in thrombosis pathogenesis, but they are also part of innate immunity, which initiates and accelerate many inflammatory conditions. In some contexts, their immune functions are protective, while in others they contribute to adverse inflammatory outcomes. Platelets express numerous receptors and contain hundreds of secretory molecules that are crucial for platelet functional responses. The capacity of platelets to produce and secrete cytokines, chemokines and related molecules, under the control of specific intracellular pathways, is intimately related to their key role in inflammation. They are also able to intervene in tissue regeneration and repair because they produce pro-angiogenic mediators. Due to this characteristic platelets are involved in cancer progression and spreading. In this review we discuss the complex role of platelets, which bridges haemostasis, inflammation and immune response both in physiological and pathological conditions. © 2017 John Wiley & Sons Ltd.

  7. Platelet activation and aggregation

    DEFF Research Database (Denmark)

    Jensen, Maria Sander; Larsen, O H; Christiansen, Kirsten


    This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated...

  8. Serotonergic mechanisms enhance platelet-mediated thrombogenicity. (United States)

    Galan, Ana M; Lopez-Vilchez, Irene; Diaz-Ricart, Maribel; Navalon, Fulgencio; Gomez, Esther; Gasto, Cristobal; Escolar, Gines


    Although it is generally acknowledged that serotonin (5-HT) is a weak agonist for human platelets, recent information suggests an association between serotonergic mechanisms and cardiovascular risk. We investigated the action of 5-HT on adhesive, cohesive and procoagulant properties of human platelets. Impact of 5-HT on whole blood coagulation and thrombin generation was measured by modified thromboelastometry (TEM) and specific fluorogenic assays. We evaluated the effects of 5-HT on thrombus formation in an in-vitro model of thrombosis using human flowing blood. In platelet-rich plasma (PRP), 5-HT favoured the expression of CD62-P, and procoagulant molecules on platelet membranes. These effects were potentiated in the presence of Ca(++) and/or ADP. Incubation with 5-HT accelerated clotting times and augmented clot strength in whole blood TEM, and enhanced thrombin generation in PRP. In perfusion studies, 5-HT significantly increased fibrin deposition at low shear (300s(-1)) and enhanced platelet thrombus formation on the damaged vascular surface at high shear (1,200s(-1)). Selective inhibition of serotonin reuptake (SSRI) attenuated effects of 5-HT on platelet activation and downregulated the prothrombotic tendencies observed in the previous experimental conditions. In general, reductions of thrombogenic patterns observed with SSRI were more evident under shear conditions (aggregation and perfusion systems) and less evident under steady conditions (TEM and thrombin generation assays). In conclusion, 5-HT is not a weak agonist for human platelets; instead it accentuates platelet activation, potentiates procoagulant responses on human blood and increases thrombogenesis on damaged vascular surfaces. The remarkable antithrombotic actions achieved through SSRI deserve further mechanistic and clinical investigations.

  9. Platelets and infections—complex interactions with bacteria

    Directory of Open Access Journals (Sweden)



    Full Text Available Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-Like Receptors but also integrins conventionally described in the hemostatic response, such as GPIIb-IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of Neutrophil Extracellular Traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet-bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the

  10. Aggregation of human platelets and adhesion of Streptococcus sanguis. (United States)

    Herzberg, M C; Brintzenhofe, K L; Clawson, C C


    Platelet vegetations or thrombi are common findings in subacute bacterial endocarditis. We investigated the hypothesis that human platelets selectively bind or adhere strains of Streptococcus sanguis and Streptococcus mutans and aggregate, as a result, into an in vitro thrombus. Earlier ultrastructural studies suggested that aggregation of platelets over time by Staphylococcus aureus was preceded in order by adhesion and platelet activation. We uncoupled the adhesion step from activation and aggregation in our studies by incubating streptococci with platelet ghosts in a simple, quantitative assay. Adhesion was shown to be mediated by protease-sensitive components on the streptococci and platelet ghosts rather than cell surface carbohydrates or dextrans, plasma components, or divalent cations. The same streptococci were also studied by standard aggregometry techniques. Platelet-rich plasma was activated and aggregated by certain isolates of S. sanguis. Platelet ghosts bound the same strains selectively under Ca2+- and plasma-depleted conditions. Fresh platelets could activate after washing, but Ca2+ had to be restored. Aggregation required fresh platelets in Ca2+-restored plasma and was inducible by washed streptococcal cell walls. These reactions in the binding and aggregometry assays were confirmed by transmission electron microscopy. Surface microfibrils on intact S. sanguis were identified. These appendages appeared to bind S. sanguis to platelets. The selectivity of adhesion of the various S. sanguis strains to platelet ghosts or Ca2+- and plasma-depleted fresh washed platelets was similar for all donors. Thus, the platelet binding site was expressed widely in the population and was unlikely to be an artifact of membrane aging or preparation. Since selective adhesion of S. sanguis to platelets was apparently required for aggregation, it is suggested that functionally defined receptors for ligands on certain strains of S. sanguis may be present on human

  11. A preliminary study on the expression and clinical value of platelet-derived growth factor BB, hypoxia inducible factor-1α and C-C motif chemokine receptor-2 in peripheral blood during the pathogenesis of Graves' disease. (United States)

    Liu, Ying; Tang, Jinglan; Hu, Qiaohong; Lu, Kefeng; Hou, Chunjie


    Platelet-derived growth factor BB (PDGF-BB) plays an important role in the development of GD (Graves' disease). However, it is still unknown whether PDGF-BB is expressed in peripheral blood and whether the expression of PDGF-BB contributes to GD. We aim to study the expression of PDGF-BB, hypoxia inducible factor (HIF)-1α and C-C motif chemokine receptor (CCR)-2 in peripheral blood of patients with GD and explore its effect and potential mechanism in pathogenesis. 41 patients with GD (GD group) and forty-five healthy people (control group) were chosen. The concentration of PDGF-BB and HIF-1α in peripheral blood specimens were detected and compared between the two groups. The expression of CCR2 in macrophages in the peripheral blood specimens were examined using FCM (Flow Cytometry). Both PDGF-BB and HIF-1α were expressed in human peripheral blood from the two groups. Compared with specimens from healthy people, there were statistically increased concentrations of PDGF-BB and HIF-1α in the GD group (P BB through HIF-1α signal, and the high expression of PDGF-BB may be involved in the pathogenesis of GD.

  12. Low differentiated microvascular density and low expression of platelet-derived growth factor-BB (PDGF-BB) predict distant metastasis and poor prognosis in clear cell renal cell carcinoma. (United States)

    Qi, Lifeng; Du, Jun; Zhang, Zhenting; Diao, Lei; Chen, Xusheng; Yao, Xin


    To examine the prognostic significance of the expression of platelet-derived growth factor-BB (PDGF-BB) and differentiated microvascular density (MVD) in patients with clear cell renal cell carcinoma (ccRCC). We used the vascular marker cluster of differentiation 34 (CD34) to identify tumour blood vessels. The expression of PDGF-BB and CD34 was detected by immunohistochemistry (IHC) in tissue microarrays (TMAs) from 100 ccRCCs. Prognostic effects of individual parameters were calculated using Cox regression models and Harrell's concordance index (c-index). Higher grade and more advanced stage ccRCCs had significantly less PDGF-BB expression and differentiated MVD (P BB expression was an independent prognostic factor for longer survival, and moreover, the final model built by the addition of PDGF-BB expression improved the predictive accuracy for disease-free survival (c-index 0.707) compared with the clinicopathological-based model (c-index 0.695). PDGF-BB expression was positively associated with differentiated MVD assessed by Spearman correlation and factor analysis (r = 0.634, P BB is as a novel and promising prognostic marker and antiangiogenic therapeutic target for the treatment of ccRCC. © 2013 The Authors. BJU International © 2013 BJU International.

  13. Platelet function in dogs

    DEFF Research Database (Denmark)

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


    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...... applied. However, the importance of these breed differences remains to be investigated. The PFA-100 method with Col + Epi as agonists, and ADP-induced platelet aggregation appear to be sensitive to ASA in dogs....

  14. Platelet counts and mean platelet volume amongst elderly Nigerians ...

    African Journals Online (AJOL)

    determining reference values of Platelet Counts, Mean Platelet Volume and the relationship between the Platelet Count and Mean Platelet Volume. These parameters were determined from 400 healthy elderly subjects comprising 210 males and 190 females. with a mean age of 69.4±7.9 years . 400 young adults were used ...

  15. Oxidative stress induced modulation of platelet integrin α2bβ3 expression and shedding may predict the risk of major bleeding in heart failure patients supported by continuous flow left ventricular assist devices. (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


    Oxidative stress and platelet integrin α2bβ3 plays important role in the process of hemostasis and thrombosis. We hypothesized that device-induced patient specific oxidative stress and integrin α2bβ3 shedding may be linked to major bleeding complication (MBC) in heart failure (HF) patients supported by continuous flow left ventricular assist devices (CF-LVADs). We recruited 47patients implanted with CF-LVADs and 15 healthy volunteers. Fourteen patients developed MBC (bleeder group) within one month after implantation while others were considered non-bleeder group (n=33). Oxidative stresses were evaluated by measuring reactive oxygen species (ROS) in platelets, superoxide dismutase (SOD) activity, total antioxidant capacity (TAC) and oxidized low density lipoprotein (oxLDL). Assessments of α2bβ3 were carried out using flow cytometry and ELISA. Biomarkers of oxidative stress and α2bβ3 shedding (decreased surface expression and higher plasma levels) were found to be preexisting condition in all HF patients prior to CF-LVAD implantation compared to the healthy volunteers. Significantly elevated levels of ROS and oxLDL; concomitant depletion of SOD and TAC; and α2bβ3 shedding were observed in the bleeder group temporarily in comparison to the non-bleeder group after CF-LVAD implantation. A significantly strong association between α2bβ3 shedding and biomarkers of oxidative stress was observed; suggesting a potential role of oxidative stress in platelet integrin shedding leading to MBC after CF-LVAD implantation. Moreover, a receiver operating characteristic (ROC) analysis indicated that the likelihood of MBC data from Integrin α2bβ3 shedding had a predictive power of MBC in CF-LVAD patients. Oxidative stress might play a potential role in accelerating α2bβ3 shedding and platelet dysfunction, resulting in MBC in CF-LVAD patients. Integrin α2bβ3 shedding may be used to refine bleeding risk stratification in CF-LVAD patients. Copyright © 2017 Elsevier Ltd

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

    Energy Technology Data Exchange (ETDEWEB)

    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))


    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. Correlation between Platelet Gelsolin and Platelet Activation Level in Acute Myocardial Infarction Rats and Intervention Effect of Effective Components of Chuanxiong Rhizome and Red Peony Root

    Directory of Open Access Journals (Sweden)

    Yue Liu


    Full Text Available The biological role of platelet gelsolin in platelet activation of acute myocardial infarction is not defined. In order to provide a potential new antiplatelet target for Chinese medicine and to elucidate the contribution of Xiongshao capsule, the effective components of Chuanxiong rhizome and red peony root, in this study, we randomly allocated Sprague Dawley rats to left anterior descending coronary artery ligation or sham surgery and different drug prophylaxis as control. We found that gelsolin is highly expressed in platelet rich plasma and lowly expressed in platelet poor plasma, accompanied by the high platelet activation level in model rats; plasma actin filaments and mean fluorescence intensity (MFI of platelet calcium ion increased and plasma vitamin D binding protein decreased in model rats. Xiongshao capsule could inhibit the gelsolin expression in platelet rich plasma and ischemic heart tissue simultaneously and reduce the level of plasma F-actin and MFI of platelet calcium ion. Our study concludes that platelet gelsolin is an important contributor to platelet activation, and platelet gelsolin inhibition may form a novel target for antiplatelet therapy. Xiongshao capsule may be a promising Chinese medicine drug for antiplatelet and aspirin-like cardioprotection effect.

  18. Rethinking platelet function: thrombocytopenia induced immunodeficiency in critical illness

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Johansson, Per Ingemar


    traditional innate immune cells, platelets are recruited immediately into injured and inflamed tissue, they release immune mediators, express and shed immunologically active membrane receptors, they interact with other immune cells and they recognize and clear pathogens. We hypothesize that thrombocytopenia...

  19. Inhibiting GPIbα shedding preserves post-transfusion recovery and hemostatic function of platelets after prolonged storage (United States)

    Chen, Wenchun; Liang, Xin; Syed, Anum K.; Jessup, Paula; Church, William R.; Ware, Jerry; Josephson, Cassandra D.; Li, Renhao


    Objectives The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor GPIbα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed. Approach and Results Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα (hTg) were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored hTg platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently 5G6 Fab-stored 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. Conclusions Specific inhibition of GPIbα shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be utilized to optimize platelet storage conditions. PMID:27417583

  20. In vivo quantitation of platelet deposition on human peripheral arterial bypass grafts using indium-111-labeled platelets. Effect of dipyridamole and aspirin

    Energy Technology Data Exchange (ETDEWEB)

    Pumphrey, C.W.; Chesebro, J.H.; Dewanjee, M.K.; Wahner, H.W.; Hollier, L.H.; Pairolero, P.C.; Fuster, V.


