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

Science.gov (United States)

Robert, Amélie; Boyer, Lucie; Pineault, Nicolas

2011-03-01

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

Fabricating bio-inspired micro/nano-particles by polydopamine coating and surface interactions with blood platelets

Energy Technology Data Exchange (ETDEWEB)

Ye, Wei [Jiangsu Provincial Key Lab for Interventional Medical Devices, Huaiyin Institute of Technology, Huaian 223003 (China); State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Shi, Qiang, E-mail: shiqiang@ciac.ac.cn [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Hou, Jianwen; Gao, Jian; Li, Chunming; Jin, Jing; Shi, Hengchong [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Yin, Jinghua, E-mail: yinjh@ciac.ac.cn [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)

2015-10-01

Graphical abstract: The particles or particle aggregations activate the blood platelets and provide the physical adhesive sites for platelets adhesion. - Highlights: • Particles with varied sizes and surface properties were fabricated by facile polydopamine (PDA) coating on polystyrene microsphere. • The direct interaction between PDA particles and blood platelets was qualitatively investigated. • The knowledge on platelet–particle interactions provided the basic principle to select biocompatible micro/nano-particles in biomedical field. - Abstract: Although bio-inspired polydopamine (PDA) micro/nano-particles show great promise for biomedical applications, the knowledge on the interactions between micro/nano-particles and platelets is still lacking. Here, we fabricate PDA-coated micro/nano-particles and investigate the platelet–particle surface interactions. Our strategy takes the advantage of facile PDA coating on polystyrene (PS) microsphere to fabricate particles with varied sizes and surface properties, and the chemical reactivity of PDA layers to immobilize fibrinogen and bovine serum albumin to manipulate platelet activation and adhesion. We demonstrate that PS particles activate the platelets in the size-dependent manner, but PDA nanoparticles have slight effect on platelet activation; PS particles promote platelet adhesion while PDA particles reduce platelet adhesion on the patterned surface; Particles interact with platelets through activating the glycoprotein integrin receptor of platelets and providing physical sites for initial platelet adhesion. Our work sheds new light on the interaction between platelets and particles, which provides the basic principle to select biocompatible micro/nano-particles in biomedical field.

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

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Uras, Fikriye; Küçük, Burhanettin; Bingöl Özakpınar, Özlem; Demir, Ahmet Muzaffer

2015-03-05

Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Signal transduction by the platelet-derived growth factor receptor

International Nuclear Information System (INIS)

Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

1988-01-01

The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

Platelet adhesion studies on dipyridamole coated polyurethane surfaces

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Aldenhoff Y. B.J.

2003-06-01

Full Text Available Surface modification of polyurethanes (PUs by covalent attachment of dipyridamole (Persantinregistered is known to reduce adherence of blood platelets upon exposure to human platelet rich plasma (PRP. This effect was investigated in further detail. First platelet adhesion under static conditions was studied with four different biomaterial surfaces: untreated PU, PU immobilised with conjugate molecule 1, PU immobilised with conjugate molecule 2, and PU immobilised with conjugate molecule 3. In PU immobilised with 1 dipyridamole is directly linked to the surface, in PU immobilised with 2 there is a short hydrophilic spacer chain in between the surface and the dipyridamole, while conjugate molecule 3 is merely the spacer chain. Scanning electron microscopy (SEM was used to characterise platelet adhesion from human PRP under static conditions, and fluorescence imaging microscopy was used to study platelet adhesion from whole blood under flow. SEM experiments encompassed both density measurements and analysis of the morphology of adherent platelets. In the static experiments the surface immobilised with 2 showed the lowest platelet adherence. No difference between the three modified surfaces emerged from the flow experiments. The surfaces were also incubated with washed blood platelets and labeled with Oregon-Green Annexin V. No capture of Oregon-Green Annexin V was seen, implying that the adhered platelets did not expose any phosphatidyl serine at their exteriour surface.

Identification of a second putative receptor of platelet activating factor on human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Hwang, S.B.

1987-01-01

Due to multiple molecular species of platelet activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors on human leukocytes and platelets. Human PMN leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (Kd) of 4.7 (+/- 1.4) x 10 -10 M. The maximal number (B/sub max/) of receptor sites was estimated to be 3.13 (+/- 1.4) x 10 -13 mol/mg protein. They compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One antagonist (Ono-6240) was found to be 8 times less potent at inhibiting the [ 3 H]PAF specific receptor binding to human leukocytes than to human platelets. Mg 2+ , Ca 2+ and K + ions potentiated the [ 3 H]PAF specific binding in both systems. Na + ions inhibited the [ 3 H]PAF specific binding to human platelets but showed no effects in human leukocytes. K + ions decreased the Mg 2+ -potentiated [ 3 H]PAF binding in human leukocytes but showed no effects in human platelets. These results suggest that the PAF specific receptors in human leukocytes are different structurally and possibly functionally from the receptors identified in human platelets

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation.

Science.gov (United States)

Barton, J F; Hardy, A R; Poole, A W; Mundell, S J

2008-03-01

Thromboxane A(2) and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y(1) but not P2Y(12) receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y(12)-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.

Renal cells activate the platelet receptor CLEC-2 through podoplanin

Science.gov (United States)

Christou, Charita M.; Pearce, Andrew C.; Watson, Aleksandra A.; Mistry, Anita R.; Pollitt, Alice Y.; Fenton-May, Angharad E.; Johnson, Louise A.; Jackson, David G.; Watson, Steve P.; O'Callaghan, Chris A.

2009-01-01

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. PMID:18215137

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

International Nuclear Information System (INIS)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface

NARCIS (Netherlands)

Weeterings, Cees; de Groot, Philip G.; Adelmeijer, Jelle; Lisman, Ton

2008-01-01

Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we

Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression

International Nuclear Information System (INIS)

Mori, Hideki; Koyama, Tsukasa; Yamashita, Itaru

1991-01-01

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets. This method involves the measurement of the accumulation of [ 3 H]-inositol-1-phosphate (IP-1) as an index of Pl hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression

Imaging thrombus with radiolabelled monoclonal antibody to platelets

Energy Technology Data Exchange (ETDEWEB)

Peters, A.M.; Lavender, J.P.; Needham, S.G.; Loutfi, I.; Snook, D.; Epenetos, A.A.; Lumley, P.; Keery, R.J.; Hogg, N.

1986-12-13

A study was conducted evaluating a method of imaging thrombus with platelets radiolabelled with a /sup 111/In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. when the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface, platelet function was undisturbed. P256 was radiolabelled with /sup 111/In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The /sup 111/In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third had chronic deep venous thrombosis and was scintigraphically negative.

Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

International Nuclear Information System (INIS)

Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H.

1990-01-01

The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[ 3 H]ethylcarboxamidoadenosine [( 3 H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [ 3 H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

Science.gov (United States)

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

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured 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.

Encapsulation of Platelet in Kefiran Polymer and Detection of Bioavailability of Immobilized Platelet in Probiotic Kefiran as A New Drug for Surface Bleeding

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Anahita Jenab

2015-11-01

Full Text Available Background : Kefir contains lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, Acetobacter and Streptococcus and yeasts (Kluyveromyces, Torula, Candida, Saccharomyces .Kefiran is the polysaccharide produced by lactic acid bacteria in kefir.Methods : Kefiran was prepared from milk containing 0.5% fat and 10 grams kefir grains and was separated from kefir by ethanol (0.02 gram following entrapping the platelets to this polymer. Ligand of the platelet-polysaccharide was studied by FTIR.Results : FTIR results showed that the bands of C-O and C-O-C connections were formed and the polysaccharides had been attached to the receptors of the platelet glycoproteins (GP Ib,GPIIb / IIIa. Stability and encapsulation of the platelet and kefiran were assessed by Coulter Counter. Encapsulation of the platelets by polysaccharide at the beginning caused to reduce the number of platelets following by releasing of 50% of the platelets after 3 hours.Conclusion : The platelets were encapsulated in kefiran polymer and detected for bioavailability as new drug for surface bleeding. Also, kefiran has antimicrobial and antifungal properties. On the other hand, the existence of nisin in kefiran could be useful as an antibacterial lantibiotic. 

Sulfatides partition disabled-2 in response to platelet activation.

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Karen E Drahos

Full Text Available BACKGROUND: Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2, which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB domain by competing with fibrinogen for alphaIIbbeta3 integrin receptor binding by an unknown mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Using protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB, specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (K(d of 0.6 microM as estimated by surface plasmon resonance (SPR analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process. CONCLUSIONS/SIGNIFICANCE: Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.

Dual roles for hepatic lectin receptors in the clearance of chilled platelets

DEFF Research Database (Denmark)

Rumjantseva, Viktoria; Grewal, Prabhjit K; Wandall, Hans H

2009-01-01

-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell...... transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold....

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

Science.gov (United States)

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-Hara, Tomoko; Fujita, Naoya

2014-08-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Modified expression of surface glyconjugates in stored human platelets

International Nuclear Information System (INIS)

Dhar, A.; Ganguly, P.

1987-01-01

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 0 or 22 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 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

Modified expression of surface glyconjugates in stored human platelets

Energy Technology Data Exchange (ETDEWEB)

Dhar, A.; Ganguly, P.

1987-05-01

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.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

Science.gov (United States)

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

2016-01-01

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

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

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Clive Metcalfe

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

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets

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Marcinkiewicz C

2017-05-01

Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis

Imaging thrombus with radiolabelled monoclonal antibody to platelets

International Nuclear Information System (INIS)

Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

1988-01-01

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

International Nuclear Information System (INIS)

Thibonnier, M.

1987-01-01

Tritiated vasopressin ([ 3 H]AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with [ 3 H]AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, [ 3 H]AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with [ 3 H]AVP is a suitable tool for the purification of the human platelet vasopressin receptor

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

Science.gov (United States)

Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Platelet alpha 2-adrenergic receptors in major depressive disorder. Binding of tritiated clonidine before and after tricyclic antidepressant drug treatment

International Nuclear Information System (INIS)

Garcia-Sevilla, J.A.; Zis, A.P.; Hollingsworth, P.J.; Greden, J.F.; Smith, C.B.

1981-01-01

The specific binding of tritiated (3H)-clonidine, an alpha 2-adrenergic receptor agonist, to platelet membranes was measured in normal subjects and in patients with major depressive disorder. The number of platelet alpha 2-adrenergic receptors from the depressed group was significantly higher than that found in platelets obtained from the control population. Treatment with tricyclic antidepressant drugs led to significant decreases in the number of platelet alpha 2-adrenergic receptors. These results support the hypothesis that the depressive syndrome is related to an alpha 2-adrenergic receptor supersensitivity and that the clinical effectiveness of tricyclic antidepressant drugs is associated with a decrease in the number of these receptors

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

National Research Council Canada - National Science Library

Czapiga, Meggan; Gao, Ji-Liang; Kirk, Allen; Lekstrom-Himes, Julie

2005-01-01

Activated platelets participate in inflammatory and microbicidal processes by upregulation of surface selectins, shedding of CD40 ligand, and release of platelet microbicidal proteins and microparticles...

Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

NARCIS (Netherlands)

De Keyser, J; Wilczak, N

1997-01-01

Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

Science.gov (United States)

Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

Directory of Open Access Journals (Sweden)

Venkateswarlu Kanamarlapudi

Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin

International Nuclear Information System (INIS)

Guha Thakurta, Sanjukta; Miller, Robert; Subramanian, Anuradha

2012-01-01

Surfaces that preferentially bind human serum albumin (HSA) were generated by grafting albumin-binding linear peptide (LP1) onto silicon surfaces. The research aim was to evaluate the adsorption pattern of proteins and the adhesion of platelets from platelet-poor plasma and platelet-rich plasma, respectively, by albumin-binding surfaces under physiological shear rate (96 and 319 s −1 ) conditions. Bound proteins were quantified using enzyme-linked immunosorbent assays (ELISAs) and two-dimensional gel electrophoresis. A ratio of ∼1000:100:1 of adsorbed HSA, human immunoglobulin (HIgG) and human fibrinogen (HFib) was noted, respectively, on LP1-functionalized surfaces, and a ratio of ∼5:2:1 of the same was noted on control surfaces, as confirmed by ELISAs. The surface-adsorbed von Willebrand factor was undetectable by sensitive ELISAs. The amount of adhered platelets correlated with the ratio of adsorbed HSA/HFib. Platelet morphology was more rounded on LP1-functionalized surfaces when compared to control surfaces. The platelet adhesion response on albumin-binding surfaces can be explained by the reduction in the co-adsorption of other plasma proteins in a surface environment where there is an excess of albumin molecules, coupled with restrictions in the conformational transitions of other surface-adsorbed proteins into hemostatically active forms. (paper)

Identification and characterization of a putative human platelet thromboxane A2/prostaglandin H2 receptor

International Nuclear Information System (INIS)

Saussy, D.L. Jr.

1986-01-01

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

Surface modification of platelet concentrate bags to reduce biofilm formation and transfusion sepsis.

Science.gov (United States)

Wilson-Nieuwenhuis, Joels S T; Dempsey-Hibbert, Nina; Liauw, Christopher M; Whitehead, Kathryn A

2017-12-01

Bacterial contamination of blood products poses a major risk in transfusion medicine, including transfusions involving platelet products. Although testing systems are in place for routine screening of platelet units, the formation of bacterial biofilms in such units may decrease the likelihood that bacteria will be detected. This work determined the surface properties of p-PVC platelet concentrate bags and investigated how these characteristics influenced biofilm formation. Serratia marcescens and Staphylococcus epidermidis, two species commonly implicated in platelet contamination, were used to study biofilm growth. The platelet concentrate bags were physically flattened to determine if reducing the surface roughness altered biofilm formation. The results demonstrated that the flattening process of the platelet bags affected the chemistry of the surface and reduced the surface hydrophobicity. Flattening of the surfaces resulted in a reduction in biofilm formation for both species after 5 days, with S. marcescens demonstrating a greater reduction. However, there was no significant difference between the smooth and flat surfaces following 7 days' incubation for S. marcescens and no significant differences between any of the surfaces following 7 days' incubation for S. epidermidis. The results suggest that flattening the p-PVC surfaces may limit potential biofilm formation for the current duration of platelet storage time of 5 days. It is hoped that this work will enhance the understanding of how surface properties influence the development of microbial biofilms in platelet concentrate bags in order to devise a solution to discourage biofilm formation. Copyright © 2017 Elsevier B.V. All rights reserved.

The interaction of thrombin with platelet protease nexin

International Nuclear Information System (INIS)

Knupp, C.L.