    Indium-111-labeled autologous platelets, injected 48 hours after operation, were used to evaluate the thrombogenicity of prosthetic material and the effect of platelet inhibitor therapy in vivo. Dacron double-velour (Microvel) aortofemoral artery bifurcation grafts were placed in 16 patients and unilateral polytetrafluoroethylene femoropopliteal grafts were placed in 10 patients. Half the patients in each group received platelet inhibitors before operation (dipyridamole, 100 mg 4 times a day) and after operation (dipyridamole, 75 mg, and acetylsalicylic acid, 325 mg 3 times a day); the rest of the patients served as control subjects. Five-minute scintigrams of the graft region were taken with a gamma camera interfaced with a computer 48, 72, and 96 hours after injection of the labeled platelets. Platelet deposition was estimated from the radioactivities of the grafts and expressed as counts per 100 pixels per microcurie injected. Dipyridamole and aspirin therapy significantly reduced the number of platelets deposited on Dacron grafts and prevented platelet accumulation over 3 days. With the small amount of platelet deposition on polytetrafluoroethylene femoropopliteal artery grafts even in control patients, platelet inhibitor therapy had no demonstrable effect on platelet deposition on these grafts. It is concluded that (1) platelet deposition on vascular grafts in vivo can be quantitated by noninvasive methods, and (2) dipyridamole and aspirin therapy reduced platelet deposition on Dacron aortofemoral artery grafts.

  1. Platelets as a Novel Source of Pro-Inflammatory Chemokine CXCL14

    Directory of Open Access Journals (Sweden)

    Alexander Witte


    Full Text Available Objective: Platelets are a major source of chemokines. Here, we demonstrate for the first time that platelets express significant amounts of CXCL14 and disclose powerful effects of platelet-derived CXCL14 on monocyte and endothelial migration. Methods: The expression of CXCL14 in platelets and in the supernatant of activated platelets was analysed by immunoblotting, ELISA, and flow cytometry. The effect of platelet-derived CXCL14 on monocyte migration was evaluated using a modified Boyden chamber. The effect of CXCL14 on monocyte phagocytosis was tested by using fluorochrome-labelled E.coli particles. The effect of platelet-derived CXCL14 on endothelial migration was explored by the use of an endothelial scratch assay. Results: Hitherto unrecognized expression of CXCL14 in human and murine platelets was uncovered by immunoblotting. Activation with platelet agonists such as adenosine-di-phosphate (ADP, collagen-related peptide (CRP, or thrombin-receptor activating peptide (TRAP, increased CXCL14 surface expression (flow cytometry and release into the supernatant (immunoblotting, ELISA. Since CXCL14 is known to be chemotactic for CD14+ monocytes, we investigated the chemotactic potential of platelet-derived CXCL14 on human monocytes. Activated platelet supernatant induced monocyte migration, which was counteracted upon neutralization of platelet-derived CXCL14 as compared to IgG control. Blocking of the chemokine receptor CXCR4, but not CXCR7, reduced the number of migratory monocytes towards recombinant CXCL14, suggesting the involvement of CXCR4 in the CXCL14-directed monocyte chemotaxis. Recombinant CXCL14 enhanced the phagocytic uptake of E.coli particles by monocytes. In scratch assays with cultured endothelial cells (HUVECs, platelet-derived CXCL14 counteracted the pro-angiogenic effects of VEGF, supporting its previously recognized angiostatic potential. Conclusions: Platelets are a relevant source of CXCL14. Platelet-derived CXCL14 at the

  2. Congenital platelet function defects (United States)

    Arnold DM, Patriquin C, Toltl LJ, Nazi I, Smith J, Kelton J. Diseases of platelet number: immune thrombocytopenia, neonatal alloimmune thrombocytopenia, and posttransfusion purpura. In: Hoffman R, Benz EJ ...

  3. Preaggregation reactions of platelets. (United States)

    Gear, A R


    Whether platelet volume increases during the morphological changes preceding aggregation has been investigated. Previous research is controversial; resistive-counting techniques reveal an increase, centrifugal methods do not. Platelets were sized with a computerized, resistive-particle counter before and after incubation with adenosine diphosphate (ADP). Resistive volume increased by 14% (p less than 0.001) in the absence of EDTA, and only 7% in its presence (ADP, 10 micro M). EDTA inhibited platelet volume changes, whether these were shrinking induced by warming or swelling by ADP. Handling of platelets, such as during centrifugation, also caused particle swelling. Particle density decreased after ADP exposure, without release of serotonin, suggesting uptake of water. Platelet shape was experimentally manipulated to test the hypothesis that resistive volume changes stem from artifacts of particle shape. Scanning electron microscopy confirmed that colchicine, chlorpromazine, and a temperature cycle of 0 degrees to 37 degrees all caused extensive alteration from the disc shape. Subsequent exposure to ADP increased resistive volume, and in the case of chlorpromazine, no long pseudopodia were extruded. It is concluded that preaggregation reactions of platelets can be associated with an increase in particle volume, and that earlier research based on centrifugation and the presence of ETA failed to reveal the increase because of inhibitory and apparent swelling effects.

  4. Effect of clopidogrel discontinuation at 1 year after drug eluting stent placement on soluble CD40L, P-selectin and C-reactive protein levels : DECADES (Discontinuation Effect of Clopidogrel After Drug Eluting Stent): a multicenter, open-label study

    NARCIS (Netherlands)

    Wykrzykowska, Joanna J.; Warnholtz, Ascan; de Jaeger, Peter; Curzen, Nick; Oldroyd, Keith G.; Collet, Jean Philippe; Ten Berg, Jurrien M.; Rademaker, Tessa; Goedhart, Dick; Lissens, Jurgen; Kint, Peter-Paul; Serruys, Patrick W.


    Antiplatelet therapy with clopidogrel has been shown to reduce major adverse cardiac events in acute coronary syndromes and after percutaneous interventions. This effect is not only due to its anti-platelet effect but also possibly due to an anti-inflammatory effect. The effect of clopidogrel

  5. Platelet-derived growth factor BB promotes the migration of bone marrow-derived mesenchymal stem cells towards C6 glioma and up-regulates the expression of intracellular adhesion molecule-1. (United States)

    Cheng, Peng; Gao, Zhi-Qiang; Liu, Yun-Hui; Xue, Yi-Xue


    Recent studies have indicated that bone marrow-derived mesenchymal stem cells (BMSCs) have the capacity of migrating towards gliomas. However, few data are available about the molecular mechanism responsible for this migratory capacity. The aim of our study was to investigate the role of platelet-derived growth factor BB (PDGFBB) in the migration of BMSCs towards C6 glioma and evaluate the effect of PDGFBB on the migrating capacity and intercellular adhesion molecule-1 (ICAM-1) expression of BMSCs. The chemokinetic activity of BMSCs in response to C6 glioma-conditioned medium and recombinant rat PDGFBB was analyzed by in vitro migration assay. The effect of PDGFBB on the expression of ICAM-1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Our data showed that C6 glioma-conditioned medium significantly increased the migration of BMSCs, which could be partially blocked by a PDGFBB neutralizing antibody. Recombinant rat PDGFBB enhanced the migration of BMSCs in a concentration-dependent way from 5 to 50ng/ml. Moreover, RT-PCR and immunofluorescence showed that 12h of 20ng/ml PDGFBB incubation could up-regulate the ICAM-1 expression of BMSCs. Our data also revealed that SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), significantly decreased the PDGFBB-induced migration and ICAM-1 expression of BMSCs. These results demonstrate that PDGFBB contributes to the migration of BMSCs towards C6 glioma and up-regulates the expression of ICAM-1, and that p38MAPK is an important signaling molecule correlating with the signal transduction of PDGFBB-induced migration and ICAM-1 expression of BMSCs.

  6. Assessing blood platelets as RNA biomarker source for prostate cancer. (United States)

    Hänze, Jörg; Jakubowski, Peter; Heers, Hendrik; Hegele, Axel; Timmesfeld, Nina; Hofmann, Rainer; Olbert, Peter J


    Blood platelets may offer as RNA biomarker source for cancer as recently described for an oncogenic transcript in glioma patients and for PCA3 in prostate cancer (PCa) patients. Here, we elaborated on this aspect for PCa. PCA3 and other PCa-associated RNA markers were measured in platelets of PCa patients (cases) and healthy subjects (controls) in comparison to PCa cell lines by relative quantitative RT-PCR. The RNA markers displayed heterogeneous expression patterns in cell lines and platelets, however, without significant differences between cases and controls. The data do not support platelets as a profitable RNA source for early detection of PCa. Nonetheless, certain PCa-derived RNA markers in platelets may merit further investigation as potential prognostic biomarkers for PCa.

  7. Clinical uses of radiolabeled platelets

    Energy Technology Data Exchange (ETDEWEB)

    Datz, F.L.; Christian, P.E.; Baker, W.J.


    Platelets were first successfully radiolabeled in 1953. At that time, investigators were primarily interested in developing a technique to accurately measure platelet life span in both normal and thrombocytopenic patients. Studies using platelets labeled with /sup 51/Cr have shown shortened platelet survival times in a number of diseases including idiopathic thrombocytopenic purpura, coronary artery disease, and diabetes mellitus. More recently, labels such as /sup 111/In have been developed that allow in vivo imaging of platelets. Indium-111 platelets are being used to better understand the pathophysiology of atherosclerosis, thrombophlebitis, pulmonary embolism and clotting disorders, and to improve the clinical diagnosis of these diseases.

  8. Correlation between Cardiorespiratory Fitness and Platelet Function in Healthy Women. (United States)

    Heber, Stefan; Assinger, Alice; Pokan, Rochus; Volf, Ivo


    Low cardiorespiratory fitness (CRF) represents a major risk factor for atherosclerosis, and platelets play a key role in the development of this chronic inflammatory disease. Therefore, the purpose of this study was to assess the relationship between CRF and platelet function. CRF and different aspects of platelet function were assessed in healthy, young, nonsmoking women. Results were compared between groups of low (LF), medium (MF) and high CRF (HF). Measurements were repeated in group LF after a supervised endurance training program lasting two menstrual cycles and obtained results were compared with groups MF and HF. CRF was quantified by maximal oxygen consumption (V˙O2max) determined by an incremental treadmill exercise test. V˙O2max criteria for groups were (mL·min·kg bodyweight): LF 55. Platelet activation state and platelet reactivity were assessed by basal and agonist-induced surface expression of CD62P and CD40L as well as the intraplatelet amount of reactive oxygen species. In group LF, basal platelet activation as well as agonist-induced platelet reactivity were increased compared with groups MF and HF. Between groups MF and HF parameters of platelet function were roughly equal despite a pronounced difference regarding CRF. Exercise training improved CRF in group LF and aligned platelet function to levels observed in groups MF and HF, although CRF still markedly differed. Low levels of CRF favor a proinflammatory platelet phenotype. A relatively low dose of exercise is sufficient to normalize platelet function, whereas superior levels of physical activity and CRF do not provide any further substantial benefit, but also no appreciable adverse effects.

  9. Expression, purification, and analysis of three recombinant ECD disintegrins (r-colombistatins) from P-III class snake venom metalloproteinases affecting platelet aggregation and SK-MEL-28 cell adhesion. (United States)

    Suntravat, Montamas; Helmke, Thomas J; Atphaisit, Chairat; Cuevas, Esteban; Lucena, Sara E; Uzcátegui, Nestor L; Sánchez, Elda E; Rodriguez-Acosta, Alexis


    Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases. Copyright © 2016. Published by Elsevier Ltd.

  10. Platelet serotonin transporter function predicts default-mode network activity.

    Directory of Open Access Journals (Sweden)

    Christian Scharinger

    Full Text Available The serotonin transporter (5-HTT is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence.A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD activity and platelet Vmax.The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity.This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation.

  11. Potential of pomegranate fruit extract (Punica granatum Linn. to increase vascular endothelial growth factor and platelet-derived growth factor expressions on the post-tooth extraction wound of Cavia cobaya

    Directory of Open Access Journals (Sweden)

    Intan Nirwana


    Full Text Available Background: Pomegranates fruit extracts have several activities, among others, anti-inflammatory, antibacterial, and antioxidants that have the main content punicalagin and ellagic acid. Pomegranate has the ability of various therapies through different mechanisms. Vascular endothelial growth factor (VEGF function was to form new blood vessels produced by various cells one of them was macrophages. Platelet-derived growth factor (PDGF was a growth factor proven chemotactic, increased fibroblast proliferation and collagen matrix production. In addition, VEGF and PDGF synergize in their ability to vascularize tissues. The PDGF function was to stabilize and regulate maturation of new blood vessels. Activities of pomegranate fruit extract were observed by measuring the increased of VEGF and PDGF expression as a marker of wound healing process. Aim: To investigate the potential of pomegranate extracts on the tooth extraction wound to increase the expression of VEGF and PDGF on the 4th day of wound healing process. Materials and Methods: This study used 12 Cavia cobaya, which were divided into two groups, namely, the provision of 3% sodium carboxymethyl cellulose and pomegranate extract. The 12 C. cobaya would be executed on the 4th day, the lower jaw of experimental animals was taken, decalcified about 30 days. The expression of VEGF and PDGF was examined using immunohistochemical techniques. The differences of VEGF and PDGF expression were evaluated statistically using t-test. Results: Statistically analysis showed that there were significant differences between control and treatment groups (p<0.05. Conclusion: Pomegranate fruit extract administration increased VEGF and PDGF expression on post-tooth extraction wound.

  12. The Platelet and Platelet Function Testing in Liver Disease

    NARCIS (Netherlands)

    Hugenholtz, Greg G. C.; Porte, Robert J.; Lisman, Ton

    Patients who have liver disease commonly present with alterations in platelet number and function. Recent data have questioned the contribution of these changes to bleeding complications in these patients. Modern tests of platelet function revealed compensatory mechanisms for the decreased platelet

  13. Mean Platelet Volume (MPV), Platelet Distribution Width (PDW ...

    African Journals Online (AJOL)

    Background: Thrombocytopenia has been shown to predict mortality. We hypothesize that platelet indices may be more useful prognostic indicators. Our study subjects were children one month to 14 years old admitted to our hospital. Aim: To determine whether platelet count, plateletcrit (PCT), mean platelet volume (MPV) ...

  14. Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

    Directory of Open Access Journals (Sweden)

    Zainab S Al-Hosni


    Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

  15. Platelet PI3Kγ Contributes to Carotid Intima-Media Thickening under Severely Reduced Flow Conditions.

    Directory of Open Access Journals (Sweden)

    Cuiping Wang

    Full Text Available Studies have begun to focus on the emerging function of platelets as immune and inflammatory cells that initiate and accelerate vascular inflammation. Phosphoinositide 3-kinase gamma (PI3Kγ is critically involved in a number of inflammatory and autoimmune diseases. This study aims to investigate the contribution of platelet PI3Kγ to vascular remodeling under flow severely reduced conditions. Mouse partial left carotid artery ligation with adoptive transfer of activated, washed wild-type or PI3Kγ-/- platelets was used as the model. Intima-media area, leukocyte recruitment, and proinflammatory mediator expression were assessed. In vitro PI3Kγ-/- platelets were used to verify the effect of PI3Kγ on platelet activation, interaction with leukocytes, and endothelial cells. Mice injected with activated platelets showed a significant increase in intima-media thickening, recruitment of neutrophils (at 3 d and macrophages (at 21 d, and intercellular adhesion molecule-1, vascular cell adhesion molecule-1, tumor necrosis factor alpha, and interleukin-6 expression (at 3 d in the flow-reduced area. These effects were abrogated by platelet PI3Kγ deficiency. Circulating platelet-leukocyte aggregates were reduced in PI3Kγ-/- mice after partial ligation. In vivo data confirmed that PI3Kγ mediated Adenine di-Phosphate -induced platelet activation through the Akt and p38 MAP kinase signaling pathways. Moreover, platelet PI3Kγ deficiency reduced platelet-leukocyte aggregation and platelet-endothelial cell (EC interaction. These findings indicate that platelet PI3Kγ contributes to platelet-mediated vascular inflammation and carotid intima-media thickening after flow severely reduced. Platelet PI3Kγ may be a new target in the treatment of vascular diseases.

  16. In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution. (United States)

    Diedrich, B; Sandgren, P; Jansson, B; Gulliksson, H; Svensson, L; Shanwell, A


    Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.

  17. Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

    NARCIS (Netherlands)

    Dercksen, M. W.; Gerritsen, W. R.; Rodenhuis, S.; Dirkson, M. K.; Slaper-Cortenbach, I. C.; Schaasberg, W. P.; Pinedo, H. M.; von dem Borne, A. E.; van der Schoot, C. E.


    Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very

  18. Nickel nanoparticles enhance platelet-derived growth factor-induced chemokine expression by mesothelial cells via prolonged mitogen-activated protein kinase activation. (United States)

    Glista-Baker, Ellen E; Taylor, Alexia J; Sayers, Brian C; Thompson, Elizabeth A; Bonner, James C


    Pleural diseases (fibrosis and mesothelioma) are a major concern for individuals exposed by inhalation to certain types of particles, metals, and fibers. Increasing attention has focused on the possibility that certain types of engineered nanoparticles (NPs), especially those containing nickel, might also pose a risk for pleural diseases. Platelet-derived growth factor (PDGF) is an important mediator of fibrosis and cancer that has been implicated in the pathogenesis of pleural diseases. In this study, we discovered that PDGF synergistically enhanced nickel NP (NiNP)-induced increases in mRNA and protein levels of the profibrogenic chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2), and the antifibrogenic IFN-inducible CXC chemokine (CXCL10) in normal rat pleural mesothelial 2 (NRM2) cells in vitro. Carbon black NPs (CBNPs), used as a negative control NP, did not cause a significant increase in CCL2 or CXCL10 in the absence or presence of PDGF. NiNPs prolonged PDGF-induced phosphorylation of the mitogen-activated protein kinase family termed extracellular signal-regulated kinases (ERK)-1 and -2 for up to 24 hours, and NiNPs also synergistically increased PDGF-induced hypoxia-inducible factor (HIF)-1α protein levels in NRM2 cells. Inhibition of ERK-1,2 phosphorylation with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, blocked the synergistic increase in CCL2, CXCL10, and HIF-1α levels induced by PDGF and NiNPs. Moreover, the antioxidant, N-acetyl-L-cysteine (NAC), significantly reduced HIF-1α, ERK-1,2 phosphorylation, and CCL2 protein levels that were synergistically increased by the combination of PDGF and NiNPs. These data indicate that NiNPs enhance the activity of PDGF in regulating chemokine production in NRM2 cells through a mechanism involving reactive oxygen species generation and prolonged activation of ERK-1,2.

  19. Biology of Platelet Purinergic Receptors and Implications for Platelet Heterogeneity

    Directory of Open Access Journals (Sweden)

    Milka Koupenova


    Full Text Available Platelets are small anucleated cells present only in mammals. Platelets mediate intravascular hemostatic balance, prevent interstitial bleeding, and have a major role in thrombosis. Activation of platelet purinergic receptors is instrumental in initiation of hemostasis and formation of the hemostatic plug, although this activation process becomes problematic in pathological settings of thrombosis. This review briefly outlines the roles and function of currently known platelet purinergic receptors (P1 and P2 in the setting of hemostasis and thrombosis. Additionally, we discuss recent novel studies on purinergic receptor distribution according to heterogeneous platelet size, and the possible implication of this distribution on hemostatic function.

  20. Platelet hemostasis in patients with metabolic syndrome and type 2 diabetes mellitus: cGMP- and NO-dependent mechanisms in the insulin-mediated platelet aggregation

    Directory of Open Access Journals (Sweden)

    Tatiana E Suslova


    Full Text Available Patients with metabolic syndrome (MetS and type 2 diabetes mellitus (T2DM have high risk of microcirculation complications and microangiopathies. An increase in thrombogenic risk is associated with platelet hyperaggregation, hypercoagulation, and hyperfibrinolysis. Factors leading to platelet activation in MetS and T2DM comprise insulin resistance, hyperglycemia, non-enzymatic glycosylation, oxidative stress, and inflammation. This review discusses the role of nitric oxide (NO in the regulation of platelet adhesion and aggregation processes. Nitric oxide is synthesized both in endotheliocytes, smooth muscle cells, macrophages, and platelets. Modification of platelet NO-synthase (NOS activity in MetS patients can play a central role in the manifestation of platelet hyperactivation. Metabolic changes, accompanying T2DM, can lead to an abnormal NOS expression and activity in platelets. Hyperhomocysteinemia, often accompanying T2DM, is a risk factor for cardiovascular accidents. Homocysteine can reduce NO production by platelets. This review provides data on the insulin effects in platelets. Decrease in a number and sensitivity of the insulin receptors on platelets in T2DM can cause platelet hyperactivation. Various intracellular mechanisms of anti-aggregating insulin effects are discussed. Anti-aggregating effects of insulin are mediated by a NO-induced elevation of cGMP and upregulation of cAMP- and cGMP-dependent pathways. The review presents data suggesting an ability of platelets to synthesize humoral factors stimulating thrombogenesis and inflammation. Proinflammatory cytokines are considered as markers of T2DM and cardiovascular complications and are involved in the development of dyslipidemia and insulin resistance. The article provides an evaluation of NO-mediated signaling pathway in the effects of cytokines on platelet aggregation. The effects of the proinflammatory cytokines on functional activity of platelets are demonstrated.

  1. Participação das plaquetas no processo de fibrose dos pacientes com esquistossomose mansônica Participation of platelets in the process of fibrosis in patients with mansonic schistosomiasis

    Directory of Open Access Journals (Sweden)

    Sheila Soares


    Full Text Available O objetivo deste estudo foi avaliar a ativação plaquetária através da P-selectina e o conteúdo de PDGF-AB e TGFbeta1, nos pacientes com esquistossomose que desenvolveram fibrose (F3, naqueles que não tiveram esta manifestação (F0 e nos controles (C. Os resultados mostraram que a percentagem de P-selectina nas plaquetas sem estímulo de agonistas foi de 10,6% nos F3; 11,1% nos FO, e 6,3% nos C e após a adição de ADP/adrenalina, foi de 44%; 25,3% e 42%, respectivamente. A dosagem do PDGF-AB e TGFbeta1 por plaquetas foi de 11,016ng/dL (F3; 3,172 ng/dL (F0 e 5,01ng/dL (C e, (0,012ng/dL (F3; 5,27ng/dL (F0 e 4,66ng/dL (C, respectivamente. Em relação à P-selectina, concluímos que as plaquetas dos pacientes com esquistossomoses, apesar de estarem pré ativadas, mantiveram-se responsivas aos agonistas. O TFGbeta1 não apresentou diferença entre os três grupos, enquanto o PDGF-AB foi significantemente maior no grupo F3, sugerindo a participação deste no desenvolvimento da fibrose.The aim of this study was to evaluate platelet activation through P-selectin, and PDGF-AB and TGFbeta1 content, in schistosomiasis patients who developed fibrosis (F3 and who did not present this (F0, and in a control group (C. The results showed that the percentage of P-selectin in platelets without agonist stimulation was 10.6% in F3, 11.1% in F0 and 6.3% in C. After the addition of ADP/adrenaline, the percentages were 44%, 25.3% and 42%, respectively. The PDGF-AB and TGFbeta1 contents per platelet were 11,016ng/dl (F3, 3,172ng/dl (F0 and 5.01ng/dl (C and 0,012ng/dl (F3, 5.27ng/dl (F0 and 4.66ng/dl (C, respectively. Concerning the P-selectin, we can conclude that platelets from patients with schistosomiasis continued to be responsive to agonists, despite being pre-activated. There were no differences in TGFbeta1 between the groups, but the PDGF-AB content was significantly higher in F3. This suggests that PDGF-AB may have some participation in the development

  2. Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface

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    LaMarre Jonathan


    Full Text Available Abstract Background Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF and platelet derived growth factor (PDGF families and anti-angiogenic factor thrombospondin-1 (TSP-1 and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. Methods Mesometrial endometrium was collected from non-pregnant gilts (n = 8. Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd 20 (n = 8 and gd 50 (n = 8. At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. Results At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p Conclusions We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor

  3. Mean Platelet Volume

    African Journals Online (AJOL)

    Department of Chest Disease, Bolu, Turkey. E-mail: Telephone number: +903742534618. Fax number: +903742534615 effective and should have wide spread acceptance. At present, none of the available diagnostic tests meets all these criteria. The mean platelet volume (MPV) is potentially one of.