1989-01-01

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible

Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation.

Science.gov (United States)

Cox, D; Kerrigan, S W; Watson, S P

2011-06-01

It has become clear that platelets are not simply cell fragments that plug the leak in a damaged blood vessel; they are, in fact, also key components in the innate immune system, which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the cells that respond first to a site of injury, they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets, which usually triggers platelet activation and the secretion of antimicrobial peptides. The main platelet receptors that mediate these interactions are glycoprotein (GP)IIb-IIIa, GPIbα, FcγRIIa, complement receptors, and TLRs. This process may involve direct interactions between bacterial proteins and the receptors, or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement, and IgG. Here, we review the variety of interactions between platelets and bacteria, and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis. © 2011 International Society on Thrombosis and Haemostasis.

Biomimetic Fluorocarbon Surfactant Polymers Reduce Platelet Adhesion on PTFE/ePTFE Surfaces

Science.gov (United States)

Wang, Shuwu; Gupta, Anirban Sen; Sagnella, Sharon; Barendt, Pamela M.; Kottke-Marchant, Kandice; Marchant, Roger E.

2010-01-01

We describe a series of fluorocarbon surfactant polymers designed as surface-modifying agents for improving the thrombogenicity of ePTFE vascular graft materials by the reduction of platelet adhesion. The surfactant polymers consist of a poly(vinyl amine) backbone with pendent dextran and perfluoroundecanoyl branches. Surface modification is accomplished by a simple dip-coating process in which surfactant polymers undergo spontaneous surface-induced adsorption and assembly on PTFE/ePTFE surface. The adhesion stability of the surfactant polymer on PTFE was examined under dynamic shear conditions in PBS and human whole blood with a rotating disk system. Fluorocarbon surfactant polymer coatings with three different dextran to perfluorocarbon ratios (1:0.5, 1:1 and 1:2) were compared in the context of platelet adhesion on PTFE/ePTFE surface under dynamic flow conditions. Suppression of platelet adhesion was achieved for all three coated surfaces over the shear-stress range of 0–75 dyn/cm2 in platelet-rich plasma (PRP) or human whole blood. The effectiveness depended on the surfactant polymer composition such that platelet adhesion on coated surfaces decreased significantly with increasing fluorocarbon branch density at 0 dyn/cm2. Our results suggest that fluorocarbon surfactant polymers can effectively suppress platelet adhesion and demonstrate the potential application of the fluorocarbon surfactant polymers as non-thrombogenic coatings for ePTFE vascular grafts. PMID:19323880

Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

Science.gov (United States)

Whiss, P A; Andersson, R G G

2002-07-01

At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg(2+) and Ca(2+) are essential for platelet adhesion and activation, but Mg(2+) can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg(2+) increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca(2+) induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn(2+) elicited dose-dependent adhesion only to collagen and fibrinogen. Zn(2+), Ni(2+) and Cu(2+) increased the adhesion of platelets independently of the surface. Ca(2+) dose-dependently inhibited adhesion elicited by Mg(2+) to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg(2+)-dependent platelet adhesion to collagen and Ca(2+)-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression.

Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion.

Science.gov (United States)

Owaynat, Hadil; Yermolenko, Ivan S; Turaga, Ramya; Lishko, Valeryi K; Sheller, Michael R; Ugarova, Tatiana P

2015-12-01

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2015-04-01

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Glu- and Lys-forms of plasminogen differentially affect phosphatidylserine exposure on the platelet surface

Directory of Open Access Journals (Sweden)

D. D. Zhernossekov

2017-04-01

Full Text Available Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood. However, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells. We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion. The aim of this work was to investigate the influence of Glu- and Lys-form of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker. Human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years by gel-filtration on Sepharose 2B. Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5. Glu- and Lys-plasminogen have different impact on the platelet functioning. Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombin- and collagen-activated human platelets. Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation. Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes. The observed different effects of Glu- and Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.

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

DEFF Research Database (Denmark)

Hansen, Klaus; Rönnstrand, L; Rorsman, C

1997-01-01

The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha...

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

International Nuclear Information System (INIS)

Torr, S.R.; Winters, K.J.; Santoro, S.A.; Sobel, B.E.

1990-01-01

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

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

Science.gov (United States)

Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1988-01-01

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A) + RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion

Energy Technology Data Exchange (ETDEWEB)

Gao, Bin [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@hotmail.com [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China); Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Lu, Jian; Zhang, Li; Zhao, Miao; Shi, Changcan; Khan, Musammir [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Guo, Jintang [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China)

2013-07-01

In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices. - Highlights: • MPC was successfully grafted onto polycarbonate urethane surface via Michael reaction. • High concentration of PC functional groups on the surface via TAEA molecule • Biomimetic surface modification • The modified surface showed high hydrophilicity and anti-platelet adhesion.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

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Chen Qian

Full Text Available Platelet-derived growth factor receptor alpha (PDGFRα is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures.To address the temporal requirement of Pdgfra in embryonic development.We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies.Current study showed that (i conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5 resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives.Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b if mutations / sequence variations of these regulatory elements cause these anomalies.

Affinity column for purification of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor

International Nuclear Information System (INIS)

Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

1987-01-01

The TXA 2 /PGH 2 receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific 3 H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA 2 /PGH 2 receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor

Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

International Nuclear Information System (INIS)

Wu Guoxin; Li Jianyong; Ruan Changgeng

1991-08-01

Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

The surface blistering kinetics and the H-platelet evolution in H-implanted germanium

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Yang Fan; Zhang Xuanxiong; Ye Tianchun; Zhuang Songlin

2012-01-01

The surface blistering phenomenon produced in H-implanted Ge by a series of low temperature annealing processes was investigated. The kinetic plot of the onset of blistering contains a break point that separates the straight-line plot into two parts, with two distinct slopes based on the calculated activation energy from the different temperature regions for 3×10 16 cm -2 and 5×10 16 cm -2 H-implanted doses. This plot indicates the existence of distinct, temperature dependent mechanisms, probably caused by the release of different types of H-platelets. The turning direction (from low to high temperature) of the Arrhenius plot with the break point is contrary to that of other known materials. The formation and evolution of the H-platelets under the Ge surface was revealed by TEM (transmission electron microscopy). The TEM results demonstrate that the 〈0 0 1〉 platelets parallel to the sample surface are first produced by a low H implantation dose; however, the vertical 〈0 1 0〉 platelets perpendicular to the sample surface form later as the H implantation dose increases. The H-platelets combine with each other, becoming micro-cracks. The {1 1 1} and {3 1 1} micro-cracks serve as interconnections between the 〈0 0 1〉-oriented micro-cracks below the substrate surface. Finally, the accumulated H 2 pressure in the cracks deforms the surface to generate Ge surface exfoliation.

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

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Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

2008-07-01

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

Platelets and infections—complex interactions with bacteria

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Hind eHAMZEH-COGNASSE

2015-02-01

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

Deposition of Fibrinogen on the Surface of in vitro Thrombi Prevents Platelet Adhesion

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Owaynat, Hadil; Yermolenko, Ivan S.; Turaga, Ramya; Lishko, Valeryi K.; Sheller, Michael R.; Ugarova, Tatiana P.

2015-01-01

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorpt...

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

Science.gov (United States)

Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

2009-03-01

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

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Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1999-01-01

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary

Platelet fibrinogen binding in Basset Hound Hereditary Thrombopathy

International Nuclear Information System (INIS)

Patterson, W.; Estry, D.; Schwartz, K.; Bell, T.

1986-01-01

Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) display a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa). In order to investigate the possibility of a functionally abnormal GPIIb-IIIa complex, which might be unable to bind fibrinogen after stimulation, fibrinogen binding in BHT was evaluated. Two canine fibrinogen preparations were used, one from BHT dogs and one from normal control dogs, as well as a human fibrinogen preparation. Platelets from BHT and normal dogs were activated with 1 x 10 -5 M ADP in the presence of 125 I-labeled fibrinogen and the surface bound radioactivity quantitated. For all fibrinogen preparations, the amount of fibrinogen bound by BHT platelets was not significantly different than that bound by normal dog platelets. BHT platelets bound 23,972 +/- 3612 and normal dog platelets bound 23,033 +/- 3971 molecules of fibrinogen per platelet. The BHT platelet aggregation defect does not seem to be caused by a functionally abnormal GP IIb-IIIa complex, since BHT platelets bind normal amounts of fibrinogen. The results suggest that fibrinogen binding is not sufficient for platelet aggregation, and other factors, perhaps receptor mobility and membrane phospholipid content should be investigated in BHT

Platelet thrombosis in cardiac-valve prostheses

International Nuclear Information System (INIS)

Dewanjee, M.K.

1989-01-01

The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs

Platelet thrombosis in cardiac-valve prostheses

Energy Technology Data Exchange (ETDEWEB)

Dewanjee, M.K.

1989-01-01

The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs.

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets.

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Nisar, Shaista; Daly, Martina E; Federici, Augusto B; Artoni, Andrea; Mumford, Andrew D; Watson, Stephen P; Mundell, Stuart J

2011-11-17

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

Exchangeable splenic platelet pool in response to intravenous infusion of isoprenaline

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Freden, K; Olsson, L B; Suurkula, M; Kutti, J

1978-01-01

8 healthy volunteers and 4 asplenic subjects, in whom autologous platelets had been labelled with radioactive sodium chromate, received intravenous infusions of isoprenaline in a dose of 0.03 ..mu..g x kg/sup -1/ x min/sup -1/ over a period of 6 min. In the former group these infusions caused a significant decrease in the concentration of labelled as well as unlabelled platelets in the peripheral blood. Body surface countings showed that a significant increase in the count rate over the spleen occurred concomitantly with the decrease in the circulating platelet-bound radioactivity (PBR). In the group of asplenic subjects no change in PBR occurred. It is concluded that adrenergic beta-receptor stimulation causes a transitory trapping of platelets in the exchangeable splenic platelet pool.

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: Production of a platelet-derived growth factor-like mitogen and expression of a gene for a platelet-derived growth factor receptor--a preliminary study

International Nuclear Information System (INIS)

Birinyi, L.K.; Warner, S.J.; Salomon, R.N.; Callow, A.D.; Libby, P.

1989-01-01

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor

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

Science.gov (United States)

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

2016-08-01

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

Platelets and their chemokines in atherosclerosis – clinical applications

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Philipp evon Hundelshausen

2014-08-01

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

Reaction of [3H]-taurine maleimide with platelet surface thiols

International Nuclear Information System (INIS)

Karl, D.W.; Mills, D.C.B.

1986-01-01

Taurine Maleimide (2-maleimidoethanesulfonate, TM) was synthesized from [2- 3 H]-taurine and methoxycarbonylmaleimide (MCM). The yield of a 1 μmol synthesis approached 100% (based on taurine) when MCM was used in 4-fold excess. The product (TM*) was purified by ion exchange chromatography. TM* reacted irreversibly with thiol groups on the surface of washed human platelets, leading to incorporation of radioactivity into platelet pellets. Incorporation was blocked by cysteine, mercuribenzenesulfonate (MBS), dithiobisnitrobenzoate, and N-ethylmaleimide, but not by taurine or by inhibitors of anion transport. Reaction of TM* with platelets showed the dependence on time and concentration characteristics of a bimolecular reaction. The number of reactive sites ranged from 1 to 5 x 10 5 /platelet, and the apparent rate constant from 1 to 3 x 10 3 /(M x min). TM was less effective than MBS as an inhibitor of platelet aggregation induced by several agents. TM had no effect on the uptake of serotonin, taurine, or phosphate by the platelets, processes which are sensitive to MBS. These differences, considered with the similarity in size and charge of TM and MBS, suggest that classes of thiols defined as exofacial by their accessibility to MBS can differ substantially in their reactivity with other impermeant reagents

Identification of Aspergillus fumigatus Surface Components That Mediate Interaction of Conidia and Hyphae With Human Platelets.

Science.gov (United States)

Rambach, Günter; Blum, Gerhard; Latgé, Jean-Paul; Fontaine, Thierry; Heinekamp, Thorsten; Hagleitner, Magdalena; Jeckström, Hanna; Weigel, Günter; Würtinger, Philipp; Pfaller, Kristian; Krappmann, Sven; Löffler, Jürgen; Lass-Flörl, Cornelia; Speth, Cornelia

2015-10-01

Platelets were recently identified as a part of innate immunity. They are activated by contact with Aspergillus fumigatus; putative consequences include antifungal defense but also thrombosis, excessive inflammation, and thrombocytopenia. We aimed to identify those fungal surface structures that mediate interaction with platelets. Human platelets were incubated with Aspergillus conidia and hyphae, isolated wall components, or fungal surface mutants. Interaction was visualized microscopically; activation was quantified by flow cytometry of specific markers. The capacity of A. fumigatus conidia to activate platelets is at least partly due to melanin, because this effect can be mimicked with "melanin ghosts"; a mutant lacking melanin showed reduced platelet stimulating potency. In contrast, conidial hydrophobin masks relevant structures, because an A. fumigatus mutant lacking the hydrophobin protein induced stronger platelet activation than wild-type conidia. A. fumigatus hyphae also contain surface structures that interact with platelets. Wall proteins, galactomannan, chitin, and β-glucan are not the relevant hyphal components; instead, the recently identified fungal polysaccharide galactosaminogalactan potently triggered platelet activation. Conidial melanin and hydrophobin as well as hyphal galactosaminogalactan represent important pathogenicity factors that modulate platelet activity and thus might influence immune responses, inflammation, and thrombosis in infected patients. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A discrepancy between platelet alpha 2-receptor density and functional circulatory changes in hypertensives

International Nuclear Information System (INIS)

Mores, N.; Martire, M.; Pistritto, G.; Cardillo, C.; Folli, G.