  4. A novel bioreactor and culture method drives high yields of platelets from stem cells. (United States)

    Avanzi, Mauro P; Oluwadara, Oluwasijibomi E; Cushing, Melissa M; Mitchell, Maxwell L; Fischer, Stephen; Mitchell, W Beau


    Platelet (PLT) transfusion is the primary treatment for thrombocytopenia. PLTs are obtained exclusively from volunteer donors, and the PLT product has only a 5-day shelf life, which can limit supply and result in PLT shortages. PLTs derived from stem cells could help to fill this clinical need. However, current culture methods yield far too few PLTs for clinical application. To address this need, a defined, serum-free culture method was designed using a novel bioreactor to increase the yield of PLTs from stem cell-derived megakaryocytes. CD34 cells isolated from umbilical cord blood were expanded with a variety of reagents and on a nanofiber membrane using serum-free medium. These cells were then differentiated into megakaryocytic lineage by culturing with thrombopoietin and stem cell factor in serum-free conditions. Polyploidy was induced by addition of Rho kinase inhibitor or actin polymerization inhibitor to the CD41 cells. A novel bioreactor was developed that recapitulated aspects of the marrow vascular niche. Polyploid megakaryocytes that were subjected to flow in the bioreactor extended proPLTs and shed PLTs, as confirmed by light microscopy, fluorescence imaging, and flow cytometry. CD34 cells were expanded 100-fold. CD41 cells were expanded 100-fold. Up to 100 PLTs per input megakaryocyte were produced from the bioreactor, for an overall yield of 10(6) PLTs per input CD34 cell. The PLTs externalized P-selectin after activation. Functional PLTs can be produced ex vivo on a clinically relevant scale using serum-free culture conditions with a novel stepwise approach and an innovative bioreactor. © 2015 AABB.

  5. A Novel Platelet Activating Factor Receptor Antagonist Reduces Cell Infiltration and Expression of Inflammatory Mediators in Mice Exposed to Desiccating Conditions after PRK

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    Salomon Esquenazi


    Results. Confocal microscopy showed an increased number of reflective structures in the corneal epithelium after PRK and exposure to DE in eyes treated with vehicle as compared to eyes treated with LAU-0901. Significant decrease of COX-2 and Arginase I expression and reduced alpha SMA cells was observed after PRK and exposure to DE in eyes treated with LAU-0901. Discussion: Exposure of mice to a DE after PRK increases the epithelial turnover rate. PAF is involved in the inflammatory cell infiltration and expression of inflammatory cytokines that follow PRK under DE.

  6. Comparison of platelet counting technologies in equine platelet concentrates. (United States)

    O'Shea, Caitlin M; Werre, Stephen R; Dahlgren, Linda A


    (1) To compare the performance of 4 platelet counting technologies in equine platelet concentrates and (2) to evaluate the ability of the Magellan platelet rich plasma (PRP) system to concentrate equine platelets. Experimental study to assess method agreement. Adult mixed breed horses (n = 32). Acid citrate dextrose-A anti-coagulated whole blood was collected and PRP produced using the Magellan system according to the manufacturer's instructions. Platelets were quantified using 4 counting methods: optical scatter (Advia 2120), impedance (CellDyn 3700), hand counting, and fluorescent antibody flow cytometry. Platelet concentrations were compared using Passing and Bablok regression analyses and mixed model ANOVA. Significance was set at P CellDyn 3700. Systematic and proportional biases were observed between these 2 automated methods when analyzed by regression analysis of the larger sample size. No bias (systematic or proportional) was observed among any of the other counting methods. Despite the bias detected between the 2 automated systems, there were no significant differences on average among the 4 counting methods evaluated, based on the ANOVA. The Magellan system consistently generated high platelet concentrations as well as higher than expected WBC concentrations. The Magellan system delivered desirably high platelet concentrations; however, WBC concentrations may be unacceptably high for some orthopedic applications. All 4 platelet counting methods tested were equivalent on average and therefore suitable for quantifying platelets in equine PRP used for clinical applications. © Copyright 2014 by The American College of Veterinary Surgeons.

  7. Storage of platelets: effects associated with high platelet content in platelet storage containers. (United States)

    Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell


    A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

  8. Activating stimuli induce platelet microRNA modulation and proteome reorganisation. (United States)

    Cimmino, Giovanni; Tarallo, Roberta; Nassa, Giovanni; De Filippo, Maria Rosaria; Giurato, Giorgio; Ravo, Maria; Rizzo, Francesca; Conte, Stefano; Pellegrino, Grazia; Cirillo, Plinio; Calabro, Paolo; Öhman, Tiina; Nyman, Tuula A; Weisz, Alessandro; Golino, Paolo


    Platelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology.

  9. Platelet accumulation in abdominal aortic aneurysm and the effect of antiplatelet drugs; Assessment by indium platelet scintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Etami, Hideki (Osaka-Minami National Hospital, Kawachi-nagano (Japan)); Kimura, Kazufumi; Isaka, Yoshinari (and others)


    A dual tracer technique using {sup 111}In labeled platelets and {sup 99m}Tc labeled human serum albumin was applied to evaluate the in vivo thrombogenicity in 12 cases with abdominal aortic aneurysm (AAA) and the effect of antiplatelet drug on the thrombogenicity. The magnitude of platelet accumulation at AAA was expressed as the ratio of radioactivity of {sup 111}In platelets on the vascular wall to those in the blood pool (PAI; platelet accumulation index). Of the 12 patients with AAA, 11 had positive studies on baseline imaging and 1 had equivocally positive image. The PAI value (Mean{+-}SD) over the AAA was 53.8{+-}34.1% as compared to -8.6{+-}4.4% in the control group (p<0.01). Seven patients with an AAA and positive baseline images were restudied during platelet active drug with 325 mg of aspirin. During treatment with aspirin, of 7 patients, 5 had positive images, of which 3 were decreased and others unchanged compared to baseline studies, 1 equivocally positive one and one negative one. The PAI value during treatment (21.9{+-}18.6%) was significantly decreased compared to those in baseline study (52.1{+-}23.9%). Our results suggest that the method used for platelet imaging in the present study may be useful for studying the in vivo thrombogenicity and the effect of platelet active drugs in AAA. (author).

  10. Effects of hormones on platelet aggregation. (United States)

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


    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.

  11. Extension of platelet concentrate storage. (United States)

    Simon, T L; Nelson, E J; Carmen, R; Murphy, S


    Extension of the storage time of platelet concentrates in a satellite bag which is part of a new blood bag system was studied by reinfusing autologous 51Cr-labeled platelets into normal volunteers, and measuring postinfusion platelet counts and bleeding times in patients requiring platelet transfusions. This satellite bag, made of polyvinylchloride plasticized with a new agent, was found to protect platelet concentrates against fall of pH better than other containers studied. This protection was felt to be due to the greater gas permeability of the new plastic. Mean in vivo recovery and half-life (greater than 31% and 3.3 days, respectively) of autologous reinfused platelets were satisfactory following 5 days of storage. Following 7 days of storage, mean recovery was 41 percent and half-life was 2.8 days. Peripheral platelet count increments in patients following platelet transfusions with concentrates stored 4 to 7 days in the new plastic were comparable to increments following transfusion of platelets stored 2 to 3 days in the other plastics studied. Bleeding times shortened in three of four patients receiving platelet concentrates stored from 4 to 6 days in the new plastic. Platelet concentrates stored in the new bag at 20 to 24 degrees C with flat-bed or elliptical agitation could be transfused for up to 5 days following phlebotomy with acceptable clinical results. The new plastic container is promising for storage of platelet concentrates for up to 7 days. Due to the higher pH of 50-ml platelet concentrates stored in bags made with the new plastic, the concentrates were superior at any storage interval to those stored in bags made of the other plastics studied.

  12. 脑心通胶囊对老年人2型糖尿病颈动脉内膜中层厚度和血浆β血小板球蛋白P选择素及纤溶酶原激活物抑制剂1的影响%The effect of Naoxintong capsule treatment on carotid artery intima-media thickness, serum beta thromboglobulin,P-selectin and plasminogen activator inhibitor-1 in elderly type 2 diabetic patients with subclinical atherosclerotic vascular disease

    Institute of Scientific and Technical Information of China (English)

    王俊; 程森华; 杨晓翠; 孙国香; 徐更华; 王益波


    目的 探讨脑心通胶囊治疗老年2型糖尿病血管病变患者对其颈动脉内膜中层厚度(IMT)、血浆β-血小板球蛋白(β-TG)、P-选择素(CD62p)和纤溶酶原激活物抑制剂-1(PAI-1)的影响.方法 回顾性研究,选择医院2014年6月至2015年5月收治的110例老年2型糖尿病血管病变患者为研究对象.数字抽签随机分为观察组和对照组(各55例),入选患者均给予常规治疗,观察组患者同时给予脑心通胶囊治疗.对比分析两组患者血浆β-TG、CD62p和PAI-1水平变化. 结果 治疗前,两组患者颈动脉IMT、PAI-1、β-TG和CD62p水平比较,差异无统计学意义(均P>0.05).治疗后,观察组与对照组患者比较,颈动脉IMT(1.31±0.26)mm比(1.44±0.26)mm,差异有统计学意义(t=4.058,P<0.05);观察组患者血浆PAI-1、β-TG和CD62p水平与对照组比较,PAI-1(2.23±0.48)μg/L比(2.56±0.61)μg/L、β TG(29.76±10.24) μg/L比(35.98±10.35)μg/L,CD62p(162.3±21.5)ng/L比(176.96±20.3)ng/L,差异有统计学意义(t值分别为3.965、11.293、14.624,均P<0.05). 结论 脑心通胶囊治疗2型糖尿病亚临床血管病变患者有利于减少颈动脉内膜中层厚度,降低血小板释放活性,减轻患者机体氧化应激反应,从而减轻老年2型糖尿病患者血管病变.%Objective To investigate the effects of Naoxintong Capsule treatment on the carotid artery intima media thickness(IMT),plasma beta thromboglobulin(beta TG),P-selectin(CD62p)and plasminogen activator inhibitor 1 (PAI 1)in elderly patients with type 2 diabetes mellitus and subclinical atherosclerotic vascular disease.Methods In retrospective study,110 cases of elderly patients with type 2 diabetic vascular diseases were selected and admitted as study subjects from June 2014 to May 2015.They were randomized into observation group and control group(n=55,each).All patients were given routine treatment.The patients in the observation group were treated with Naoxintong Capsule.The levels of

  13. Platelet aggregation following trauma

    DEFF Research Database (Denmark)

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


    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...

  14. RGS10 Negatively Regulates Platelet Activation and Thrombogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole R Hensch

    Full Text Available Regulators of G protein signaling (RGS proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT. Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.

  15. Rethinking platelet function: thrombocytopenia induced immunodeficiency in critical illness

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Johansson, Per Ingemar


    Thrombocytopenia in critical illness predicts a poor clinical outcome. Apart from its role in microvascular thrombus formation, it is widely anticipated that this association is indirect rather than causal. Emerging evidence however indicates that platelets are also immune competent cells. Like...... traditional innate immune cells, platelets are recruited immediately into injured and inflamed tissue, they release immune mediators, express and shed immunologically active membrane receptors, they interact with other immune cells and they recognize and clear pathogens. We hypothesize that thrombocytopenia...... per se results in immunodeficiency through loss of platelet-mediated immune functions, and propose that thrombocytopenia induced immunodeficiency in critical illness in part explain the negative predictive value of low or declining platelet count. We propose that rethinking the risks...

  16. Mammary tumors that become independent of the type I insulin-like growth factor receptor express elevated levels of platelet-derived growth factor receptors

    Directory of Open Access Journals (Sweden)

    Campbell Craig I


    Full Text Available Abstract Background Targeted therapies are becoming an essential part of breast cancer treatment and agents targeting the type I insulin-like growth factor receptor (IGF-IR are currently being investigated in clinical trials. One of the limitations of targeted therapies is the development of resistant variants and these variants typically present with unique gene expression patterns and characteristics compared to the original tumor. Results MTB-IGFIR transgenic mice, with inducible overexpression of the IGF-IR were used to model mammary tumors that develop resistance to IGF-IR targeting agents. IGF-IR independent mammary tumors, previously shown to possess characteristics associated with EMT, were found to express elevated levels of PDGFRα and PDGFRβ. Furthermore, these receptors were shown to be inversely expressed with the IGF-IR in this model. Using cell lines derived from IGF-IR-independent mammary tumors (from MTB-IGFIR mice, it was demonstrated that PDGFRα and to a lesser extent PDGFRβ was important for cell migration and invasion as RNAi knockdown of PDGFRα alone or PDGFRα and PDGFRβ in combination, significantly decreased tumor cell migration in Boyden chamber assays and suppressed cell migration in scratch wound assays. Somewhat surprisingly, concomitant knockdown of PDGFRα and PDGFRβ resulted in a modest increase in cell proliferation and a decrease in apoptosis. Conclusion During IGF-IR independence, PDGFRs are upregulated and function to enhance tumor cell motility. These results demonstrate a novel interaction between the IGF-IR and PDGFRs and highlight an important, therapeutically relevant pathway, for tumor cell migration and invasion.

  17. Increase in platelet non-integrin type I collagen receptor in patients with systemic sclerosis. (United States)

    Chiang, Thomas M; Takayama, Hiroshi; Postlethwaite, Arnold E


    Microvascular injury is one of the major pathogenetic processes involved in systemic sclerosis (SSc). Interaction of the platelet types I and III collagen receptors with their respective ligand in the exposed subendothelial stroma as a result of ongoing microvascular injury in SSc patients results in platelet activation and aggregation with the release of mediators, which contribute to vascular damage and inflammation. We have found that there is a twofold increase in radiolabeled type I collagen binding to washed platelets from patients with SSc compared to platelets obtained from normal volunteers. Western blot analyses showed that the non-integrin platelet type I collagen receptor protein (65 kDa) is increased dramatically in lysates of platelet from patients with SSc. However, the integrin (alpha(2)beta(1)) and other non-integrin receptors such as glycoprotein VI, glycoprotein IV, and the platelet receptor for type III collagen remain unchanged. In addition, platelet lysates from rheumatic disease controls (rheumatoid arthritis, osteoarthritis, gout, and systemic lupus erythematosus) do not show any significant increases. There is no nitrotyrosylation on 65 kDa in patients with SSc compared to controls, suggesting this might also contribute to binding of CI to the 65-kDa CIR. These results suggest that there is a specific increase in the number of platelet type I collagen receptors in SSc patient's platelets. In addition, the activity of nitric oxide synthase is decreased in patients' platelet lysates compared to controls. The increase in platelet expression of the 65-kDa non-integrin platelet type I collagen receptor may explain the enhanced aggregation of platelets from patients with SSc to CI in vitro and microvascular thrombosis in the disease in vivo.

  18. Supporting roles of platelet thrombospondin-1 and CD36 in thrombus formation on collagen. (United States)

    Kuijpers, Marijke J E; de Witt, Susanne; Nergiz-Unal, Reyhan; van Kruchten, Roger; Korporaal, Suzanne J A; Verhamme, Peter; Febbraio, Maria; Tjwa, Marc; Voshol, Peter J; Hoylaerts, Marc F; Cosemans, Judith M E M; Heemskerk, Johan W M


    Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus. © 2014 American Heart Association, Inc.

  19. Platelet immunology in fungal infections. (United States)

    Speth, Cornelia; Rambach, Günter; Lass-Flörl, Cornelia


    Up to date, perception of platelets has changed from key players in coagulation to multitaskers within the immune network, connecting its most diverse elements and crucially shaping their interplay with invading pathogens such as fungi. In addition, antimicrobial effector molecules and mechanisms in platelets enable a direct inhibitory effect on fungi, thus completing their immune capacity. To precisely assess the impact of platelets on the course of invasive fungal infections is complicated by some critical parameters. First, there is a fragile balance between protective antimicrobial effects and detrimental reactions that aggravate the fungal pathogenesis. Second, some platelet effects are exerted indirectly by other immune mediators and are thus difficult to quantify. Third, drugs such as antimycotics, antibiotics, or cytostatics, are commonly administered to the patients and might modulate the interplay between platelets and fungi. Our article highlights selected aspects of the complex interactions between platelets and fungi and the relevance of these processes for the pathogenesis of fungal infections.

  20. The role of platelet and endothelial GARP in thrombosis and hemostasis.

    Directory of Open Access Journals (Sweden)

    Elien Vermeersch

    Full Text Available Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32 is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish.To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice.Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice.Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected.Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

  1. Orthobiologics and platelet rich plasma

    National Research Council Canada - National Science Library

    Dhillon, Mandeep S; Behera, Prateek; Patel, Sandeep; Shetty, Vijay


    ... in many chronic musculoskeletal ailments. Investigators have published results of laboratory as well as clinical studies, using orthobiologics like platelet rich plasma, stem cells, autologous conditioned serum etc...

  2. Novel aspects of platelet aggregation

    Directory of Open Access Journals (Sweden)

    Roka-Moya Y. M.


    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.

  3. In vitro viability effects on apheresis and buffy-coat derived platelets administered through infusion pumps

    Directory of Open Access Journals (Sweden)

    Sandgren P


    Full Text Available Per Sandgren,1,2 Veronica Berggren,3 Carl Westling,1,2 Viveka Stiller1 1Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, 2Department of Laboratory Medicine, Karolinska Institutet, 3Department of Neonatology, Karolinska University Hospital, Stockholm, SwedenBackground: Different infusion pump systems as well as gravity infusion have been widely used in neonatal transfusion. However, the limited number of published studies describing the use of infusion pumps on platelets illustrates the necessity for more robust data.Methods: To evaluate the potential in vitro effects on the cellular, metabolic, functional and phenotypic properties of platelets, we set up a four-arm paired study simultaneously comparing the use of different infusion pumps (Alaris® CC/GP with unexposed platelets. The platelet units (n=8 were either produced by the apheresis technique and suspended in 100% plasma or derived from buffy coats to yield platelet units stored in approximately 30% plasma and 70% SSP+. Fresh and 5-day old platelets were tested.Results: Regardless of the production system or storage time used, no significant differences were observed in glucose and lactate concentration, pH, adenosine triphosphate levels, response to extent of shape change, hypotonic shock response reactivity, and CD62P expression. Similarly, no differences were observed in expression of the conformational epitope on glycoprotein IIb/IIIa, determined using procaspase-activating compound 1, or in the expression of CD42b and platelet-endothelial cell adhesion molecule-1 in a comparison between platelets administered through infusion pumps versus unexposed platelets.Conclusion: Using Alaris CC/GP infusion pumps had no influence on the cellular, functional, and phenotypic in vitro properties of platelets. This fact seems not to be affected by different production systems or storage time.Keywords: platelets, neonatal platelet transfusion

  4. Isolation of Platelet-Derived Extracellular Vesicles

    NARCIS (Netherlands)

    Aatonen, Maria; Valkonen, Sami; Böing, Anita; Yuana, Yuana; Nieuwland, Rienk; Siljander, Pia


    Platelets participate in several physiological functions, including hemostasis, immunity, and development. Additionally, platelets play key roles in arterial thrombosis and cancer progression. Given this plethora of functions, there is a strong interest of the role of platelet-derived

  5. miR-326 targets antiapoptotic Bcl-xL and mediates apoptosis in human platelets.

    Directory of Open Access Journals (Sweden)

    Shifang Yu

    Full Text Available Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.

  6. Increased platelet activation in early symptomatic versus asymptomatic carotid stenosis and relationship with microembolic status: Results from the Platelets And Carotid Stenosis (PACS) Study.

    LENUS (Irish Health Repository)

    Kinsella, Ja


    BACKGROUND: Cerebral microembolic signals (MES) may predict increased stroke risk in carotid stenosis. However, the relationship between platelet counts or platelet activation status and MES in symptomatic versus asymptomatic carotid stenosis has not been comprehensively assessed. SETTING: University teaching hospitals. METHODS: This prospective, pilot observational study assessed platelet counts and platelet activation status, and the relationship between platelet activation and MES in asymptomatic versus early (≤4 weeks after TIA\\/stroke) and late phase (≥3 months) symptomatic moderate or severe (≥50%) carotid stenosis patients. Full blood count measurements were performed, and whole blood flow cytometry was used to quantify platelet surface activation marker expression (CD62P and CD63) and circulating leucocyte-platelet complexes. Bilateral simultaneous transcranial Doppler ultrasound monitoring of the middle cerebral arteries was performed for 1 hour to classify patients as MES-positive or MES-negative. RESULTS: Data from 31 asymptomatic patients were compared with 46 symptomatic patients in the early phase, and 35 of these patients followed up to the late phase after symptom onset. The median platelet count (211 vs. 200 x 10(9) \\/L; p=0.03) and the median% lymphocyte-platelet complexes were higher in early symptomatic than asymptomatic patients (2.8 vs. 2.4%, p=0.001). The% lymphocyte-platelet complexes was higher in early symptomatic than asymptomatic patients with ≥70% carotid stenosis (p=0.0005), and in symptomatic patients recruited within 7 days of symptom onset (p=0.028). Complete TCD data were available in 25 asymptomatic and 31 early phase symptomatic, and 27 late phase symptomatic patients. 12% of asymptomatic versus 32% of early phase symptomatic (p=0.02) and 19% of late phase symptomatic patients (p=0.2) were MES-positive. Early symptomatic MES-negative patients had a higher% lymphocyte-platelet complexes than asymptomatic MES

  7. Expression of platelet-derived growth factor (PDGF) in the epididymis and analysis of the epididymal development in PDGF-A, PDGF-B, and PDGF receptor beta deficient mice. (United States)

    Basciani, Sabrina; Mariani, Stefania; Arizzi, Mario; Brama, Marina; Ricci, Andrea; Betsholtz, Christer; Bondjers, Cecilia; Ricci, Giulia; Catizone, Angela; Galdieri, Michela; Spera, Giovanni; Gnessi, Lucio


    The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) alpha- and beta-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-beta loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-alpha immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-beta were absent. The cellular localization of PDGF-A within the epithelium and the alpha-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-alpha system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR alpha- and beta-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-alpha mRNAs were seen by reverse transcription-polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-beta were almost not detectable. During the first days of life, PDGF-B and PDGFR-beta genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-beta, and PDGFR-alpha were constant with the only exception of a progressive decrease of PDGFR-alpha in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-beta -/- mice were available, and no differences

  8. Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis

    DEFF Research Database (Denmark)

    Rönnstrand, L; Siegbahn, A; Rorsman, C


    ., Siegbahn, A. , Rorsman, C., Engström, U., Wernstedt, C., Heldin, C.-H., and Rönnstrand, L. (1996) EMBO J. 15, 5299-5313). Here we show that the increased chemotaxis correlates with increased activation of phospholipase C-gamma1 (PLC-gamma1), measured as inositol-1,4, 5-trisphosphate release. By two......-dimensional phosphopeptide mapping, the increase in phosphorylation of PLC-gamma1 was shown not to be selective for any site, rather a general increase in phosphorylation of PLC-gamma1 was seen. Specific inhibitors of protein kinase C, bisindolylmaleimide (GF109203X), and phosphatidylinositol 3-kinase (PI3-kinase), LY294002......, did not affect the activation of PLC-gamma1. To assess whether increased activation of PLC-gamma1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wild-type or a catalytically compromised version of PLC-gamma1 under a tetracycline...

  9. Mean Platelet Volume and Platelet Immunofluorescence as Indicators of Platelet Compatibility. (United States)


    2.5 ml of 1.2% EDTA in 0.9% NaCl. The platelet- rich plasm (PRP) was isolated by centrifugation at 280 X g for 5 mimmter. The PRP from all the blood...samples was pooled, and the platelets were concentrated by cpntrifugation at 1000 X g for 10 minutes. The platelet- poor plasma (PPP) was removed and -6.4% 2 shrinking 18. TABLE 2 .60A PLATLET _RLUME OF A+ OR 0+ DONOR PLATELETS TREATED WITH EITHER &U,&06OS S O SRUMNFRN AN AOIMMUNIZED TR

  10. Effects of cancer on platelets. (United States)

    van Es, Nick; Sturk, Auguste; Middeldorp, Saskia; Nieuwland, Rienk


    The main function of circulating platelets is to stop bleeding upon vascular injury by the formation of a hemostatic plug. The presence of cancer results in numerical and functional abnormalities of platelets. Thrombocytosis is commonly observed in cancer patients and is associated with decreased survival. Conversely, thrombocytopenia has been shown to have antimetastatic effects in experimental models. Tumor cells also can induce changes in the platelet activation status, both in direct and indirect manners. Direct tumor cell-induced platelet aggregation enables the formation of a cloak of aggregated platelets around circulating tumor cells (CTCs) that shields them from attacks by the immune system and facilitates metastasis to distant sites. Cancer also can induce platelet activation in various indirect ways. Tumor cells shed small extracellular vesicles that expose the transmembrane protein tissue factor (TF)--the initiator of the extrinsic coagulation cascade. The abundant presence of TF in the circulation of cancer patients can result in local generation of thrombin, the most potent platelet activator. Another pathway of indirect platelet activation is by increased formation of neutrophil extracellular traps in the presence of tumor-secreted granulocyte colony-stimulating factor (G-CSF). Last, tumor cells may regulate the selective secretion of angiogenic proteins from platelet granules, which enables the tumor to stimulate and stabilize the immature neovasculature in the tumor environment. Since there is little doubt that the cancer-induced platelet alterations are beneficial to tumor growth and dissemination, it could be worthwhile to intervene in the underlying mechanisms for anticancer purposes. Antiplatelet and anticoagulant agents that inhibit platelet activation and thrombin generation can potentially slow cancer progression, although the clinical evidence thus far is not unequivocal. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Platelet Rich Plasma in Periodontal Therapy

    National Research Council Canada - National Science Library

    S Sathya Priya Eshwar; Dhayanand John Victor; S Sangeetha; PSG Prakash


    Keywords: Growth factors, Platelet concentrates, Platelet Rich Plasma, Regenerative medicine, Tissue engineering Introduction The goal of periodontal therapy is to improve periodontal health and thereby...