1990-01-01

To investigate whether differences exist in peripheral alpha 2-adrenoceptors between normotensive and hypertensive subjects, we determined platelet alpha 2-adrenoceptor density in 10 (7 males) untreated essential hypertensives (mean age of 51.1 years, range of 44-59 years) and in 10 age- and sex-matched normotensive controls. Moreover, in hypertensive patients, we examined the relationship between receptor density and cardiovascular reactivity to mental arithmetic, static handgrip, and bicycle exercise, to verify the hypothesis that alpha 2-adrenoceptors might play a role in modulation of hemodynamic response to sympathetic stimuli. alpha 2-Adrenoceptor density, as calculated by binding of [3H]yohimbine to platelets, was significantly higher in essential hypertensives (314.8 +/- 38.7 fmol/mg) than in normotensive subjects (213.6 +/- 34.7 fmol/mg) (p less than 0.05), whereas receptor affinity was similar in both groups (4.0 +/- 0.5 nM hypertensives, 4.3 +/- 0.5 nM normotensives; p greater than 0.05). Mental arithmetic increased mean arterial pressure (MAP) by 21.5% from basal values and heart rate (HR) by 13.2%. During isometric exercise, MAP increased by 38.1% and HR by 24.7%, while during bicycle ergometry, mean increases in MAP and HR from baseline were of 27.2 and 54.3%, respectively. No correlation was found between platelet alpha 2-adrenoceptor density and percent changes in MAP induced by all tests, or between adrenoceptors and absolute basal and peak MAP values. Our findings suggest that in hypertensive patients, peripheral alpha 2-adrenoceptors are increased with respect to matched normotensives, but these receptors seem not to be involved in the modulation of cardiovascular adaptation to enhanced sympathetic activity

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Molecular insight into human platelet antigens: structural and evolutionary conservation analyses offer new perspective to immunogenic disorders.

Science.gov (United States)

Landau, Meytal; Rosenberg, Nurit

2011-03-01

Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the receptor for von Willebrand factor; and 3) integrin α2β1, which functions as the collagen receptor. We analyzed the structural location and the evolutionary conservation of the residues associated with the HPAs to characterize the features that induce immunologic responses but do not cause inherited diseases. We found that all HPAs reside in positions located on the protein surface, apart from the ligand-binding site, and are evolutionary variable. Disease-causing mutations often reside in highly conserved and buried positions. In contrast, the HPAs affect residues on the protein surface that were not conserved throughout evolution; this explains their naive effect on the protein function. Nonetheless, the HPAs involve substitutions of solvent-exposed positions that lead to altered interfaces on the surface of the protein and might present epitopes foreign to the immune system. © 2010 American Association of Blood Banks.

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

Science.gov (United States)

El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

2005-12-15

The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

Platelets and the innate immune system: Mechanisms of bacterial-induced platelet activation.

OpenAIRE

Cox, Dermot; Kerrigan, Steven W; Watson, Steve

2011-01-01

It has become clear that platelets are not simply cell fragments that can plug the leak in a damaged blood vessel, they are in fact key components in the innate immune system which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the first responding cell to a site of injury they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets which usually triggers platelet ac...

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

Science.gov (United States)

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Characterization of cutaneous vascular permeability induced by platelet-activating factor in guinea pigs and rats and its inhibition by a platelet-activating factor receptor antagonist

International Nuclear Information System (INIS)

Hwang, S.B.; Li, C.L.; Lam, M.H.; Shen, T.Y.

1985-01-01

Mechanisms of platelet-activating factor (PAF)-induced increases of cutaneous vascular permeability in guinea pigs and in rats were further explored. PAF so far is the most potent vasoactive mediator, being more than 1000-fold more potent than histamine and bradykinin in both species. In guinea pigs, there is a time delay of 5 to 10 minutes before PAF action, whereas, in the rat, the increased vasopermeability occurs immediately following the intradermal PAF injection. Relative vasoactive potencies of PAF and several structure-related analogues in both species correlate very well with their relative inhibition of the binding of 3 H-PAF to specific receptor sites on isolated rabbit platelet plasma membranes and their aggregatory abilities of rabbit platelets. Furthermore, the PAF-induced cutaneous vascular permeability is inhibitable by a competitive specific PAF receptor antagonist, kadsurenone, suggesting that binding of PAF to its specific receptor site is the first step to initiate its action of increased cutaneous vascular permeability. Several pure cyclooxygenase inhibitors, including indomethacin, diflunisal, and flurbiprofen, and the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, but not the histamine antagonists, inhibit the PAF-induced vasopermeability in guinea pigs. The inhibition by indomethacin or BW755C can be fully reversed by coinjection intradermally with PAF and prostaglandin E1 but not leukotriene B4. Also, prostaglandin E1 but not leukotriene B4 enhances the guinea pig in vivo response to PAF in this model. However, in rats, none of the cyclooxygenase inhibitors, histamine antagonists, or BW755C inhibit the PAF effect of cutaneous phenomena

The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.

Science.gov (United States)

Ohlmann, Philippe; Lecchi, Anna; El-Tayeb, Ali; Müller, Christa E; Cattaneo, Marco; Gachet, Christian

2013-03-01

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.

Platelet Glycoprotein lb-1X and Malignancy

Science.gov (United States)

2011-09-01

initiate coagulation, resulting in the formation of a fibrin - rich tumor cell- platelet emboli (Figure 1). Many of these coagulation factor-tumor cell...the tumor cell in a fibrin - rich web. (13;23;24) During this process, the platelet integrin receptor, aIIb 3, serves as a receptor linking fibrin ... platelets , and tumor cells into a fibrin rich clot normally associated with a thrombus. (25;25) Indeed, it can be speculated the fibrin - rich clot

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

OpenAIRE

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protei...

Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.

Science.gov (United States)

Aungraheeta, Riyaad; Conibear, Alexandra; Butler, Mark; Kelly, Eamonn; Nylander, Sven; Mumford, Andrew; Mundell, Stuart J

2016-12-08

Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca 2+ release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. © 2016 by The American Society of Hematology.

The use of quartz crystal microbalance with dissipation (QCM-D) for studying nanoparticle-induced platelet aggregation

Science.gov (United States)

Santos-Martinez, Maria Jose; Inkielewicz-Stepniak, Iwona; Medina, Carlos; Rahme, Kamil; D’Arcy, Deirdre M; Fox, Daniel; Holmes, Justin D; Zhang, Hongzhou; Radomski, Marek Witold

2012-01-01

Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D) has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast and scanning helium ion microscopy. PMID:22275839

Effects of hormones on platelet aggregation.

Science.gov (United States)

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

2014-04-01

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

Transcriptomic landscape of acute promyelocytic leukemia reveals aberrant surface expression of the platelet aggregation agonist Podoplanin.

Science.gov (United States)

Lavallée, Vincent-Philippe; Chagraoui, Jalila; MacRae, Tara; Marquis, Miriam; Bonnefoy, Arnaud; Krosl, Jana; Lemieux, Sébastien; Marinier, Anne; Pabst, Caroline; Rivard, Georges-Étienne; Hébert, Josée; Sauvageau, Guy

2018-02-23

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.

Platelets in Immune Response to Virus and Immunopathology of Viral Infections

Directory of Open Access Journals (Sweden)

Eugenio D. Hottz

2018-04-01

Full Text Available Platelets are essential effector cells in hemostasis. Aside from their role in coagulation, platelets are now recognized as major inflammatory cells with key roles in the innate and adaptive arms of the immune system. Activated platelets have key thromboinflammatory functions linking coagulation to immune responses in various infections, including in response to virus. Recent studies have revealed that platelets exhibit several pattern recognition receptors (PRR including those from the toll-like receptor, NOD-like receptor, and C-type lectin receptor family and are first-line sentinels in detecting and responding to pathogens in the vasculature. Here, we review the main mechanisms of platelets interaction with viruses, including their ability to sustain viral infection and replication, their expression of specialized PRR, and activation of thromboinflammatory responses against viruses. Finally, we discuss the role of platelet-derived mediators and platelet interaction with vascular and immune cells in protective and pathophysiologic responses to dengue, influenza, and human immunodeficiency virus 1 infections.

Platelet-activating factor increases platelet-dependent glycoconjugate secretion from tracheal submucosal gland

International Nuclear Information System (INIS)

Sasaki, T.; Shimura, S.; Ikeda, K.; Sasaki, H.; Takishima, T.

1989-01-01

Using isolated glands from feline trachea, we examined the effect of platelet-activating factor (PAF) on radiolabeled glycoconjugate release and glandular contraction by measuring induced tension in the absence or presence of platelets. PAF alone did not produce any significant glandular contraction nor any significant change in glycoconjugate release from isolated glands. In the presence of purified platelets containing no plasma, PAF (10(-8) to 10(-5) M) produced significant glycoconjugate secretion in a dose-dependent fashion, but it produced no significant glandular contraction. PAF-evoked glycoconjugate secretion was time dependent, reaching a peak response of 277% of control 15-30 min after the exposure of isolated glands to 10(-5) M PAF in the presence of platelets and returning to 135% of controls at 2 h. Platelets alone did not produce any significant stimulation in glycoconjugate release. CV-3988, a known PAF antagonist, inhibited the secretory response to PAF. Methysergide, a known antagonist to receptors for 5-hydroxytryptamine, did not alter PAF-evoked glycoconjugate secretion. Both indomethacin and SQ 29,548, a thromboxane receptor antagonist, abolished the PAF-evoked glycoconjugate secretion from isolated submucosal glands. Epithiomethanothromboxane A2, a stable thromboxane A2 analogue, produced a significant increase in glycoconjugate secretion in a dose-dependent fashion. These findings indicate that PAF increases glycoconjugate release in the presence of platelets and that the increase is dependent on some aspect of platelet function, namely thromboxane generation

The binding of fibrinogen to platelets in diabetes mellitus

International Nuclear Information System (INIS)

Minno, G. di; Cerbone, A.M.; Iride, C.; Mancini, M.

1986-01-01

Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125 I-fibrinogen than platelets from normal controls. Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thrombaxane A 2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from none-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinophatic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from none-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis. (Author)

Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter

NARCIS (Netherlands)

Afink, G. B.; Nistér, M.; Stassen, B. H.; Joosten, P. H.; Rademakers, P. J.; Bongcam-Rudloff, E.; van Zoelen, E. J.; Mosselman, S.

1995-01-01

Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R

Platelets as a Novel Source of Pro-Inflammatory Chemokine CXCL14

Directory of Open Access Journals (Sweden)

Alexander Witte

2017-03-01

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

Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins.

Science.gov (United States)

Macaulay, Iain C; Tijssen, Marloes R; Thijssen-Timmer, Daphne C; Gusnanto, Arief; Steward, Michael; Burns, Philippa; Langford, Cordelia F; Ellis, Peter D; Dudbridge, Frank; Zwaginga, Jaap-Jan; Watkins, Nicholas A; van der Schoot, C Ellen; Ouwehand, Willem H

2007-04-15

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation.

Science.gov (United States)

Alberio, Lorenzo; Ravanat, Catherine; Hechler, Béatrice; Mangin, Pierre H; Lanza, François; Gachet, Christian

2017-06-02

The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

Science.gov (United States)

Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

2017-01-03

The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

Science.gov (United States)

Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

2018-01-01

Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

OpenAIRE

Jarvis, Gavin E.; Best, Denise; Watson, Steve P.

2004-01-01

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induc...

[Effect of losartan on human platelet activation by thromboxane A2].

Science.gov (United States)

Guerra, J I; Montón, M; Rodríguez-Feo, J A; Farré, J; Jiménez, A M; Núñez, A; Gómez, J; Rico, L; Marcos, P; Castilla, C; Sánchez De Miguel, L; Casado, S; López-Farré, A

2000-04-01

Previous studies have demonstrated that losartan, an AT-1 receptor antagonist of angiotensin II (Ang II) could block the receptor of thromboxane A2 (TXA2) in the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. Platelets were obtained from 15 healthy men between the age 26 and 40. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by a synthetic TXA2 analogue, U46619 (5 x 10(-6) mol/l). The U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-response manner. Only a high dose of EXP 3174 (5 10-5 mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I-converting inhibitor failed to modify U46619-induced platelet aggregation. Despite the platelets expressing AT-1 type receptors, of Ang II exogenous Ang II did not modify platelet aggregation induced by U46619. The binding of U46619 to platelets was competitively inhibited by losartan in dose-dependent manner. However, only a high dose of EXP 3174 reduced the binding of U46619. Captopril failed to modify the binding of U46619 to platelets. Losartan decreased platelet aggregation by a TXA2-dependent mechanism. EXP 3174 showed a lesser potency than losartan to reduce TXA2-platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced TXA2-dependent platelet activation independently of the blockade of AT-1 receptors.

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

International Nuclear Information System (INIS)

Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

1989-01-01

The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

OpenAIRE

Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

2014-01-01

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the pro...

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets.

Science.gov (United States)

Crider, J Y; Xu, S X; Sharif, N A

2001-01-01

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.

Of von Willebrand factor and platelets.

Science.gov (United States)

Bryckaert, Marijke; Rosa, Jean-Philippe; Denis, Cécile V; Lenting, Peter J

2015-01-01

Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

International Nuclear Information System (INIS)

Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

1987-01-01

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel.

Science.gov (United States)

Nam, Jeonghun; Lim, Hyunjung; Kim, Dookon; Shin, Sehyun

2011-10-07

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

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Fang Rao

Full Text Available Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ. We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg or increased (120, 180, 240 mmHg hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg. The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs. These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

Science.gov (United States)

Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

Platelet-vessel wall interaction in health and disease

NARCIS (Netherlands)

Löwenberg, E. C.; Meijers, J. C. M.; Levi, M. [=Marcel M.

2010-01-01

Upon vessel wall injury platelets rapidly adhere to the exposed subendothelial matrix which is mediated by several cellular receptors present on platelets or endothelial cells and various adhesive proteins such as von Willebrand factor, collagen and fibrinogen. Subsequent platelet activation results

Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

NARCIS (Netherlands)

van der Wal, E.

2010-01-01

Storage of platelets at low temperature reduces bacterial growth and might better preserve the haemostatic function of platelets than current procedures. Incubation at 0C is known to expose ?-N-acetyl-D-glucosamine-residues on glycoprotein (GP)Ibalpha inducing receptor-clustering and platelet

Novel agents for anti-platelet therapy

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Ji Xuebin

2011-11-01

Full Text Available Abstract Anti-platelet therapy plays an important role in the treatment of patients with thrombotic diseases. The most commonly used anti-platelet drugs, namely, aspirin, ticlopidine, and clopidogrel, are effective in the prevention and treatment of cardio-cerebrovascular diseases. Glycoprotein IIb/IIIa antagonists (e.g., abciximab, eptifibatide and tirofiban have demonstrated good clinical benefits and safety profiles in decreasing ischemic events in acute coronary syndrome. However, adverse events related to thrombosis or bleeding have been reported in cases of therapy with glycoprotein IIb/IIIa antagonists. Cilostazol is an anti-platelet agent used in the treatment of patients with peripheral ischemia, such as intermittent claudication. Presently, platelet adenosine diphosphate P2Y(12 receptor antagonists (e.g., clopidogrel, prasugrel, cangrelor, and ticagrelor are being used in clinical settings for their pronounced protective effects. The new protease-activated receptor antagonists, vorapaxar and atopaxar, potentially decrease the risk of ischemic events without significantly increasing the rate of bleeding. Some other new anti-platelet drugs undergoing clinical trials have also been introduced. Indeed, the number of new anti-platelet drugs is increasing. Consequently, the efficacy of these anti-platelet agents in actual patients warrants scrutiny, especially in terms of the hemorrhagic risks. Hopefully, new selective platelet inhibitors with high anti-thrombotic efficiencies and low hemorrhagic side effects can be developed.

Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

International Nuclear Information System (INIS)

Burroughs, S.F.; Johnson, G.J.

1990-01-01

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of [14C]-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with [3H]-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist [3H]-U46619 and antagonist [3H]-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ([Ca2+]i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in [Ca2+]i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane

In vitro investigation of protein adsorption and platelet adhesion on inorganic biomaterial surfaces

Energy Technology Data Exchange (ETDEWEB)

Yan Huang [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Lue Xiaoying [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China)], E-mail: luxy@seu.edu.cn; Ma Jingwu [State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096 (China); Nan Huang [Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031 (China)], E-mail: nhuang@263.com

2008-11-15

The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the ratio of adsorbed albumin (Alb) to IgG (R{sub A/I}) on DLC is larger than Ti, which coincide with the sequence of the ratio of interfacial tension between solid surface and Alb ({gamma}{sub S,Alb}) to interfacial tension between surface and IgG ({gamma}{sub S,IgG}) ({gamma}{sub S,Alb}/{gamma}{sub S,IgG}). The DLC film has a preferential adsorption for Alb. The results suggest that the ratio of {gamma}{sub S,Alb}/{gamma}{sub S,IgG} may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood compatibility of DLC seems to be better than Ti.

Platelet "first responders" in wound response, cancer, and metastasis.

Science.gov (United States)

Menter, David G; Kopetz, Scott; Hawk, Ernest; Sood, Anil K; Loree, Jonathan M; Gresele, Paolo; Honn, Kenneth V

2017-06-01

Platelets serve as "first responders" during normal wounding and homeostasis. Arising from bone marrow stem cell lineage megakaryocytes, anucleate platelets can influence inflammation and immune regulation. Biophysically, platelets are optimized due to size and discoid morphology to distribute near vessel walls, monitor vascular integrity, and initiate quick responses to vascular lesions. Adhesion receptors linked to a highly reactive filopodia-generating cytoskeleton maximizes their vascular surface contact allowing rapid response capabilities. Functionally, platelets normally initiate rapid clotting, vasoconstriction, inflammation, and wound biology that leads to sterilization, tissue repair, and resolution. Platelets also are among the first to sense, phagocytize, decorate, or react to pathogens in the circulation. These platelet first responder properties are commandeered during chronic inflammation, cancer progression, and metastasis. Leaky or inflammatory reaction blood vessel genesis during carcinogenesis provides opportunities for platelet invasion into tumors. Cancer is thought of as a non-healing or chronic wound that can be actively aided by platelet mitogenic properties to stimulate tumor growth. This growth ultimately outstrips circulatory support leads to angiogenesis and intravasation of tumor cells into the blood stream. Circulating tumor cells reengage additional platelets, which facilitates tumor cell adhesion, arrest and extravasation, and metastasis. This process, along with the hypercoagulable states associated with malignancy, is amplified by IL6 production in tumors that stimulate liver thrombopoietin production and elevates circulating platelet numbers by thrombopoiesis in the bone marrow. These complex interactions and the "first responder" role of platelets during diverse physiologic stresses provide a useful therapeutic target that deserves further exploration.

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

Directory of Open Access Journals (Sweden)

Andrew Yee

Full Text Available von Willebrand factor (VWF tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes

Science.gov (United States)

Rowley, Jesse W.; Oler, Andrew J.; Tolley, Neal D.; Hunter, Benjamin N.; Low, Elizabeth N.; Nix, David A.; Yost, Christian C.; Zimmerman, Guy A.

2011-01-01

Inbred mice are a useful tool for studying the in vivo functions of platelets. Nonetheless, the mRNA signature of mouse platelets is not known. Here, we use paired-end next-generation RNA sequencing (RNA-seq) to characterize the polyadenylated transcriptomes of human and mouse platelets. We report that RNA-seq provides unprecedented resolution of mRNAs that are expressed across the entire human and mouse genomes. Transcript expression and abundance are often conserved between the 2 species. Several mRNAs, however, are differentially expressed in human and mouse platelets. Moreover, previously described functional disparities between mouse and human platelets are reflected in differences at the transcript level, including protease activated receptor-1, protease activated receptor-3, platelet activating factor receptor, and factor V. This suggests that RNA-seq is a useful tool for predicting differences in platelet function between mice and humans. Our next-generation sequencing analysis provides new insights into the human and murine platelet transcriptomes. The sequencing dataset will be useful in the design of mouse models of hemostasis and a catalyst for discovery of new functions of platelets. Access to the dataset is found in the “Introduction.” PMID:21596849

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

Science.gov (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

2016-01-01

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.

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

Directory of Open Access Journals (Sweden)

Stefanie Krajewski

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

Rare platelet GPCR variants: what can we learn?

Science.gov (United States)

Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

2015-07-01

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules.

Science.gov (United States)

Nurden, A T; Horisberger, M; Savariau, E; Caen, J P

1980-10-15

Washed human platelets have been incubated with the lectins WGA, ConA and RCA1, adsorbed to different-sized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.

The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

Science.gov (United States)

Lin, Olivia A.; Karim, Zubair A.; Vemana, Hari Priya; Espinosa, Enma V. P.; Khasawneh, Fadi T.

2014-01-01

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

Influence of nitriding atmosphere on the modification of surface titanium with focus on the behavior of blood platelets adhesion

International Nuclear Information System (INIS)

Vitoriano, J.O.; Alves, C.; Braz, D.C.; Camara, R.B.G.; Rocha, H.A.O.

2014-01-01

The present study aimed to analyze the influence of surface modification of titanium on the adhesion of blood platelets, through techniques of adhesion and morphological analyzes. Discs of titanium grade II received different surface treatments with plasma of Ar + N_2 + H_2 and Ar + H_2, forming two experimental groups including only polished samples used as standard. Before and after treatment the samples were characterized according to topography, crystalline structure and wettability, using atomic force microscopy, X-ray diffraction, Raman spectroscopy and testing of sessile drop, respectively. Platelet rich plasma (PRP) was applied on the modified surfaces in a culture plates. Images obtained by electron microscopy of adhered platelets were analyzed to verify the behavior of platelets in the different experimental conditions. (author)

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Science.gov (United States)

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer

OpenAIRE

Metelli, Alessandra; Wu, Bill X; Fugle, Caroline W; Rachidi, Saleh; Sun, Shaoli; Zhang, Yongliang; Wu, Jennifer; Tomlinson, Stephen; Howe, Philip; Yang, Yi; Garrett-Mayer, Elizabeth; Liu, Bei; Li, Zihai

2016-01-01

GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGF-β which is expressed naturally by platelets and regulatory T cells. Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGF-β in the tumor microenvironment. We found that human breast, lung and colon cancers expres...

The Signaling Role of CD40 Ligand in Platelet Biology and in Platelet Component Transfusion

Science.gov (United States)

Aoui, Chaker; Prigent, Antoine; Sut, Caroline; Tariket, Sofiane; Hamzeh-Cognasse, Hind; Pozzetto, Bruno; Richard, Yolande; Cognasse, Fabrice; Laradi, Sandrine; Garraud, Olivier

2014-01-01

The CD40 ligand (CD40L) is a transmembrane molecule of crucial interest in cell signaling in innate and adaptive immunity. It is expressed by a variety of cells, but mainly by activated T-lymphocytes and platelets. CD40L may be cleaved into a soluble form (sCD40L) that has a cytokine-like activity. Both forms bind to several receptors, including CD40. This interaction is necessary for the antigen specific immune response. Furthermore, CD40L and sCD40L are involved in inflammation and a panoply of immune related and vascular pathologies. Soluble CD40L is primarily produced by platelets after activation, degranulation and cleavage, which may present a problem for transfusion. Soluble CD40L is involved in adverse transfusion events including transfusion related acute lung injury (TRALI). Although platelet storage designed for transfusion occurs in sterile conditions, platelets are activated and release sCD40L without known agonists. Recently, proteomic studies identified signaling pathways activated in platelet concentrates. Soluble CD40L is a good candidate for platelet activation in an auto-amplification loop. In this review, we describe the immunomodulatory role of CD40L in physiological and pathological conditions. We will focus on the main signaling pathways activated by CD40L after binding to its different receptors. PMID:25479079

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood.

Science.gov (United States)

Montrucchio, Giuseppe; Bosco, Ornella; Del Sorbo, Lorenzo; Fascio Pecetto, Paolo; Lupia, Enrico; Goffi, Alberto; Omedè, Paola; Emanuelli, Giorgio; Camussi, Giovanni

2003-11-01

Several studies focused on the ability of bacterial lipopolysac-charides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS. LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators. In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.

UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

NARCIS (Netherlands)

Verhaar, Robin; Dekkers, David W. C.; de Cuyper, Iris M.; Ginsberg, Mark H.; de Korte, Dirk; Verhoeven, Arthur J.

2008-01-01

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

Science.gov (United States)

Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanisms of andrographolide-induced platelet apoptosis in human platelets: regulatory roles of the extrinsic apoptotic pathway.

Science.gov (United States)

Lien, Li-Ming; Su, Cheng-Chen; Hsu, Wen-Hsien; Lu, Wan-Jung; Chung, Chi-Li; Yen, Ting-Lin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2013-11-01

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by inhibition of the p38 MAPK/(●) HO-NF-κB-ERK2 cascade. Although platelets are anucleated cells, apoptotic machinery apparatus recently has been found to regulate platelet activation and limit platelet lifespan. Therefore, we further investigated the regulatory effects of andrographolide on platelet apoptotic events. In this study, apoptotic signaling events for caspase-3, -8, and Bid were time (10-60 min)- and dose (25-100 μΜ)-dependently activated by andrographolide in human platelets. Andrographolide could also disrupt mitrochondrial membrane potential. In addition, caspase-8 inhibitor (z-IETD-fmk, 50 μΜ) was found to reverse andrographolide-induced caspase-8 activation, whereas the antagonistic anti-Fas receptor (ZB4, 500 ng/mL) and anti-tumor necrosis factor-R1 (H398, 10 µg/mL) monoclonal antibodies did not. In conclusion, this study for the first time demonstrated that andrographolide might limit platelet lifespan by initiating the caspase-8-dependent extrinsic apoptotic pathway, in spite of no direct evidence that death receptors are involved in this process proved. Overall, the various medicinal properties of andrographolide suggest its potential value in treating patients with thromboembolic disorders. Copyright © 2012 John Wiley & Sons, Ltd.

Partial purification and identification of the thrombozane A2/prostaglandin H2 receptor protein in human platelets

International Nuclear Information System (INIS)

Lim, C.T.; Kattelman, E.J.; Arora, S.K.; Venton, D.L.; Le Breton, G.C.

1986-01-01

The thromboxane A 2 /prostaglandin H 2 (TXA 2 /PGH 2 ) receptor antagonist [ 3 H]-13-azaprostanoic acid (13-APA) was used to identify and purify the platelet TXA 2 /PGH 2 receptor protein. Optimal solubilization of the 13-APA binding protein was achieved by extraction with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) detergent. Preliminary purification of the crude solubilized membrane fraction was performed by gel filtration chromatography using a Sepharose 4B column. Further purification was accomplished by high performance liquid chromatography (HPLC) using a Synchropak GPC-500 column. The HPLC protein profile revealed two protein peaks, only one of which was enriched in [ 3 H]-13-APA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this peak revealed two bands with molecular weights of 65,000 and 60,000 daltons. In binding studies using the 60,000 dalton-enriched subfraction, unlabelled 13-APA, the TXA 2 /PGH 2 mimetic U46619 and the TXA 2 /PGH 2 antagonist SQ 29,548 all competed for [ 3 H]-13-APA binding whereas TXB 2 did not compete for binding. Heat denaturation of this subfraction resulted in a complete loss of binding activity. These findings indicate that a protein of approximately 60,000 daltons represents the human platelet TXA 2 /PGH 2 receptor

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

Science.gov (United States)

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2016-11-30

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

Glycoprotein Ibα clustering in platelet storage and function

NARCIS (Netherlands)

Gitz, E.

2013-01-01

Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through ?IIb?3 Evokes Phosphatidylserine Exposure on Their Cell Surface

OpenAIRE

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyz...

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

Science.gov (United States)

Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans

Science.gov (United States)

Washington, A. Valance; Gibot, Sébastien; Acevedo, Ismael; Gattis, James; Quigley, Laura; Feltz, Robert; De La Mota, Alina; Schubert, Rebecca L.; Gomez-Rodriguez, Julio; Cheng, Jun; Dutra, Amalia; Pak, Evgenia; Chertov, Oleg; Rivera, Linette; Morales, Jessica; Lubkowski, Jacek; Hunter, Robert; Schwartzberg, Pamela L.; McVicar, Daniel W.

2009-01-01

Triggering receptor expressed on myeloid cells–like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte α-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1–/– mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1–/– mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1–/– mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1–mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation. PMID:19436112

Low multiple electrode aggregometry platelet responses are not associated with non-synonymous variants in G-protein coupled receptor genes.

Science.gov (United States)

Norman, Jane E; Lee, Kurtis R; Walker, Mary E; Murden, Sherina L; Harris, Jessica; Mundell, Stuart; J Murphy, Gavin; Mumford, Andrew D

2015-10-01

Multiple electrode aggregometry (MEA) improves prediction of thrombosis and bleeding in cardiac patients. However, the causes of inter-individual variation in MEA results are incompletely understood. We explore whether low MEA results are associated with platelet G-protein coupled receptor (GPCR) gene variants. The effects of P2Y12 receptor (P2Y12), thromboxane A2 receptor (TPα) and protease-activated receptor 1 (PAR1) dysfunction on the MEA ADP-test, ASPI-test and TRAP-test were determined using receptor antagonists. Cardiac surgery patients with pre-operative MEA results suggesting GPCR dysfunction were selected for P2Y12 (P2RY12), TPα (TBXA2R) and PAR1 (F2R) sequencing. In control blood samples, P2Y12, TPα or PAR1 antagonists markedly reduced ADP-test, ASPI-test and TRAP-test results respectively. In the 636 patients from a cohort of 2388 cardiac surgery patients who were not receiving aspirin or a P2Y12 blocker, the median ADP-test result was 75.1 U (range 4.8-153.2), ASPI-test 83.7 U (1.4-157.3) and TRAP-test 117.7 U (2.4-194.1), indicating a broad range of results unexplained by anti-platelet drugs. In 238 consenting patients with unexplained low MEA results, three P2RY12 variants occurred in 70/107 (65%) with suspected P2Y12 dysfunction and four TBXA2R variants occurred in 19/22 (86%) with suspected TPα dysfunction although the later group was too small to draw meaningful conclusions about variant frequency. All the variants were synonymous and unlikely to cause GPCR dysfunction. There were no F2R variants in the 109 cases with suspected PAR1 dysfunction. MEA results suggesting isolated platelet GPCR dysfunction were common in cardiac surgery patients, but were not associated with non-synonymous variants in P2RY12 or F2R. Copyright © 2015 Elsevier Ltd. All rights reserved.

Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

International Nuclear Information System (INIS)

Carter, M.G.; Shukla, S.D.

1987-01-01

Human platelet concentrate from the American Red Cross Blood Center was stored at 24 degree C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying 32 P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 x 10 -7 M PAF at 37 degree C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for 32 P-phosphoinositides. The percent stimulation of 32 P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage

Cystamine immobilization on TiO2 film surfaces and the influence on inhibition of collagen-induced platelet activation

International Nuclear Information System (INIS)

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

2011-01-01

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

S1P and the birth of platelets

Science.gov (United States)

Galvani, Sylvain; Rafii, Shahin; Nachman, Ralph

2012-01-01

Recent work has highlighted the multitude of biological functions of sphingosine 1-phosphate (S1P), which include roles in hematopoietic cell trafficking, organization of immune organs, vascular development, and neuroinflammation. Indeed, a functional antagonist of S1P1 receptor, FTY720/Gilenya, has entered the clinic as a novel therapeutic for multiple sclerosis. In this issue of the JEM, Zhang et al. highlight yet another function of this lipid mediator: thrombopoiesis. The S1P1 receptor is required for the growth of proplatelet strings in the bloodstream and the shedding of platelets into the circulation. Notably, the sharp gradient of S1P between blood and the interstitial fluids seems to be essential to ensure the production of platelets, and S1P appears to cooperate with the CXCL12–CXCR4 axis. Pharmacologic modulation of the S1P1 receptor altered circulating platelet numbers acutely, suggesting a potential therapeutic strategy for controlling thrombocytopenic states. However, the S1P4 receptor may also regulate thrombopoiesis during stress-induced accelerated platelet production. This work reveals a novel physiological action of the S1P/S1P1 duet that could potentially be harnessed for clinical translation. PMID:23166370

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

2017-05-01

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

Palliation of bone cancer pain by antagonists of platelet-activating factor receptors.

Directory of Open Access Journals (Sweden)

Katsuya Morita

Full Text Available Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2 protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

Science.gov (United States)

Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

2014-07-01

Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

Synergistic structures from magnetic freeze casting with surface magnetized alumina particles and platelets.

Science.gov (United States)

Frank, Michael B; Hei Siu, Sze; Karandikar, Keyur; Liu, Chin-Hung; Naleway, Steven E; Porter, Michael M; Graeve, Olivia A; McKittrick, Joanna

2017-12-01

Magnetic freeze casting utilizes the freezing of water, a low magnetic field and surface magnetized materials to make multi-axis strengthened porous scaffolds. A much greater magnetic moment was measured for larger magnetized alumina platelets compared with smaller particles, which indicated that more platelet aggregation occurred within slurries. This led to more lamellar wall alignment along the magnetic field direction during magnetic freeze casting at 75 mT. Slurries with varying ratios of magnetized particles to platelets (0:1, 1:3, 1:1, 3:1, 7:1, 1:0) produced porous scaffolds with different structural features and degrees of lamellar wall alignment. The greatest mechanical enhancement in the magnetic field direction was identified in the synergistic condition with the highest particle to platelet ratio (7:1). Magnetic freeze casting with varying ratios of magnetized anisotropic and isotropic alumina provided insights about how heterogeneous morphologies aggregate within lamellar walls that impact mechanical properties. Fabrication of strengthened scaffolds with multi-axis aligned porosity was achieved without introducing different solid materials, freezing agents or additives. Resemblance of 7:1 particle to platelet scaffold microstructure to wood light-frame house construction is framed in the context of assembly inspiration being derived from both natural and synthetic sources. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the stimulation of human platelets by stable analogues of PGH2/TXA2

International Nuclear Information System (INIS)

Morinelli, T.A.

1987-01-01

The specific effects of the TXA 2 /PGH 2 analogues, U46619 (9,11-dideoxy,9α-11α-methanoepoxy-PGF/sub 2α/), and 9,11 aza-PGH 2 , on human platelet shape change, myosin light chain phosphorylation, serotonin release, fibrinogen receptor exposure and platelet aggregation were measured and compared with binding of 3 H-U46619 to platelets. Shape change and myosin light chain phosphorylation were found to saturable and dose dependent. These two effects were competitively inhibited by specific antagonists of TXA 2 /PGH 2 receptors (BM13177 and I-PTA-OH) indicating that they are receptor mediated. Binding of 3 H-U46619 showed two components. Occupancy of high affinity binding sites correlated with platelet shape change and myosin and light chain phosphorylation. A second component with an apparent K/sub d/ of 1.46 +/- 0.47 μM, may represent a second, low-affinity site. Therefore, the platelet release reaction as not directly correlated with occupancy of high affinity receptors but could be related to the second binding component of U46619. Fibrinogen receptor exposure and platelet aggregation caused by U46619 appeared to be events mediated by the release of ADP from platelet dense granules

Thrombopoietin contributes to enhanced platelet activation in patients with unstable angina.

Science.gov (United States)

Lupia, Enrico; Bosco, Ornella; Bergerone, Serena; Dondi, Anna Erna; Goffi, Alberto; Oliaro, Elena; Cordero, Marco; Del Sorbo, Lorenzo; Trevi, Giampaolo; Montrucchio, Giuseppe

2006-12-05

We sought to investigate the potential role of elevated levels of thrombopoietin (TPO) in platelet activation during unstable angina (UA). Thrombopoietin is a humoral growth factor that does not induce platelet aggregation per se, but primes platelet activation in response to several agonists. No data concerning its contribution to platelet function abnormalities described in patients with UA are available. We studied 15 patients with UA and, as controls, 15 patients with stable angina (SA) and 15 healthy subjects. We measured TPO and C-reactive protein (CRP), as well as monocyte-platelet binding and the platelet expression of P-selectin and of the TPO receptor, c-Mpl. The priming activity of patient or control plasma on platelet aggregation and monocyte-platelet binding and the role of TPO in this effect also were studied. Patients with UA showed higher circulating TPO levels, as well as increased monocyte-platelet binding, platelet P-selectin expression, and CRP levels, than those with SA and healthy control subjects. The UA patients also showed reduced platelet expression of the TPO receptor, c-Mpl. In vitro, the plasma from UA patients, but not from SA patients or healthy controls, primed platelet aggregation and monocyte-platelet binding, which were both reduced when an inhibitor of TPO was used. Thrombopoietin may enhance platelet activation in the early phases of UA, potentially participating in the pathogenesis of acute coronary syndromes.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

Science.gov (United States)

Mammadova-Bach, Elmina; Ollivier, Véronique; Loyau, Stéphane; Schaff, Mathieu; Dumont, Bénédicte; Favier, Rémi; Freyburger, Geneviève; Latger-Cannard, Véronique; Nieswandt, Bernhard; Gachet, Christian; Mangin, Pierre H; Jandrot-Perrus, Martine

2015-07-30

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbβ3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization. © 2015 by The American Society of Hematology.

Platelet adhesion and plasma protein adsorption control of collagen surfaces by He+ ion implantation

International Nuclear Information System (INIS)

Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.

2003-01-01

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

Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

Science.gov (United States)

Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

2010-01-01

Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-12-15

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

Rac1 is essential for phospholipase C-gamma2 activation in platelets

DEFF Research Database (Denmark)

Pleines, Irina; Elvers, Margitta; Strehl, Amrei

2008-01-01

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

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

Science.gov (United States)

Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Breaking the mold: transcription factors in the anucleate platelet and platelet-derived microparticles

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Katie L Lannan

2015-02-01

Full Text Available Platelets are small anucleate blood cells derived from megakaryocytes. In addition to their pivotal roles in hemostasis, platelets are the smallest, yet most abundant, immune cell and regulate inflammation, immunity, and disease progression. Although platelets lack DNA, and thus no functional transcriptional activities, they are nonetheless rich sources of RNAs, possess an intact spliceosome, and are thus capable of synthesizing proteins. Previously, it was thought that platelet RNAs and translational machinery were remnants from the megakaryocyte. We now know that the initial description of platelets as cellular fragments is an antiquated notion, as mounting evidence suggests otherwise. Therefore, it is reasonable to hypothesize that platelet transcription factors are not vestigial remnants from megakaryoctes, but have important, if only partly understood functions. Proteins play multiple cellular roles to minimize energy expenditure for maximum cellular function; thus, the same can be expected for transcription factors. In fact, numerous transcription factors have non-genomic roles, both in platelets and in nucleated cells. Our lab and others have discovered the presence and nongenomic roles of transcription factors in platelets, such as the nuclear factor kappa β (NFκB family of proteins and peroxisome proliferator activated receptor gamma (PPARγ. In addition to numerous roles in regulating platelet activation, functional transcription factors can be transferred to vascular and immune cells through platelet microparticles. This method of transcellular delivery of key immune molecules may be a vital mechanism by which platelet transcription factors regulate inflammation and immunity. At the very least, platelets are an ideal model cell to dissect out the nongenomic roles of transcription factors in nucleated cells. There is abundant evidence to suggest that transcription factors in platelets play key roles in regulating inflammatory and

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

Directory of Open Access Journals (Sweden)

Carmen H Coxon

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

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

Science.gov (United States)

Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

2017-01-15

Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner

DEFF Research Database (Denmark)

Vinholt, Pernille Just; Nielsen, Christian; Söderström, Anna Cecilia

2017-01-01

Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor...

Nephropathy in type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

DEFF Research Database (Denmark)

Tarnow, Inge; Michelson, Alan D.; Barnard, Marc R.

2009-01-01

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation...... and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte...... controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P Type 1 diabetic nephropathy, as compared with normoalbuminuria...

Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

International Nuclear Information System (INIS)

Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

2014-01-01

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

Hypothermia and Platelet Dysfunction

National Research Council Canada - National Science Library

Michelson, A

1994-01-01

... (the GPIIb-IIIa complex). Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPIb-IX and GPIIb-IIIa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

Science.gov (United States)

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

Science.gov (United States)

Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

2015-02-17

A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis.

Science.gov (United States)

Lombard, Stephanie E; Pollitt, Alice Y; Hughes, Craig E; Di, Ying; Mckinnon, Tom; O'callaghan, Chris A; Watson, Steve P

2017-11-01

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A 2 (TxA 2 ). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.

Hypothermia and Platelet Dysfunction

National Research Council Canada - National Science Library

Michelson, Alan

1993-01-01

... (the OPlib-Illa complex).3 Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPTh-IX and OPlib-Illa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

Peroxiredoxin II is an antioxidant enzyme that negatively regulates collagen-stimulated platelet function.

Science.gov (United States)

Jang, Ji Yong; Wang, Su Bin; Min, Ji Hyun; Chae, Yun Hee; Baek, Jin Young; Yu, Dae-Yeul; Chang, Tong-Shin

2015-05-01

Collagen-induced platelet signaling is mediated by binding to the primary receptor glycoprotein VI (GPVI). Reactive oxygen species produced in response to collagen have been found to be responsible for the propagation of GPVI signaling pathways in platelets. Therefore, it has been suggested that antioxidant enzymes could down-regulate GPVI-stimulated platelet activation. Although the antioxidant enzyme peroxiredoxin II (PrxII) has emerged as having a role in negatively regulating signaling through various receptors by eliminating H2O2 generated upon receptor stimulation, the function of PrxII in collagen-stimulated platelets is not known. We tested the hypothesis that PrxII negatively regulates collagen-stimulated platelet activation. We analyzed PrxII-deficient murine platelets. PrxII deficiency enhanced GPVI-mediated platelet activation through the defective elimination of H2O2 and the impaired protection of SH2 domain-containing tyrosine phosphatase 2 (SHP-2) against oxidative inactivation, which resulted in increased tyrosine phosphorylation of key components for the GPVI signaling cascade, including Syk, Btk, and phospholipase Cγ2. Interestingly, PrxII-mediated antioxidative protection of SHP-2 appeared to occur in the lipid rafts. PrxII-deficient platelets exhibited increased adhesion and aggregation upon collagen stimulation. Furthermore, in vivo experiments demonstrated that PrxII deficiency facilitated platelet-dependent thrombus formation in injured carotid arteries. This study reveals that PrxII functions as a protective antioxidant enzyme against collagen-stimulated platelet activation and platelet-dependent thrombosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

Science.gov (United States)

Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet

Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat

Directory of Open Access Journals (Sweden)

Groneberg David A

2008-02-01

Full Text Available Abstract Background Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity. Methods The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique. Results Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity. Conclusion Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.

The Orientation of Nanoscale Apatite Platelets in Relation to Osteoblastic-Osteocyte Lacunae on Trabecular Bone Surface.

Science.gov (United States)

Shah, Furqan A; Zanghellini, Ezio; Matic, Aleksandar; Thomsen, Peter; Palmquist, Anders

2016-02-01

The orientation of nanoscale mineral platelets was quantitatively evaluated in relation to the shape of lacunae associated with partially embedded osteocytes (osteoblastic-osteocytes) on the surface of deproteinised trabecular bone of adult sheep. By scanning electron microscopy and image analysis, the mean orientation of mineral platelets at the osteoblastic-osteocyte lacuna (Ot.Lc) floor was found to be 19° ± 14° in the tibia and 20° ± 14° in the femur. Further, the mineral platelets showed a high degree of directional coherency: 37 ± 7% in the tibia and 38 ± 9% in the femur. The majority of Ot.Lc in the tibia (69.37%) and the femur (74.77%) exhibited a mean orientation of mineral platelets between 0° and 25°, with the largest fraction within a 15°-20° range, 17.12 and 19.8% in the tibia and femur, respectively. Energy dispersive X-ray spectroscopy and Raman spectroscopy were used to characterise the features observed on the anorganic bone surface. The Ca/P (atomic %) ratio was 1.69 ± 0.1 within the Ot.Lc and 1.68 ± 0.1 externally. Raman spectra of NaOCl-treated bone showed peaks associated with carbonated apatite: ν1, ν2 and ν4 PO4(3-), and ν1 CO3(2-), while the collagen amide bands were greatly reduced in intensity compared to untreated bone. The apatite-to-collagen ratio increased considerably after deproteinisation; however, the mineral crystallinity and the carbonate-to-phosphate ratios were unaffected. The ~19°-20° orientation of mineral platelets in at the Ot.Lc floor may be attributable to a gradual rotation of osteoblasts in successive layers relative to the underlying surface, giving rise to the twisted plywood-like pattern of lamellar bone.