  12. Sequential regulation of the small GTPase Rap1 in human platelets

    NARCIS (Netherlands)

    Franke, B; van Triest, M; de Bruijn, KMT; van Willligen, G; Nieuwenhuis, HK; Negrier, C; Akkerman, JWN; Bos, JL

    Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with alpha-thrombin. For this activation, a phospholipase C-mediated increase in intracellular

  13. Platelets release thrombopoietin (Tpo) upon activation: another regulatory loop in thrombocytopoiesis?

    NARCIS (Netherlands)

    Folman, C. C.; Linthorst, G. E.; van Mourik, J.; van Willigen, G.; de Jonge, E.; Levi, M. [=Marcel M.; de Haas, M.; von dem Borne, A. E.


    Thrombopoietin is produced at a constant rate by the liver and kidney and is removed from the circulation upon binding and subsequent uptake via the Tpo receptor, c-Mpl, expressed by platelets and mega-karyocytes. Apart from uptake, this study shows that platelets can also function as a storage pool

  14. Transcriptomic profiling of platelet senescence and platelet extracellular vesicles. (United States)

    Pienimaeki-Roemer, Annika; Konovalova, Tatiana; Musri, Melina M; Sigruener, Alexander; Boettcher, Alfred; Meister, Gunter; Schmitz, Gerd


    Platelets (PLTs) are derived from megakaryocytes during PLT shedding. Senescent or activated PLTs are expanded in vascular and neurological diseases and release PLT extracellular vesicles (PL-EVs). A systematic analysis of regular messenger RNA (mRNA) and small RNA composition in PLTs and PL-EVs during in vitro PLT senescence has not yet been published. We isolated PLTs, total PL-EVs, and PL-EV subsets on Days 0 and 5 from human stored donor platelet concentrates. Isolated mRNA species and microRNA (miRNA) species were analyzed by microarrays and deep sequencing. Correlation of mRNA and miRNA species (miR) and miRNA target analyses were performed using bioinformatics. During in vitro PLT senescence, residual PLT mRNA species were decreased and partially converted to miRNA species. Residual mRNAs included encoded genes relevant for atherosclerosis, inflammation (matrix metallopeptidase 14 [MMP-14], granulin [GRN], angiopoietin like 2 [ANGPTL2]), and neurotransmission (dopamine receptor 2 [DRD2], γ-aminobutyric acid type A receptor ρ3 [GABRR3]). Compared with senescent PLTs, PL-EVs have up-regulated their miRNA species involved in "diabesity" and in vascular and metabolic disease (miR-144-3p, miR-486-5p, miR-142-5p, miR-451a, miR-25-3p, miR-145-5p, and let-7f-5p). The 100 highest expressed PL-EV miRNA species determined by microarrays were compared with the 100 highest expressed PL-EV miRNA species detected by deep sequencing. This approach resulted in 66 overlaps. The regulated miRNAs (assessed by both methods) were related to neurological disorders, including targets for Alzheimer's disease (e.g., β-site amyloid precursor protein APP-cleaving enzyme 1 [BACE1], translocase of outer mitochondrial membrane 40 homolog [TOMM40], neuron navigator 3 [NAV3]). During in vitro senescence, PLTs degrade large RNA species. Concomitantly, they up-regulate a distinct set of known small RNA species involved in atherosclerosis, inflammation, and neurodegeneration. PL-EVs enrich

  15. Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1. (United States)

    Lucidi, Cynthia de A; Takahira, Regina K; Gerlach, John A; Davis, John M; Schwartz, Kenneth A; Scott, Michael A


    In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes. © 2011 American Society for Veterinary Clinical Pathology.

  16. Platelet aggregation, secretion, and coagulation changes in children with asthma. (United States)

    Buyukyilmaz, Gonul; Soyer, Ozge U; Buyuktiryaki, Betul; Alioglu, Bulent; Dallar, Yildiz


    The chronic inflammation in asthma evolves by cells including eosinophils, mast cells and lymphocytes. Despite their principal function in hemostasis, platelets contribute to pathogenesis of asthma that activation of platelets occurs following antigen provocation and during asthma attack. Our aim was to evaluate the platelet functions and other hemostatic features of children with asthma, both during symptom-free period and asthma attack. We enrolled patients with asthma attack (n = 33), mild intermittent asthma (n = 18), mild persistent asthma (n = 15) and healthy children (n = 20). Demographic characteristics and disease-related features were noted. Platelet aggregation and secretion tests (expressed as ATP release) were performed by lumiaggregometer method by stimulation with collagen, epinephrine, ADP, thrombin, ristocetin and arachidonic acid. Plasma levels of D-dimer, factor VIII (FVIII) and von Willebrand factor (vWF) were assessed. There were no differences in platelet aggregation induced by agonists between study groups. ATP release from platelets of patients with asthma exacerbation induced by ADP was lower compared with mild intermittent asthma (P coagulation mechanisms might be critical for asthma pathogenesis.

  17. Effects of cancer on platelets

    NARCIS (Netherlands)

    van Es, Nick; Sturk, Auguste; Middeldorp, Saskia; Nieuwland, Rienk


    The main function of circulating platelets is to stop bleeding upon vascular injury by the formation of a hemostatic plug. The presence of cancer results in numerical and functional abnormalities of platelets. Thrombocytosis is commonly observed in cancer patients and is associated with decreased

  18. Role of platelets in inflammation

    Directory of Open Access Journals (Sweden)

    Kadir Serkan Yalçın


    Full Text Available Inflammation, which is extremely useful process for humanbody is the response of living vascular tissues topathological phenomena that removes the pathogens andinitiates the healing procedure. Microorganisms, physicaltrauma, chemical, mechanical, irradiation, or thermal injury,ischemia and immune reactions are most commoncauses of this exceptionally important event for humanbody. Platelets are non-nucleated cells in blood that producedin bone marrow as derived from megakaryocytes.Apart from stop bleeding and achieving hemostasis thereare incredibly important roles of platelets in inflammation.Platelets contain important mediators for inflammationlike neutrophils or macrophages and can alter the courseof mechanism. In this article changing platelet function ininflammation and the effect of these functions to the processof inflammation will be discussed.Key words: Platelet, inflammation, cytokines

  19. Treatment of buffy coat platelets in platelet additive solution with the mirasol(®) pathogen reduction technology system. (United States)

    Castrillo, Azucena; Cardoso, Marcia; Rouse, Lindsay


    The Mirasol pathogen reduction technology (PRT) system uses riboflavin and ultraviolet light and is currently approved and used in Europe for the treatment of platelets and plasma. Mirasol treatment is intended to reduce the infectious pathogen load and to inactivate leukocytes in blood products. Our objective was to evaluate buffy coat platelet concentrates (BCPCs) prepared with platelet additive solution (PAS) and treated with the Mirasol system and to examine the effects on platelet cell quality during storage. 26 BCPCs were prepared and split, creating 13 paired control and test units. The test units were treated with the Mirasol system and the platelet quality was assessed in all units over 7 days of storage. All products met the incoming specifications for Mirasol treatment, and the pH of all Mirasol-treated BCPCs in PAS met the requirements of the Council of Europe guidelines throughout storage. Analysis of lactate production and glucose consumption rates, CD62p expression and cytokines indicates enhanced cellular metabolism in treated platelets, but the levels were within previously published ranges. While Mirasol-treated BCPCs in PAS had increased metabolism and activation compared to controls, the results indicate that these units can be stored for 7 days with acceptable cell quality.

  20. Platelet indices in SGA newborns. (United States)

    Wasiluk, A; Dabrowska, M; Osada, J; Jasinska, E; Laudanski, T; Redzko, S


    The current study objective was to compare blood platelet indices in full-term small-for-gestational-age newborns (SGA) and full-term appropriate-for-gestational-age newborns (AGA). We introduced to our study 61 SGA newborns (31 females and 30 males) and 70 eutrophic infants (32 females and 38 males). The SGA newborns were divided into two groups: those weighing less than the 5th centile: 35 infants (16 females and 19 males) and those between the 5th and 10th centiles: 26 infants (15 females and 11 males). Platelet indices were estimated in blood samples collected from the umbilical artery. SGA demonstrated a decreased count of blood platelets (238×103/μ) as compared with AGA (286×103/μL), p=0.0001. Platelet hematocrit (PTC) also showed differences in both groups (SGA=0.19% vs. AGA=0.22%; p=0.0005). Mean platelet volume (MPV) was higher in SGA (8.25fl) as compared with AGA (7.84fl); p=0.008. Large platelet count (LPLT) was higher in AGA 6.26% vs. SGA=4.75%; p=0.01. Platelet distribution width (PDW) was found to be nearly the same (SGA=47%, AGA=46%). PDW was higher in SGA newborns SGA infants between the 5th and 10th centiles (52%); p=0.008. A decreased blood platelet count, platelet hematocrit and large metabolically active platelet count, which in addition to reduced synthesis and excessive consumption of coagulation factors in states of hiperclotting is characteristic of IUGR, enhances the possibility of bleeding complications and increases the risk of infections. From a clinical point of view, it is important to take into consideration the degree of intrauterine hypotrophy during the evaluation of hemostatic disorders.

  1. Cellular source of human platelet secretory phospholipase A2. (United States)

    Emadi, S; Mirshahi, M; Elalamy, I; Nicolas, C; Vargaftig, B B; Hatmi, M


    Platelets are one source of the group II extracellular form of phospholipase A2 (sPLA2) which is involved in the amplification of local and systemic inflammation. Although sPLA2 protein has been described in human platelets, its presence in human megakaryocytes has not been yet established. We demonstrated in this study that the human erythroleukaemia (HEL) cell line, which has megakaryoblastic features, constitutively expresses sPLA2. Using an anti-rhsPLA2 monoclonal antibody (mAb BA11) and dot-blot detection, we showed that HEL cells and platelets release sPLA2 into incubation medium upon stimulation by thrombin. Similar results were obtained for sPLA2 activity detected by a spectrofluorescence assay. Enzymatic activity was abolished by mAb BA11 and by protamine. In both cell types, although released, the major part of sPLA2 remained in the cell pellet, and was probably adsorbed at non-specific membrane sites. Double labelling experiments using mAb BA11 and an anti-GPIIb antiserum revealed the presence of sPLA2 in human bone-marrow megakaryocytes. The use of reverse transcription-polymerase chain reaction conjugated with hybridization analysis demonstrated the presence of mRNA encoding for sPLA2 in platelets and HEL cells. Expression of sPLA2 in platelets and megakaryocytes at both transcriptional and post-translational levels strongly argues in favour of a megakaryocytic origin of platelet sPLA2 and rules out a role for endocytosis of the enzyme from plasma by circulating platelets.

  2. Alzheimer disease and platelets: how’s that relevant

    Directory of Open Access Journals (Sweden)

    Catricala Silvia


    Full Text Available Abstract Alzheimer Disease (AD is the most common neurodegenerative disorder worldwide, and account for 60% to 70% of all cases of progressive cognitive impairment in elderly patients. At the microscopic level distinctive features of AD are neurons and synapses degeneration, together with extensive amounts of senile plaques and neurofibrillars tangles. The degenerative process probably starts 20–30 years before the clinical onset of the disease. Senile plaques are composed of a central core of amyloid β peptide, Aβ, derived from the metabolism of the larger amyloid precursor protein, APP, which is expressed not only in the brain, but even in non neuronal tissues. More than 30 years ago, some studies reported that human platelets express APP and all the enzymatic activities necessary to process this protein through the same pathways described in the brain. Since then a large number of evidence has been accumulated to suggest that platelets may be a good peripheral model to study the metabolism of APP, and the pathophysiology of the onset of AD. In this review, we will summarize the current knowledge on the involvement of platelets in Alzheimer Disease. Although platelets are generally accepted as a suitable model for AD, the current scientific interest on this model is very high, because many concepts still remain debated and controversial. At the same time, however, these still unsolved divergences mirror a difficulty to establish constant parameters to better defined the role of platelets in AD.

  3. Immunochemical characterization of platelet-specific alloantigens

    NARCIS (Netherlands)

    Mulder, A.; van Leeuwen, E. F.; Veenboer, G. J.; Tetteroo, P. A.; von dem Borne, A. E.