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation

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V Milleret

2011-05-01

Full Text Available Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm, patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Science.gov (United States)

Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

1993-11-01

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Platelet Glycoprotein Ib-IX and Malignancy

Science.gov (United States)

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Glycans and glycosylation of platelets: current concepts and implications for transfusion

DEFF Research Database (Denmark)

Sørensen, Anne Louise; Hoffmeister, Karin M; Wandall, Hans H

2008-01-01

by the alphaMbeta2 hepatic lectin receptor. Capping the exposed beta-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown...... that galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets. SUMMARY: Room temperature storage of platelet products increases...... the risk of transfusion-mediated sepsis and accelerates platelet deterioration, limiting platelet shelf life. Recent evidence suggests that glycoengineering of platelets might allow for their cold storage, significantly improving the quality of platelet products....

Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

Science.gov (United States)

Lee, Jin Ju; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Lee, Hu Jang; Min, Wongi; Her, Moon; Rhee, Man Hee; Watarai, Masahisa; Chang, Hong Hee; Kim, Suk

2016-04-21

Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

Science.gov (United States)

Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

2014-05-01

The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

Platelet-Derived Microvesicles in Cardiovascular Diseases

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Maria T. K. Zaldivia

2017-11-01

Full Text Available Microvesicles (MVs circulating in the blood are small vesicles (100–1,000 nm in diameter derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs, the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their “cargo” of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

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Nathan J. Sniadecki

2011-12-01

Full Text Available Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

Platelet-activating factor podoplanin: from discovery to drug development.

Science.gov (United States)

Takemoto, Ai; Miyata, Kenichi; Fujita, Naoya

2017-06-01

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin-CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

The use of quartz crystal microbalance with dissipation (QCM-D for studying nanoparticle-induced platelet aggregation

Directory of Open Access Journals (Sweden)

Santos-Martinez MJ

2012-01-01

Full Text Available Maria Jose Santos-Martinez1–3, Iwona Inkielewicz-Stepniak1,4, Carlos Medina1, Kamil Rahme5,6, Deirdre M D'Arcy1, Daniel Fox3, Justin D Holmes3,5, Hongzhou Zhang3, Marek Witold Radomski3,51School of Pharmacy and Pharmaceutical Sciences, 2School of Medicine, 3Center for Research on Adaptive Nanostructures and Nanodevices, Trinity College Dublin, Dublin, Ireland; 4Department of Medicinal Chemistry, Medical University of Gdansk, Gdansk, Poland; 5Materials and Supercritical Fluids Group, Department of Chemistry and the Tyndall National Institute, University College Cork, Cork, Ireland; 6Department of Sciences, Faculty of Natural and Applied Science, Notre Dame University, Zouk Mosbeh, LebanonAbstract: Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by

PLATELET ADHESION TO POLYURETHANE UREA UNDER PULSATILE FLOW CONDITIONS

Science.gov (United States)

Navitsky, Michael A.; Taylor, Joshua O.; Smith, Alexander B.; Slattery, Margaret J.; Deutsch, Steven; Siedlecki, Christopher A.; Manning, Keefe B.

2014-01-01

Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s−1. The aim of the current work is to determine platelet adhesion properties to the polyurethane urea surface as a function of time varying shear exposure. A rotating disk system is used to study the influence of steady and pulsatile flow conditions (e.g. cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments retain the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk is rotated in platelet rich bovine plasma for two hours with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow is found to exponentially decay with increasing shear rate. Adhesion levels are found to depend upon peak platelet flux and shear rate regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices. PMID:24721222

Crosstalk between Platelets and the Immune System: Old Systems with New Discoveries

Directory of Open Access Journals (Sweden)

Conglei Li

2012-01-01

Full Text Available Platelets are small anucleate cells circulating in the blood. It has been recognized for more than 100 years that platelet adhesion and aggregation at the site of vascular injury are critical events in hemostasis and thrombosis; however, recent studies demonstrated that, in addition to these classic roles, platelets also have important functions in inflammation and the immune response. Platelets contain many proinflammatory molecules and cytokines (e.g., P-selectin, CD40L, IL-1β, etc., which support leukocyte trafficking, modulate immunoglobulin class switch, and germinal center formation. Platelets express several functional Toll-like receptors (TLRs, such as TLR-2, TLR-4, and TLR-9, which may potentially link innate immunity with thrombosis. Interestingly, platelets also contain multiple anti-inflammatory molecules and cytokines (e.g., transforming growth factor-β and thrombospondin-1. Emerging evidence also suggests that platelets are involved in lymphatic vessel development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2. Besides the active contributions of platelets to the immune system, platelets are passively targeted in several immune-mediated diseases, such as autoimmune thrombocytopenia, infection-associated thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. These data suggest that platelets are important immune cells and may contribute to innate and adaptive immunity under both physiological and pathological conditions.

The Non-Hemostatic Aspects of Transfused Platelets

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Caroline Sut

2018-02-01

Full Text Available Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR. In this review, we focus on the inflammatory potential of platelet components (PCs and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs—particularly on a critically ill patient’s context—could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization. This is linked to the platelets’ propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions.

The Non-Hemostatic Aspects of Transfused Platelets

Science.gov (United States)

Sut, Caroline; Tariket, Sofiane; Aubron, Cécile; Aloui, Chaker; Hamzeh-Cognasse, Hind; Berthelot, Philippe; Laradi, Sandrine; Greinacher, Andreas; Garraud, Olivier; Cognasse, Fabrice

2018-01-01

Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR). In this review, we focus on the inflammatory potential of platelet components (PCs) and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs—particularly on a critically ill patient’s context—could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization). This is linked to the platelets’ propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions. PMID:29536007

Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

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Daniela eWeth

2015-04-01

Full Text Available At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P. It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/µl, 106/µl, 107/µl and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1-/-, S1P3-/-. Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralisation of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIb)β₃ evokes phosphatidylserine exposure on their cell surface.

Science.gov (United States)

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Effect of losartan on human platelet activation.

Science.gov (United States)

Guerra-Cuesta, J I; Montón, M; Rodríguez-Feo, J A; Jiménez, A M; González-Fernández, F; Rico, L A; García, R; Gómez, J; Farré, J; Casado, S; López-Farré, A

1999-03-01

Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.

Are the changes in the peripheral brain-derived neurotrophic factor levels due to platelet activation?

Science.gov (United States)

Serra-Millàs, Montserrat

2016-01-01

Brain-derived neurotrophic factor (BDNF) plays an important role in central nervous system development, neurogenesis and neuronal plasticity. BDNF is also expressed in several non-neuronal tissues, and it could play an important role in other processes, such as cancer, angiogenesis, etc. Platelets are the major source of peripheral BDNF. However, platelets also contain high amounts of serotonin; they express specific surface receptors during activation, and a multitude of pro-inflammatory and immunomodulatory bioactive compounds are secreted from the granules. Until recently, there was insufficient knowledge regarding the relationship between BDNF and platelets. Recent studies showed that BDNF is present in two distinct pools in platelets, in α-granules and in the cytoplasm, and only the BDNF in the granules is secreted following stimulation, representing 30% of the total BDNF in platelets. BDNF has an important role in the pathophysiology of depression. Low levels of serum BDNF have been described in patients with major depressive disorder, and BDNF levels increased with chronic antidepressant treatment. Interestingly, there is an association between depression and platelet function. This review analyzed studies that evaluated the relationship between BDNF and platelet activation and the effect of treatments on both parameters. Only a few studies consider this possible confounding factor, and it could be very important in diseases such as depression, which show changes in both parameters. PMID:27014600

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

Science.gov (United States)

Brzoska, Tomasz; Suzuki, Yuko; Mogami, Hideo; Sano, Hideto; Urano, Tetsumei

2013-01-01

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment. PMID:23383331

Binding of thrombin-activated platelets to a fibrin scaffold through α(IIbβ₃ evokes phosphatidylserine exposure on their cell surface.

Directory of Open Access Journals (Sweden)

Tomasz Brzoska

Full Text Available Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIbβ₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.

Science.gov (United States)

Gilio, Karen; Munnix, Imke C A; Mangin, Pierre; Cosemans, Judith M E M; Feijge, Marion A H; van der Meijden, Paola E J; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O; Jackson, Shaun P; Heemskerk, Johan W M

2009-12-04

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Molecular insight into human platelet antigens: structural and evolutionary conservation analyses offer new perspective to immunogenic disorders

OpenAIRE

Landau, Meytal; Rosenberg, Nurit

2011-01-01

BACKGROUND: Human platelet antigens (HPAs) are polymorphisms in platelet membrane glycoproteins (GPs) that can stimulate production of alloantibodies once exposed to foreign platelets (PLTs) with different HPAs. These antibodies can cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and PLT transfusion refractoriness. Most HPAs are localized on the main PLT receptors: 1) integrin αIIbβ3, known as the fibrinogen receptor; 2) the GPIb-IX-V complex that functions as the recepto...

Equipoise in the enhanced supression of the platelet IIb/IIIa receptor with integrilin trial (ESPRIT): a critical appraisal.

Science.gov (United States)

Mann, Howard; London, Alex John; Mann, Jeffrey

2005-01-01

Enhanced Supression of the Platelet IIb/IIIa Receptor with Integrilin Trial (ESPRIT) was a multicenter randomized controlled clinical trial in which participants were randomized between eptifibatide and placebo. A "clinical hold" was initially placed on the trial by the US Food and Drug Administration (FDA), which was concerned about the placebo-only control arm. The hold was lifted after additional information concerning the use of platelet glycoprotein IIb/IIIa inhibitors in clinical practice, derived from a survey of interventional cardiologists, was provided. The trial's principal investigator and colleagues have described how these issues were resolved, and advance a claim of equipoise for the trial. In this critical appraisal we examine the information and arguments proffered in support of the trial design and conclude that they evidence a misunderstanding of equipoise. We believe that a placebo-only control arm was not justified by the information provided by the trialists.

A Biomarker to Differentiate between Primary and Cocaine-Induced Major Depression in Cocaine Use Disorder: The Role of Platelet IRAS/Nischarin (I1-Imidazoline Receptor

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Benjamin Keller

2017-12-01

Full Text Available The association of cocaine use disorder (CUD and comorbid major depressive disorder (MDD; CUD/MDD is characterized by high prevalence and poor treatment outcomes. CUD/MDD may be primary (primary MDD or cocaine-induced (CUD-induced MDD. Specific biomarkers are needed to improve diagnoses and therapeutic approaches in this dual pathology. Platelet biomarkers [5-HT2A receptor and imidazoline receptor antisera selected (IRAS/nischarin] were assessed by Western blot in subjects with CUD and primary MDD (n = 16 or CUD-induced MDD (n = 9; antidepressant free, AD−; antidepressant treated, AD+ and controls (n = 10 at basal level and/or after acute tryptophan depletion (ATD. Basal platelet 5-HT2A receptor (monomer was reduced in comorbid CUD/MDD subjects (all patients: 43% compared to healthy controls, and this down-regulation was independent of AD medication (decreases in AD−: 47%, and in AD+: 40%. No basal differences were found for IRAS/nischarin contents in AD+ and AD− comorbid CUD/MDD subjects. The comparison of IRAS/nischarin in the different subject groups during/after ATD showed opposite modulations (i.e., increases and decreases in response to low plasma tryptophan levels with significant differences discriminating between the subgroups of CUD with primary MDD and CUD-induced MDD. These specific alterations suggested that platelet IRAS/nischarin might be useful as a biomarker to discriminate between primary and CUD-induced MDD in this dual pathology.

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.

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

International Nuclear Information System (INIS)

Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

1988-01-01

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

International Nuclear Information System (INIS)

Peters, A.M.; Lavender, J.P.

1983-01-01

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of ''abnormal'' platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy

Kinetics of platelets in dogs with thrombocytopenia induced by antiglycoprotein IIb/IIIa receptor monoclonal antibody

International Nuclear Information System (INIS)

Hosono, Makoto; Sone, Naoaki; Endo, Keigo; Saga, Tsuneo; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Yasunaga, Kojiro; Konishi, Junji

1995-01-01

To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111 In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 μg/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 μg/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia

Activated platelets enhance IL-10 secretion and reduce TNF-α secretion by monocytes

DEFF Research Database (Denmark)

Gudbrandsdottir, Sif; Hasselbalch, Hans C; Nielsen, Claus H

2013-01-01

), Escherichia coli LPS, or intact Porphyromonas gingivalis. Addition of platelets activated by thrombin-receptor-activating peptide enhanced IL-10 production induced by LPS (p gingivalis (p ....05), and P. gingivalis (p gingivalis-stimulated cultures (p ... of activated platelets. Adherence of platelets increased TG- and TT-induced IL-10 secretion by monocytes (p gingivalis (p

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

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Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

The life cycle of platelet granules.

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Sharda, Anish; Flaumenhaft, Robert

2018-01-01

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Evaluation of the TEG® platelet mappingTM assay in blood donors

DEFF Research Database (Denmark)

Bochsen, Louise; Wiinberg, Bo; Kjelgaard-Hansen, Mads Jens

2007-01-01

for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. Methods In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG® Platelet MappingTM assay were determined together......Background Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG® Platelet MappingTM assay measures clot strength as maximal amplitude (MA) and enables...

Subpopulations in purified platelets adhering on glass.

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Donati, Alessia; Gupta, Swati; Reviakine, Ilya

2016-06-22

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

Stimulus-response coupling in platelets

International Nuclear Information System (INIS)

Huang, E.M.

1986-01-01

To understand the mechanism of stimulus-response coupling in platelets, the potentiating effect of succinate and lithium on platelet activation was examined. The action of succinate was immediate; preincubation with succinate did not lead to desensitization. Succinate was comparable to ADP in lowering cAMP levels previously elevated by PGl 2 . Since inhibition of cAMP is not a prerequisite for platelet activation, the mechanism of potentiation of succinate remains undefined. Lithium has also been shown to inhibit adenylate cyclase in PGl 2 -pretreated platelets. Lithium, however, can also inhibit inositol phosphate (InsP) phosphatase and lead to an accumulation of InsP. In human platelets, lithium also enhanced the thrombin-induced accumulation of [ 3 H]inositol-labelled inositol trisphosphate (InsP 3 ), and inositol bisphosphate (InsP 2 ). One hour after thrombin addition, all 3 inositol phosphates returned to near basal levels. In the presence of lithium, while labelled InsP 2 and InsP 3 returned to their respective basal levels, the InsP level remained elevated, consistent with the known inhibitory effect of lithium on InsP phosphatase. In thrombin-stimulated platelets prelabeled with [ 32 P]phosphate, lithium led to a decrease in labelled phosphatidylinositol 4-phosphate (PtdIns4P) as well as an enhanced production of labelled lysophosphatidylinositol, suggesting multiple effects of lithium on platelet phosphoinositide metabolism. These observed effects, however, occurred too slowly to be the mechanism by which lithium potentiated agonist-induced platelet activation. To study the agonist-receptor interaction, the effect of the specific, high affinity thrombin inhibitor, hirudin, on thrombin-induced accumulation of [ 3 H]inositol-labelled inositol phosphates was studied

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

DEFF Research Database (Denmark)

Tricoci, Pierluigi; Huang, Zhen; Held, Claes

2012-01-01

Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation.......Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation....

Podoplanin enhances lung cancer cell growth in vivo by inducing platelet aggregation.

Science.gov (United States)

Miyata, Kenichi; Takemoto, Ai; Okumura, Sakae; Nishio, Makoto; Fujita, Naoya

2017-06-22

Podoplanin/Aggrus, known as a platelet aggregation-inducing factor, is frequently overexpressed in lung squamous cell carcinomas (LSCC) and glioblastomas among other tumours, and its expression has been reported to be correlated with poor prognosis. However, the contribution of podoplanin to malignant progression has been elusive. Here we demonstrate that in podoplanin-positive LSCC cells, their growth was abrogated by podoplanin knockout in vivo but not in vitro. Conversely, ectopic expression of podoplanin promoted cell growth in vivo and facilitated intratumoral platelet activation. Consistently, LSCC cells evoked podoplanin-mediated platelet aggregation (PMPA), and the releasates from platelets during PMPA promoted the growth of LSCC cells in vitro. Phospho-receptor-tyrosine-kinase array analysis revealed that epidermal growth factor receptor (EGFR) phosphorylation of LSCC cells was responsible for the growth promotion induced by platelet releasates. Treatment with an antiplatelet agent or podoplanin-neutralizing antibody depressed the growth of an LSCC tumour xenograft via suppression of EGFR phosphorylation. These results suggested that podoplanin in LSCC enhanced cell growth by inducing PMPA in vivo and contributed to malignant progression.

Platelet function in stored heparinised autologous blood is not superior to in patient platelet function during routine cardiopulmonary bypass.

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Rolf C G Gallandat Huet

Full Text Available BACKGROUND: In cardiac surgery, cardiopulmonary bypass (CPB and unfractionated heparin have negative effects on blood platelet function. In acute normovolemic haemodilution autologous unfractionated heparinised blood is stored ex-vivo and retransfused at the end of the procedure to reduce (allogeneic transfusion requirements. In this observational study we assessed whether platelet function is better preserved in ex vivo stored autologous blood compared to platelet function in the patient during CPB. METHODOLOGY/PRINCIPAL FINDING: We measured platelet aggregation responses pre-CPB, 5 min after the start of CPB, at the end of CPB, and after unfractionated heparin reversal, using multiple electrode aggregometry (Multiplate® with adenosine diphosphate (ADP, thrombin receptor activating peptide (TRAP and ristocetin activated test cells. We compared blood samples taken from the patient with samples taken from 100 ml ex-vivo stored blood, which we took to mimick blood storage during normovolemic haemodilution. Platelet function declined both in ex-vivo stored blood as well as in blood taken from the patient. At the end of CPB there were no differences in platelet aggregation responses between samples from the ex vivo stored blood and the patient. CONCLUSION/SIGNIFICANCE: Ex vivo preservation of autologous blood in unfractionated heparin does not seem to be profitable to preserve platelet function.

Immunohistochemical examination of effects of kefir, koumiss and commercial probiotic capsules on platelet derived growth factor-c and platelet derived growth factor receptor-alpha expression in mouse liver and kidney.

Science.gov (United States)

Bakir, B; Sari, E K; Aydin, B D; Yildiz, S E

2015-04-01

We investigated using immunohistochemistry the effects of kefir, koumiss and commercial probiotic capsules on the expression of platelet derived growth factor-c (PDGF-C) and platelet derived growth factor receptor-alpha (PDGFR-α) in mouse liver and kidney. Mice were assigned to four groups: group 1 was given commercial probiotic capsules, group 2 was given kefir, group 3 was given koumiss and group 4 was untreated. After oral administration for 15 days, body weights were recorded and liver and kidney tissue samples were obtained. Hematoxylin and eosin staining was used to examine histology. PDGF-C and PDGFR-α in liver and kidney were localized using the streptavidin-biotin peroxidase complex method (ABC). We found that the weights of the mice in the kefir, koumiss and commercial probiotic capsules groups increased compared to the control group. No differences in liver and kidney histology were observed in any of the experimental groups. Kefir, koumiss and the commercial probiotic preparation increased PDGF-C and PDGFR-α expression.

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression

International Nuclear Information System (INIS)

Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X.; Walterscheid, Jeffrey P.; Ullrich, Stephen E.

2004-01-01

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependant manner. The release of biological response modifiers, particularly prostaglandin E 2 (PGE 2 ), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE 2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE 2 secretion. Jet fuel-induced PGE 2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin

The measurement of platelet activation by radioimmunoassay in asthma

International Nuclear Information System (INIS)

Wu Guoxin; Sun Jian; Li Jianyong; Ruan Changgeng

1992-02-01

Radioimmunoassay with specific monoclonal antibody was used to evaluate the platelet activation in 14 cases of acute bronchial asthma. The result showed that the number of molecules of alpha-granule membrane protein (GMP-140) which was exposed on the surface of platelet following secretion significantly increased on the surface of platelet and in plasma, while the number of molecules of glycoprotein (GP) I b and GPIII a did not change significantly; the concentration of thromboxane B 2 in plasma was raised, while the concentration of 6-keto-PGF 1a was within the normal limits; the concentrations of β-thromboglobulin (β-TG) and platelet factor 4(PF 4 ) in plasma increased significantly; the number of platelets decreased. These results strongly confirmed that the degree of platelet activation was enhanced during acute asthmatic attack. The significance of platelet activation in the pathogenesis of asthma should be further investigated

Polyphosphate nanoparticles on the platelet surface trigger contact system activation

NARCIS (Netherlands)

Verhoef, Johan J F; Barendrecht, Arjan D; Nickel, Katrin F; Dijkxhoorn, Kim; Kenne, Ellinor; Labberton, Linda; McCarty, Owen J T; Schiffelers, Raymond; Heijnen, Harry F G; Hendrickx, Antoni P A; Schellekens, Huub; Fens, Marcel H; de Maat, Steven; Renné, Thomas; Maas, Coen

2017-01-01

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII.

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

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Karl Egan

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

EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

Science.gov (United States)

Schulz, C; von Brühl, M-L; Barocke, V; Cullen, P; Mayer, K; Okrojek, R; Steinhart, A; Ahmad, Z; Kremmer, E; Nieswandt, B; Frampton, J; Massberg, S; Schmidt, R

2011-05-01

Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development. © 2011 International Society on Thrombosis and Haemostasis.

Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

International Nuclear Information System (INIS)

Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T.

1990-01-01

Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation

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

International Nuclear Information System (INIS)

Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

1987-01-01

Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [ 3 H]serotonin, or alter the dose-responsive binding of 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

Emerging Evidence for Platelets as Immune and Inflammatory Effector Cells

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Matthew Thomas Rondina

2014-12-01

Full Text Available While traditionally recognized for their roles in hemostatic pathways, emerging evidence demonstrates that platelets have previously unrecognized, dynamic roles that span the immune continuum. These newly-recognized platelet functions, including the secretion of immune mediators, interactions with endothelial cells, monocytes, and neutrophils, toll-like receptor (TLR mediated responses, and induction of neutrophil extracellular trap (NET formation, bridge thrombotic and inflammatory pathways and contribute to host defense mechanisms against invading pathogens. In this focused review, we highlight several of these emerging aspects of platelet biology and their implications in clinical infectious syndromes.

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

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Jacqueline Stockley

Full Text Available The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12 could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =, both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =. Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

Science.gov (United States)

Stockley, Jacqueline; Nisar, Shaista P; Leo, Vincenzo C; Sabi, Essa; Cunningham, Margaret R; Eikenboom, Jeroen C; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C; Watson, Steve P; Mundell, Stuart J; Daly, Martina E

2015-01-01

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

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

International Nuclear Information System (INIS)

Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

2014-01-01

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

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

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Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Role of newly formed platelets in thrombus formation in rat after clopidogrel treatment

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Kuijpers, Marijke J E; Megens, Remco T A; Nikookhesal, Elham

2011-01-01

Platelet P2Y₁₂ receptors play an important role in arterial thrombosis by stimulating thrombus growth. Both irreversibly (clopidogrel) and reversibly binding (ticagrelor, AZD6140) P2Y₁₂ antagonists are clinically used for restricted periods, but possible differences in platelet function recovery ...

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

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Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

Neer Award 2018: Platelet-derived growth factor receptor α co-expression typifies a subset of platelet-derived growth factor receptor β-positive progenitor cells that contribute to fatty degeneration and fibrosis of the murine rotator cuff.

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Jensen, Andrew R; Kelley, Benjamin V; Mosich, Gina M; Ariniello, Allison; Eliasberg, Claire D; Vu, Brandon; Shah, Paras; Devana, Sai K; Murray, Iain R; Péault, Bruno; Dar, Ayelet; Petrigliano, Frank A

2018-04-10

After massive tears, rotator cuff muscle often undergoes atrophy, fibrosis, and fatty degeneration. These changes can lead to high surgical failure rates and poor patient outcomes. The identity of the progenitor cells involved in these processes has not been fully elucidated. Platelet-derived growth factor receptor β (PDGFRβ) and platelet-derived growth factor receptor α (PDGFRα) have previously been recognized as markers of cells involved in muscle fibroadipogenesis. We hypothesized that PDGFRα expression identifies a fibroadipogenic subset of PDGFRβ + progenitor cells that contribute to fibroadipogenesis of the rotator cuff. We created massive rotator cuff tears in a transgenic strain of mice that allows PDGFRβ + cells to be tracked via green fluorescent protein (GFP) fluorescence. We then harvested rotator cuff muscle tissues at multiple time points postoperatively and analyzed them for the presence and localization of GFP + PDGFRβ + PDGFRα + cells. We cultured, induced, and treated these cells with the molecular inhibitor CWHM-12 to assess fibrosis inhibition. GFP + PDGFRβ + PDGFRα + cells were present in rotator cuff muscle tissue and, after massive tears, localized to fibrotic and adipogenic tissues. The frequency of PDGFRβ + PDGFRα + cells increased at 5 days after massive cuff tears and decreased to basal levels within 2 weeks. PDGFRβ + PDGFRα + cells were highly adipogenic and significantly more fibrogenic than PDGFRβ + PDGFRα - cells in vitro and localized to adipogenic and fibrotic tissues in vivo. Treatment with CWHM-12 significantly decreased fibrogenesis from PDGFRβ + PDGFRα + cells. PDGFRβ + PDGFRα + cells directly contribute to fibrosis and fatty degeneration after massive rotator cuff tears in the mouse model. In addition, CWHM-12 treatment inhibits fibrogenesis from PDGFRβ + PDGFRα + cells in vitro. Clinically, perioperative PDGFRβ + PDGFRα + cell inhibition may limit rotator cuff tissue degeneration and, ultimately

Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

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Sandra Mjoll Jonsdottir-Buch

Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets

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Singh, Anirudh K.; Woodiga, Shireen A.; Grau, Margaret A.

2016-01-01

ABSTRACT Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. PMID:27993975

Haplotype of platelet receptor P2RY12 gene is associated with residual clopidogrel on-treatment platelet reactivity.

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Nie, Xiao-Yan; Li, Jun-Lei; Zhang, Yong; Xu, Yang; Yang, Xue-Li; Fu, Yu; Liang, Guang-Kai; Lu, Yun; Liu, Jian; Shi, Lu-Wen

To investigate a possible association between common variations of the P2RY12 and the residual clopidogrel on-treatment platelet reactivity after adjusting for the influence of CYP2C19 tested by thromboelastography (TEG). One hundred and eighty patients with acute coronary syndrome (ACS) treated with clopidogrel and aspirin were included and platelet function was assessed by TEG. Five selected P2RY12 single nucleotide polymorphisms (SNPs; rs6798347, rs6787801, rs6801273, rs6785930, and rs2046934), which cover the common variations in the P2RY12 gene and its regulatory regions, and three CYP2C19 SNPs ( * 2, * 3, * 17) were genotyped and possible haplotypes were analyzed. The high on-treatment platelet reactivity (HTPR) prevalence defined by a platelet inhibition rate <30% by TEG in adenosine diphosphate (ADP)-channel was 69 (38.33%). Six common haplotypes were inferred from four of the selected P2RY12 SNPs (denoted H 0 to H 5 ) according to the linkage disequilibrium R square (except for rs2046934). Haplotype H 1 showed a significantly lower incidence of HTPR than the reference haplotype (H 0 ) in the total study population while haplotypes H 1 and H 2 showed significantly lower incidences of HTPR than H 0 in the nonsmoker subgroup after adjusting for CYP2C19 effects and demographic characteristics. rs2046934 (T744C) did not show any significant association with HTPR. The combination of common P2RY12 variations including regulatory regions rather than rs2046934 (T744C) that related to pharmacodynamics of clopidogrel in patients with ACS was independently associated with residual on-clopidogrel platelet reactivity. This is apart from the established association of the CYP2C19. This association seemed more important in the subgroup defined by smoking.

Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

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Mohammad Mizanur Rahman

2017-02-01

Full Text Available This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74 and pooled platelets (n=54. Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001. On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001. This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

Purinergic control of inflammation and thrombosis: Role of P2X1 receptors

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Cécile Oury

2015-01-01

Full Text Available Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/− mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/− mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.