    Immunoprecipitation was performed with platelet-specific alloantisera (anti-Zwa, -Zwb, -Baka and antiserum Luc) and 125I-labelled platelets of a panel of donors typed for these platelet-specific alloantigens. This was done by sensitization of intact, radiolabelled platelets with the antisera,

  4. Neonatal thrombocytopenia and platelets transfusion

    Directory of Open Access Journals (Sweden)

    Anil K Gupta


    Full Text Available Background: Neonates often develop thrombocytopenia at some time during hospital stay. Platelet transfusion are frequently given to them and are likely to result in unnecessary transfusion. Material and Methods: Thus, we analyzed thrombocytopenia in neonates, its prevalence, and relationship if any, between clinical condition and platelet transfusion in neonates, which would have been helpful in developing guidelines and/or protocols for platelet transfusion (and reducing the donor exposure in neonates. Results: A total of 870 neonates who were admitted in Neonatal Intensive Care Unit (NICU with various morbidities had platelets count done; of these, 146 (16.7% neonate revealed thrombocytopenia. Discussion: Low birth weight babies (P 0.009 and babies born with mother having hypertension (P 0.04 showed significant thrombocytopenia. Neonates with intrauterine growth retardation (IUGR diagnosed during antenatal screening showed lower platelet count (P 0.022. Neonates having associated illness, such as sepsis, gastrointestinal, and respiratory problems, and on vasopressor drugs were found to be associated with low platelet count. Conclusion: In our study, 16.40% of thrombocytopenic neonates required platelet transfusion either alone or with other blood component during their stay in NICU.

  5. Prophylactic platelets in dengue

    DEFF Research Database (Denmark)

    Whitehorn, James; Rodriguez Roche, Rosmari; Guzman, Maria G


    Dengue is the most important arboviral infection of humans. Thrombocytopenia is frequently observed in the course of infection and haemorrhage may occur in severe disease. The degree of thrombocytopenia correlates with the severity of infection, and may contribute to the risk of haemorrhage...... of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity...... of the responses highlights the variation in clinical practice and lack of an evidence base in this area and underscores the importance of prospective clinical trials to address this key question in the clinical management of patients with dengue....

  6. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu


    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  7. Maresin 1 induces a novel pro-resolving phenotype in human platelets. (United States)

    Lannan, K L; Spinelli, S L; Blumberg, N; Phipps, R P


    Essentials Specialized proresolving mediators (SPMs) promote the resolution of inflammation. This study sought to investigate the effects of SPMs on human platelet function. The SPM, Maresin 1, enhanced hemostatic, but suppressed inflammatory functions of platelets. SPMs uniquely regulate platelet function and may represent a new class of antiplatelet agents. Background Antiplatelet therapy is a cornerstone of modern medical practice and is routinely employed to reduce the likelihood of myocardial infarction, thrombosis and stroke. However, current antiplatelet therapies, such as aspirin, often have adverse side-effects, including increased risk of bleeding, and some patients are relatively 'aspirin-resistant'. Platelets are intimately involved in hemostasis and inflammation, and clinical consequences are associated with excessive or insufficient platelet activation. Objectives A major unmet need in the field of hematology is the development of new agents that safely prevent unwanted platelet activation in patients with underlying cardiovascular disease, while minimizing the risk of bleeding. Here, we investigate the potential of endogenously produced, specialized pro-resolving mediators (SPMs) as novel antiplatelet agents. SPMs are a recently discovered class of lipid-derived molecules that drive the resolution of inflammation without being overtly immunosuppressive. Methods Human platelets were treated with lipoxin A4, resolvin D1, resolvin D2, 17-HDHA or maresin 1 for 15 min, then were subjected to platelet function tests, including spreading, aggregation and inflammatory mediator release. Results We show for the first time that human platelets express the SPM receptors, GPR32 and ALX. Furthermore, our data demonstrate that maresin 1 differentially regulates platelet hemostatic function by enhancing platelet aggregation and spreading, while suppressing release of proinflammatory and prothrombotic mediators. Conclusions These data support the concept that SPMs

  8. The origin and function of platelet glycosyltransferases

    DEFF Research Database (Denmark)

    Wandall, Hans H; Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll


    that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results...... to function in the extracellular space. This platelet glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biological functions....

  9. The effect of pathogen reduction technology (Mirasol) on platelet quality when treated in additive solution with low plasma carryover. (United States)

    Johnson, L; Winter, K M; Reid, S; Hartkopf-Theis, T; Marschner, S; Goodrich, R P; Marks, D C


    Pathogen reduction technologies (PRT) for platelets are now compatible with both plasma and platelet additive solutions (PAS). The aim of this study was to examine the effect of PRT on the platelet storage lesion, in the presence of PAS with low plasma carryover. PRT-treated (Mirasol) and untreated buffy coat-derived platelet concentrates prepared in 28% plasma/PAS-IIIM were evaluated using in vitro cell quality parameters on days 1, 2, 5, and 7 post-collection. At day 5, there were no significant differences between control and PRT treated platelets for swirl, viability, pO(2) , pCO(2) , mean platelet volume and adenosine diphosphate-induced aggregation. PRT treatment did not affect the functional integrity of the mitochondria. However, PRT resulted in a decrease in pH and enhancement of platelet glycolysis and activation, evidenced by increased glucose consumption and lactate production rates, increased expression of CD62P, CD63, annexin V staining and increased secretion of cytokines (P < 0.05). Hypotonic shock response and aggregation in response to collagen were also significantly reduced in PRT treated platelets (P < 0.05). Despite the observed differences in platelet metabolism and activation observed following PRT treatment in PAS and low plasma carryover, the results suggest that treatment and storage of platelets in PAS is no more detrimental to platelets than treatment and storage in plasma. © 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.

  10. Contribution of blood platelets to vascular pathology in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Zhang W


    Full Text Available Wei Zhang,1,2 Wei Huang,1 Fang Jing11Department of Pharmacology, Institutes for Advanced Interdisciplinary Research, East China Normal University, Shanghai, People's Republic of China; 2Shanghai Engineering Research Center of Molecular Therapy and Pharmaceutical Innovation, Shanghai, People's Republic of ChinaAbstract: Cerebral amyloid angiopathy (CAA is a critical factor in the pathogenesis of Alzheimer's disease (AD. In the clinical setting, nearly 98% AD patients have CAA, and 75% of these patients are rated as severe CAA. It is characterized by the deposition of the β-amyloid peptide (mainly Aβ40 in the walls of cerebral vessels, which induces the degeneration of vessel wall components, reduces cerebral blood flow, and aggravates cognitive decline. Platelets are anuclear cell fragments from bone marrow megakaryocytes and their function in hemostasis and thrombosis has long been recognized. Recently, increasing evidence suggests that platelet activation can also mediate the onset and development of CAA. First, platelet activation and adhesion to a vessel wall is the initial step of vascular injury. Activated platelets contribute to more than 90% circulating Aß (mainly Aβ1-40, which in turn activates platelets and results in the vicious cycle of Aβ overproduction in damaged vessel. Second, the uncontrolled activation of platelets leads to a chronic inflammatory reaction by secretion of chemokines (eg, platelet factor 4 [PF4], regulated upon activation normal T-cell expressed and presumably secreted [RANTES], and macrophage inflammatory protein [MIP-1α], interleukins (IL-1 β, IL-7, and IL-8, prostaglandins, and CD40 ligand (CD40L. The interaction of these biological response modulators with platelets, endothelial cells, and leukocytes establishes a localized inflammatory response that contributes to CAA formation. Finally, activated platelets are the upholder of fibrin clots, which are structurally abnormal and resistant to degradation

  11. Clinical significance of receptor shedding-platelet GPVI as an emerging diagnostic and therapeutic tool. (United States)

    Chatterjee, Madhumita; Gawaz, Meinrad


    Platelet membrane bedecked with a wide array of receptors offers a platform to regulate platelet responsiveness, thrombotic propensity, inflammatory disposition, and immune reactivity under diverse pathophysiological conditions. Ectopic proteolytic cleavage of such receptors irreversibly inactivates receptor-mediated intracellular signaling governing cellular functions, further releases soluble fragments into circulation which might modulate functions of target cells. Glycoprotein VI-(GPVI) is a membrane glycoprotein expressed in platelets and megakaryocytes. Platelet GPVI surface expression is enhanced following acute ischemic events like myocardial infarction and cerebral stroke, serves as an imminent diagnostic tool independent of markers of tissue necrosis, and is associated with poor prognosis. Platelets undergo GPVI shedding and thereby contribute to soluble plasma levels of sGPVI, with distinct diagnostic and prognostic attributes. This review summarizes the functional significance and mechanistic basis whereby GPVI surface availability is up- or downregulated on platelets and the impact of GPVI in diagnostic, prognostic, and therapeutic strategies in diseases where platelets play a regulatory role. Further, we also highlight how novel non-invasive platelet-based diagnostic and therapeutic strategies have evolved utilizing GPVI for lesion-directed antithrombotic therapy or to counteract atherosclerotic disposition to ameliorate care of patients particularly in the context of cardio-cerebro-vascular medicine.

  12. Morphology of platelet Golgi apparatus and their significance after acute cerebral infarction. (United States)

    Lu, Wei; Xu, Dong; Tu, Ranran; Hu, Zhiping


    Blood samples were harvested from the antecubital vein of 20 fasting patients with acute cerebral infarction at 1, 7 and 15 days after onset to prepare blood platelet suspension. Fasting antecubital vein blood was collected from an additional 20 normal adults as controls. Under transmission tron microscope, platelet Golgi tubules and vesicles became significantly thickened, enlarged, and irregular after acute cerebral infarction. Alpha granules in platelets significantly reduced in number, especially 1 day after cerebral infarction. Under immunoelectron microscopy, a few alpha granules aggregated around Golgi tubules and vesicles after infarction. These results suggested that platelet Golgi apparatus displayed significant morphological changes, which were possibly associated with enhanced synthetic and secretory functions of activated platelets after acute cerebral infarction. This study used Golgi apparatus blocking agent Brefeldin A to block Golgi apparatus in an aim to study the effects of Golgi apparatus on CD40L expression on the surface of activated platelets. Flow cytometry revealed that CD40L expression on activated platelet surfaces decreased significantly when Golgi apparatus was blocked, which indicated that Golgi apparatus participated in the synthesis and transport of CD40L to the platelet surface.

  13. CD40 ligand (CD154) involvement in platelet transfusion reactions. (United States)

    Sahler, J; Spinelli, S; Phipps, R; Blumberg, N


    Platelet transfusions are commonly used treatments that occasionally lead to adverse reactions. Clinical trials, in vitro and animal studies have been performed to try to understand the causes of such reactions. Multiple studies have shown that the supernatant fraction of platelet concentrates contain prothrombotic and pro-inflammatory mediators. The origin of these mediators was first ascribed to white blood cells contaminating the platelet preparation. However, the accumulation of bioactive mediators after leukoreduction focused attention on platelets themselves during storage. Numerous cytokines, chemokines and prostaglandins are released in stored platelet concentrates. We have focused on a powerful mediator called soluble CD40 ligand (sCD40L, formally known as CD154) as a seminal contributor to adverse reactions. sCD40L can bind and signal the surface receptor, CD40, which is present on various types of human cells including white blood cells, vascular cells and fibroblasts. Downstream results of sCD40L/CD40 signaling include pro-inflammatory cytokine and chemokine production, prothrombotic mediator release, adherence and transmigration of leukocytes to endothelium and other undesirable vascular inflammatory events. Increased plasma levels of sCD40L can be detected in conditions such as myocardial infarction, stroke, unstable angina, high cholesterol, or other cardiovascular conditions. In retrospective studies, correlations were made between increased sCD40L levels of platelet concentrates and adverse transfusion reactions. We hypothesize that transfusion of partially activated, CD40L-expressing platelets along with sCD40L into a recipient with damaged or dysfunctional vascular tissue results in a "double-hit", thus inciting inflammation and vascular damage in the recipient. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  14. The platelet fibrinogen receptor: from megakaryocyte to the mortuary

    Directory of Open Access Journals (Sweden)

    Ferro Albert


    Full Text Available Platelets are integral to normal haemostatic function and act to control vascular haemorrhage with the formation of a stable clot. The fibrinogen receptor (glycoprotein IIb/IIIa [GPIIb/IIIa] is the most abundant platelet integrin and, by binding fibrinogen, facilitates irreversible binding of platelets to the exposed extracellular matrix and enables the cross-linking of adjacent platelets. The vital role of GPIIb/IIIa requires tight control of both its synthesis and function. After transcription from distinct domains on chromosome 17, the two subunits of the heterodimer are carefully directed through organelles with intricate regulatory steps designed to prevent the cellular expression of a dysfunctional receptor. Similarly, exquisite control of platelet activation via bidirectional signalling acts to limit the inappropriate and excessive formation of platelet-mediated thrombus. However, the enormous diversity of genetic mutations in the fibrinogen receptor has resulted in a number of allelic variants becoming established. The Pro33 polymorphism in GPIIIa is associated with increased cardiovascular risk due to a pathological persistence of outside-in signalling once fibrinogen has dissociated from the receptor. The polymorphism has also been associated with the phenomenon of aspirin resistance, although larger epidemiological studies are required to establish this conclusively. A failure of appropriate receptor function due to a diverse range of mutations in both structural and signalling domains, results in the bleeding diathesis Glanzmann's thrombasthaenia. GPIIb/IIIa inhibitors were the first rationally designed anti-platelet drugs and have proven to be a successful therapeutic option in high-risk primary coronary intervention. As our understanding of bidirectional signalling improves, more subtle and directed therapeutic strategies may be developed.

  15. Differences between mainstream and sidestream tobacco smoke extracts and nicotine in the activation and aggregation of platelets subjected to cardiovascular conditions in diabetes. (United States)

    Yin, Wei; Rubenstein, David A


    Mainstream and sidestream tobacco smoke extracts have been shown to increase platelet activation directly. Furthermore, advanced glycation end products, which are present in the diabetic vasculature, have also been shown to enhance platelet activity. However, the combined effects of these two risk factors on platelet functions remain unclear. Platelets were exposed to tobacco extracts concurrently with advanced glycation end products. Timed samples were removed to assess the extent of platelet activity. The presence of smoke extracts enhanced platelet activity as compared to control conditions, this was especially prevalent for sidestream extracts. With the addition of irreversibly glycated albumin, there was an additive effect, further enhancing platelet responses. This was at least partially regulated by α-granule release and CD41 expression. The combination of cardiovascular risk factors can significantly enhance platelet activation and aggregation, and therefore it is possible to accelerate cardiovascular diseases through the interactions of multiple cardiovascular risk factors.

  16. Complement Activation Alters Platelet Function (United States)


    been shown to participate directly in the immune response through interaction with vascular endothelium , with antigen presenting cells (APC) and...Syk inhibition decreases platelet lodging in the lungs indicating Syk is integral in platelet sequestration and organ damage. Crossing B6.lpr...adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading. Blood 110:1879-1886. 7. Lapchak, P.H., A. Ioannou, P. Rani

  17. Oral streptococci and cardiovascular disease: searching for the platelet aggregation-associated protein gene and mechanisms of Streptococcus sanguis-induced thrombosis. (United States)

    Herzberg, Mark C; Nobbs, Angela; Tao, Lin; Kilic, Ali; Beckman, Eric; Khammanivong, Ali; Zhang, Yongshu


    Pathogenic mechanisms in infective endocarditis, disseminated intravascular coagulation, and cardiovascular events involve the aggregation of platelets into thrombi. Attendant infection by oral bacteria contributes to these diseases. We have been studying how certain oral streptococci induce platelet aggregation in vitro and in vivo. Streptococcus sanguis expresses a platelet aggregation-associated protein (PAAP), which contributes little to adhesion to platelets. When specific antibodies or peptides block PAAP, S. sanguis fails to induce platelet aggregation in vitro or in vivo. We used subtractive hybridization to identify the gene encoding for PAAP. After subtraction of strain L50 (platelet aggregation-negative), four strain 133-79 specific sequences were characterized. Sequence agg4 encoded a putative collagen-binding protein (CbpA), which was predicted to contain two PAAP collagen-like octapeptide sequences. S. sanguis CbpA- mutants were constructed and tested for induction of platelet aggregation in vitro. Platelet aggregation was substantially inhibited when compared to the wild-type using platelet-rich plasma from the principal donor, but adhesion was unaffected. Other donor platelets responded normally to the CbpA- strain, suggesting additional mechanisms of response to S. sanguis. In contrast, CshA- and methionine sulfoxide reductase-negative (MsrA-) strains neither adhered nor induced platelet aggregation. CbpA was suggested to contribute to site 2 interactions in our two-site model of platelet aggregation in response to S. sanguis. Platelet polymorphisms were suggested to contribute to the thrombogenic potential of S. sanguis.

  18. Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease). (United States)

    Nofer, Jerzy-Roch; Herminghaus, Grazyna; Brodde, Martin; Morgenstern, Eberhard; Rust, Stephan; Engel, Thomas; Seedorf, Udo; Assmann, Gerd; Bluethmann, Horst; Kehrel, Beate E


    ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.

  19. Genetic determinants of platelet large-cell ratio, immature platelet fraction, and other platelet-related phenotypes. (United States)

    Pujol-Moix, Núria; Vázquez-Santiago, Miquel; Morera, Agnès; Ziyatdinov, Andrey; Remacha, Angel; Nomdedeu, Josep F; Fontcuberta, Jordi; Soria, José Manuel; Souto, Juan Carlos


    Platelets play a significant role in arterial thrombosis and are involved also in venous thrombosis. The genetic determinants of several platelet-related phenotypes have been studied previously. However, to the best of our knowledge, the genetic determinants of other platelet phenotypes have not been reported such as platelet-large-cell ratio (P-LCR) index, immature platelet fraction (IPF) parameters and overall platelet function measured through the PFA-100 system. As part of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project, 935 individuals from 35 large Spanish families, ascertained through a proband with thrombophilia, were studied. Using variance component methods, implemented in the SOLAR package, the heritability of the following sets of platelet-related phenotypes was determined: platelet count and indices, IPF, and platelet function. High heritabilities of the platelet count and index phenotypes (from 0.41 to 0.64) were found, especially for those related to platelet volume. The heritabilities of the IPF phenotypes, as a measure of platelet turnover, were the highest (from 0.65 to 0.69). The heritabilities of the platelet function phenotypes were high also (0.45 and 0.62). The covariate age influenced all of the platelet phenotypes. Smoking influenced the platelet indices related to platelet volume and all the IPF phenotypes. Venous thrombosis showed a heritability of 0.67. We did not find a genetic correlation between any of the platelet-related phenotypes and venous thrombosis. The high heritabilities found for all of the platelet phenotypes provid promising data for the identification of new genes that underly these phenotypes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations. (United States)

    Hom, Sarah; Chen, Li; Wang, Tony; Ghebrehiwet, Berhane; Yin, Wei; Rubenstein, David A


    Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

  1. A megakaryocyte with no platelets: anti-platelet antibodies, apoptosis, and platelet production. (United States)

    Perdomo, José; Yan, Feng; Chong, Beng H


    Primary immune thrombocytopenia (ITP) and drug-induced thrombocytopenia (DITP) are disorders caused primarily by the presence of anti-platelet auto-antibodies (Abs). Hematologists have traditionally seen thrombocytopenia as the result of increased destruction of Ab-coated platelets by the reticuloendothelial system. While accurate, this approach does not fully account for other laboratory observations. There is increasing evidence suggesting a significant cellular component in the etiology of both ITP and DITP. In ITP, megakaryocytes (Mks) show characteristics consistent with increased apoptosis, which correlates with a reduction in platelet production capacity. Platelet production by Mks is impaired in both the bone marrow of ITP patients and in Mks produced in vitro when treated with ITP or DITP auto-Abs. Recently, it was shown that anti GPIb/IX DITP Abs act directly on Mks and induce apoptosis, hinder differentiation, and prevent platelet production. The origin of pathological megakaryocytic apoptosis is yet to be explored in more detail but current observations imply that there is a direct contribution by anti-platelet Abs. Here we review the evidence for Ab-mediated megakaryocytic damage in ITP and DITP, examine possible molecular mechanisms and consider potential clinical implications.

  2. Comparative evaluation of the role of the adhesion molecule CD177 in neutrophil interactions with platelets and endothelium. (United States)

    Pliyev, Boris K; Menshikov, Mikhail


    Neutrophil-specific glycoprotein CD177 is expressed on a subset of human neutrophils and has been shown to be a counter-receptor for platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31). Previous studies have demonstrated that the interaction of CD177 with endothelial PECAM-1 supports neutrophil transendothelial migration resulting in preferential transmigration of the CD177-expressing neutrophil subset. As PECAM-1 is also abundantly expressed on platelets, we addressed a follow-up suggestion that CD177/PECAM-1 adhesive interaction may mediate platelet-neutrophil interactions and CD177-positive neutrophils may have a competitive advantage over CD177-negative neutrophils in binding platelets. Here, we report that CD177-positive and CD177-negative neutrophils do not differ significantly in their capacity to form platelet-neutrophil conjugates as assayed in whole blood and in mixed preparations of isolated platelets and neutrophils. Under flow conditions, neither platelet nor neutrophil activation resulted in preferential binding of platelets to CD177-expressing neutrophils. Furthermore, no significant difference was found in the ability of both neutrophil subsets to adhere to and migrate across surface-adherent activated platelets, whereas predominantly CD177-positive neutrophils migrated across HUVEC monolayers. In addition, we demonstrated that S(536) N dimorphism of PECAM-1, which affects CD177/PECAM-1 interaction, did not influence the equal capacity of the two neutrophil subsets to interact with platelets but influenced significantly the transendothelial migration of CD177-expressing neutrophils. Thus, CD177/PECAM-1 adhesive interaction, while contributing to neutrophil-endothelial cell interaction in neutrophil transendothelial migration, does not contribute to or is redundant in platelet-neutrophil interactions. © 2012 John Wiley & Sons A/S.

  3. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

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    Svensson Holm, Ann-Charlotte B., E-mail: [Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoeping University, SE-581 85 Linkoeping (Sweden); Experimental Pathology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Bengtsson, Torbjoern [Department of Biomedicine, School of Health and Medical Sciences, Oerebro University, SE-70182 Oerebro (Sweden); Grenegard, Magnus; Lindstroem, Eva G. [Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoeping University, SE-581 85 Linkoeping (Sweden)


    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  4. Platelets and infection — an emerging role of platelets in viral infection

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    Alice eAssinger


    Full Text Available Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia and several platelet function disorders increase the risk of bleeding. Over the last years more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients.Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favours platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies.All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count, but also shapes immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation and platelet-mediated modulations of innate and adaptive immune responses.

  5. Chemotherapy induced thrombocytopenia treated by four types of platelets concentrates

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    Nikolić Ljubinka I.


    Full Text Available Introduction: Serious adverse event of anticancer chemotherapy is glanulocytopenia and thrombocytopenia which can decrease efficiency of final therapy results. After many years, platelet concentrates transfusion (PCT is still researching problem without sure standpoint. The aim: To determine whether there is a difference in the clinical efficiency in the use of 4 types of platelet applied for transfusion; - to ascertain whether platelet count increase expressed as corrected count increment (CCI, is a better parameter for the evaluation of platelet transfusion efficiency than the bleeding time (Bt, as the only readily assessable in vivo platelet function related parameter. Subjects and methods: This paper is a part of academic (noncommercial IV phase observational nointervetion study. Investigation included 78 patients diagnosed with malignant lymphoma and metastatic solid tumors, transfused by platelet concentrates. Patients were devided into 4 groups, based on the type of platelet concentrates used for transfusion. Results: Patients, were transfused with total number of 647 PC units (235 units were non-leukodepleted and 412 units were leukodepleted. Mean number of PC transfusions per patient was 8.3 PC units, and 4.8 PC unit per one transfusion episode. Before PCT: platelets values were: 18.1 x109/L ±13.1, Bt 8.4±6.1min, and after PCT were 28.2 x109/L ±22.1, 4.7±4.4 min respectively ((p<0.01. Mean CCI value was 13.8±30.4. CCI was corrected in 196/129 PCT and Bt in 122/129 PCT. After supportive therapy using PCs Bt was corrected and became similar in all 4 groups. Discussion: Clinical output is the most important parameter for treatment decision because many patients can tolerate prolonged periods of profound thrombocytopenia without serious bleeding problems. Conclusion: In all 4 investigated groups of patients bleeding time was a far better parameter compared with CCI for the PC therapy efficiency. Authors suggest to be careful and follow

  6. Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study. (United States)

    Pieroni, Luisa; Finamore, Francesco; Ronci, Maurizio; Mattoscio, Domenico; Marzano, Valeria; Mortera, Stefano Levi; Quattrucci, Serena; Federici, Giorgio; Romano, Mario; Urbani, Andrea


    Platelets are of pathophysiological relevance in haemostasis, wound repair, inflammation and cardiovascular disease. We have shown that human platelets express a biologically active Cystic Fibrosis Transmembrane Conductance Regulator, which is dysfunctional in Cystic Fibrosis (CF) patients, and regulate platelet responses related to inflammation and its resolution. In order to further elucidate platelet involvement in CF inflammation, we pursued a comparative proteomic analysis of cells from healthy donors and CF patients, in association with a non-supervised comparative analysis of the Gene Ontology. Our results, showing changes in the integrin signalling in CF, support a pro-inflammatory profile of CF platelets.