Coordinated Molecular Cross-Talk between Staphylococcus aureus, Endothelial Cells and Platelets in Bloodstream Infection

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Carolina D. Garciarena

2015-12-01

Full Text Available Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the human body. Upon entry to the otherwise sterile environment of the cardiovascular system, S. aureus can lead to serious complications resulting in organ failure and death. The success of S. aureus as a pathogen in the bloodstream is due to its ability to express a wide array of cell wall proteins on its surface that recognise host receptors, extracellular matrix proteins and plasma proteins. Endothelial cells and platelets are important cells in the cardiovascular system and are a major target of bloodstream infection. Endothelial cells form the inner lining of a blood vessel and provide an antithrombotic barrier between the vessel wall and blood. Platelets on the other hand travel throughout the cardiovascular system and respond by aggregating around the site of injury and initiating clot formation. Activation of either of these cells leads to functional dysregulation in the cardiovascular system. In this review, we will illustrate how S. aureus establish intimate interactions with both endothelial cells and platelets leading to cardiovascular dysregulation.

Characterization of platelet adhesion under flow using microscopic image sequence analysis.

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Machin, M; Santomaso, A; Cozzi, M R; Battiston, M; Mazzuccato, M; De Marco, L; Canu, P

2005-07-01

A method for quantitative analysis of platelet deposition under flow is discussed here. The model system is based upon perfusion of blood platelets over an adhesive substrate immobilized on a glass coverslip acting as the lower surface of a rectangular flow chamber. The perfusion apparatus is mounted onto an inverted microscope equipped with epifluorescent illumination and intensified CCD video camera. Characterization is based on information obtained from a specific image analysis method applied to continuous sequences of microscopical images. Platelet recognition across the sequence of images is based on a time-dependent, bidimensional, gaussian-like pdf. Once a platelet is located,the variation of its position and shape as a function of time (i.e., the platelet history) can be determined. Analyzing the history we can establish if the platelet is moving on the surface, the frequency of this movement and the distance traveled before its resumes the velocity of a non-interacting cell. Therefore, we can determine how long the adhesion would last which is correlated to the resistance of the platelet-substrate bond. This algorithm enables the dynamic quantification of trajectories, as well as residence times, arrest and release frequencies for a high numbers of platelets at the same time. Statistically significant conclusions on platelet-surface interactions can then be obtained. An image analysis tool of this kind can dramatically help the investigation and characterization of the thrombogenic properties of artificial surfaces such as those used in artificial organs and biomedical devices.

Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

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Kellie J Hall

2008-09-01

Full Text Available PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/- T cells exhibit reduced activation and PKCtheta(-/- mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIbbeta(3 in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed.In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIbbeta(3 activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/- platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/- platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIbbeta(3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1 was enhanced.These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIbbeta(3 activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

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

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van Bladel Esther R

2012-09-01

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.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

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Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blood platelet kinetics and platelet transfusion.

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Aster, Richard H

2013-11-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The "acidification" approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

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Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B

2012-03-16

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

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

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

2009-01-01

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...

Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

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N Sadat Razavi Hoseini

2016-02-01

Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

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

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Söderström, Anna C; Nybo, Mads; Nielsen, Christian; Vinholt, Pernille J

2016-12-01

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. 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 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. The median percentage 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%), pcentrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

Platelet size and age determine platelet function independently

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Thompson, C.B.; Jakubowski, J.A.; Quinn, P.G.; Deykin, D.; Valeri, C.R.

1984-01-01

A study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms

Platelet activating factor activity in the phospholipids of bovine spermatozoa

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Parks, J.E.; Hough, S.; Elrod, C. (Cornell Univ., Ithaca, NY (USA))

1990-11-01

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of (3H)serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

Platelets Promote Metastasis via Binding Tumor CD97 Leading to Bidirectional Signaling that Coordinates Transendothelial Migration

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Yvona Ward

2018-04-01

Full Text Available Summary: Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR, is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread. : Tumor-initiated platelet activation promotes tissue invasion of cancer cells and metastasis. Ward et al. demonstrate that a common tumor-associated antigen, CD97, accounts for platelet activation and participates directly in LPA-mediated signal transduction leading to tumor cell invasion. CD97 promotes vascular extravasation and metastasis in pre-clinical models. Keywords: platelets, metastasis, transendothelial migration, circulating tumor cells, CD97, adhesion GPCR, LPA

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

2015-01-01

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.......036) in the Babesia-infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia-infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62...... groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia-infected dogs, specifically dogs that survived, had a significantly increased percentage...

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L.

Science.gov (United States)

Favale, Fabrizia; Messaoudi, Kahia; Varghese, Leila N; Boukour, Siham; Pecquet, Christian; Gryshkova, Vitalina; Defour, Jean Philippe; Albu, Roxana-Irina; Bluteau, Olivier; Ballerini, Paola; Leverger, Guy; Plo, Isabelle; Debili, Najet; Raslova, Hana; Favier, Remi; Constantinescu, Stefan N; Vainchenker, William

2016-12-29

The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34 + cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl -/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [ 125 I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells. © 2016 by The American Society of Hematology.

[27- Hydroxycholesterol reverses estradiol induced inhibition of platelet aggregation in postmenopausal women].

Science.gov (United States)

Rocha, Gladys; Sierralta, Walter; Valladares, Luis

2016-11-01

The decline of estrogen levels increases cardiovascular risk in women. Platelets express estrogen receptors and 17β-estradiol- (E2) can produce a protective effect on thrombus formation. The hydroxylation of cholesterol generates several sterols and 27-hydroxycholesterol (27HC) predominates in circulation. To evaluate the effect of 27HC as an endogenous antagonist of the anti-aggregating properties of E2 in platelets of postmenopausal women. Platelet function of postmenopausal women was evaluated ex-vivo. Platelets pre-incubated with 27HC in the presence or absence of E2, were stimulated with collagen. Aggregation was evaluated using turbidimetry using a Chrono-log aggregometer. Collagen-stimulated platelet aggregation was significantly inhibited by E2. The inhibitory effect of E2 on collagen-stimulated platelet aggregation was significantly reversed in the presence of 27HC. The suppressive effect of E2 on platelet aggregation is inhibited by 27HC, which could contribute to increase cardiovascular risk in postmenopausal women.

Glucose impairs aspirin inhibition in platelets through a NAD(P)H oxidase signaling pathway.

Science.gov (United States)

Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

2017-07-01

Hyperglycemia has been suggested to play a role in the increased platelet resistance to antiplatelet therapy in patients with diabetes mellitus. Exposure to high glucose impairs platelet inhibition by aspirin. It has been found that antioxidant agents reduce the effect of glucose, confirming the involvement of reactive oxygen species (ROS) in the effect of glucose. The aim of the study was to examine the mechanism of ROS increase by high glucose in aspirin-treated platelets. Platelet aggregation was measured by the optical method, and the production of ROS was detected using luminol-dependent horseradish peroxidase-enhanced chemiluminescence. We found that glucose did not affect ADP-induced platelet aggregation. However, it reduced the effect of aspirin on platelet aggregation, which was accompanied by an increase in ROS generation. The inhibition of NAD(P)H oxidase (NOX) prevented the glucose effect and ROS generation. The same result was recorded after the inhibition of p38 mitogen-activated protein kinases (p38 MAPK), phospholipase A 2 (PLA 2 ) or 12-lipoxygenase (12-LOX). The inhibition of TxA 2 receptor did not decrease the effect of glucose indicating that the effect was not caused by activation of TxA 2 receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

Storage of platelets: effects associated with high platelet content in platelet storage containers.

Science.gov (United States)

Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

2012-04-01

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.

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation.

Science.gov (United States)

Ng, Ashley P; Kauppi, Maria; Metcalf, Donald; Hyland, Craig D; Josefsson, Emma C; Lebois, Marion; Zhang, Jian-Guo; Baldwin, Tracey M; Di Rago, Ladina; Hilton, Douglas J; Alexander, Warren S

2014-04-22

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

Directory of Open Access Journals (Sweden)

Renáta Hudák

2017-01-01

Full Text Available The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA, on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1 activator, thrombin receptor activating peptide (TRAP. Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS- expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

Science.gov (United States)

Hall, Kellie J.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M.; Heemskerk, Johan W. M.; Poole, Alastair W.

2008-01-01

Background PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed. Methodology/Principal Findings In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced. Conclusions/Significance These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors. PMID:18815612

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

Science.gov (United States)

Singh, Anirudh K; Woodiga, Shireen A; Grau, Margaret A; King, Samantha J

2017-03-01

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis , like Streptococcus gordonii and Streptococcus sanguinis , binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. Copyright © 2017 American Society for Microbiology.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance

Science.gov (United States)

Cuker, Adam; Husseinzadeh, Holleh; Lebedeva, Tatiana; Marturano, Joseph E.; Massefski, Walter; Lowery, Thomas J.; Lambert, Michele P.; Abrams, Charles S.; Weisel, John W.

2016-01-01

Objectives: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). Methods: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. Results: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. Conclusions: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests. PMID:28028118

Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

2010-07-30

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

The effect of 60Co-irradiation on platelet cytoskeleton and function

International Nuclear Information System (INIS)

Zhuang Qingqi; Li Chengzhu

1987-01-01

Changes in rat platelet cytoskeleton and platelet function by 60 Co-irradiation were investigated. When platelet-rich plasma (PRP) was stimulated with ADP, PRP aggregation rates on the 3rd, 6th and 9th day after irradiation were 135%, 96% and 0% of the controls respectively. Although control rat PRP responded to collagen stimulation well, PRP from treated rats had lost the aggregation ability induced by collagen. When same amounts of platelets from the control and the treated rats were extracted with 1% triton X-100-EGTA buffer and the cytoskeleton pellet analysed by SDS-PAGE, it was found that the amount and components of the rat platelet cytoskeleton especially actin and myosin on the 3rd day after irradiation were greatly increased. Those from rat platelets on the 6th day after irradiation showed no difference from the controls, but was significantly decreased on the 9th day after irradiation. Based on these results, the authors concluded that the platelets from rats on the 3rd day after irradiation were in an active state. Exhaustion of granule contents or impairment of collagen receptors might render platelets unable to respond to collagen. Decreased platelet function on the 9th day after irradiation was due to a failure of assembly of its cytoskeleton

Clopidogrel discontinuation and platelet reactivity following coronary stenting

LENUS (Irish Health Repository)

2011-01-01

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

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

Science.gov (United States)

Tsai, W B; Grunkemeier, J M; Horbett, T A

1999-02-01

The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogen's ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule. Copyright 1999 John Wiley & Sons, Inc.

The use of autologous 111In-labelled platelets and scintigraphy to illustrate enhanced platelet activity during erection in the chacma baboon

International Nuclear Information System (INIS)

Dormehl, I.C.; Du Plessis, M.; Maree, M.; Bornman, M.S.; Du Plessis, D.J.

1984-01-01

The demonstration of thrombelastographic hypercoagulability in the penile blood during erection, and the accompanying deposition of fibrin onto the endothelial layer of the deep penile artery and trabecular surface inspired this investigation of the possible role that platelets might play in the process. The bloodpooling pattern in the penis during and after erection from electro-stimulation was studied in 9 male adult baboons (Papio ursinus) using in vivo sup(99m)Tc-labelled red blood cells and scintigraphy. Platelet activity was similarly investigated after administering autologous 111 In-labelled platelets to the baboons. The results indicate an enhanced platelet concentration with respect to blood-pooling during erection, and an entrapment of platelets after erection. (orig.) [de

Measurement of platelet aggregation, independently of patient platelet count

DEFF Research Database (Denmark)

Vinholt, P J; Frederiksen, H; Hvas, A-M

2017-01-01

with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.......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...

Radiolabeled platelets

International Nuclear Information System (INIS)

Datz, F.L.; Taylor, A.T.

1986-01-01

Initial interest in developing techniques to radiolabel platelets was spurred by the lack of an accurate method for measuring platelet life span in both normals and in thrombocytopenic patients. Early investigators could obtain only rough estimates of platelet life spans by monitoring the platelet counts of thrombocytopenic patients undergoing platelet transfusions. Labels were also sought that would allow imaging of platelets in vivo in order to better understand the pathophysiology of atherosclerosis, thrombophlebitis, and clotting disorders, and to improve the clinical diagnosis of these diseases. Two types of platelet labels were investigated: cohort (pulse) labels and random labels. Cohort labels are taken up by megakaryocytes in the bone marrow and incorporated in the DNA and other components of the forming platelet. In theory, only freshly released platelets of a uniform age are labeled. Random labels, on the other hand, tag platelets in the peripheral blood, labeling platelets of all ages

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration.

Science.gov (United States)

Cloutier, Nathalie; Allaeys, Isabelle; Marcoux, Genevieve; Machlus, Kellie R; Mailhot, Benoit; Zufferey, Anne; Levesque, Tania; Becker, Yann; Tessandier, Nicolas; Melki, Imene; Zhi, Huiying; Poirier, Guy; Rondina, Matthew T; Italiano, Joseph E; Flamand, Louis; McKenzie, Steven E; Cote, Francine; Nieswandt, Bernhard; Khan, Waliul I; Flick, Matthew J; Newman, Peter J; Lacroix, Steve; Fortin, Paul R; Boilard, Eric

2018-02-13

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays.

Science.gov (United States)

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2018-01-16

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin α IIb β 3 ), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, α IIb β 3 . Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery

DEFF Research Database (Denmark)

Schiødt, I; Jensen, Charlotte Harken; Kjaersgaard, E

2000-01-01

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell......-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood...... at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need...

Platelet deposition in rat heart allografts and the effect of a thromboxane receptor antagonist

International Nuclear Information System (INIS)

Foegh, M.L.; Khirabadi, B.S.; Ramwell, P.W.

1986-01-01

The effect of a thromboxane antagonist, L640,035 on platelet deposition in heart allografts was studied. Twenty Lewis rats received heterotopic allografts from Lewis x Brown-Norway F1 hybrid. All recipients received azathioprine (5 mg/kg/day). The rats were divided into three groups. Groups II and III were also treated daily with either the vehicle for L640,035 or L640,035 respectively. Syngeneic indium-111-labeled platelet deposition was determined in the allograft and the native heart at 6, 9, and 13 days after transplantation; group III was studied on the sixth and ninth day only. A rapidly increasing platelet deposition was seen in allografts from rats given azathioprine; whereas the thromboxane antagonist prevented the increase in platelet deposition on the ninth day

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

Science.gov (United States)

Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

2012-03-01

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

Platelet size does not correlate with platelet age.

Science.gov (United States)

Thompson, C B; Love, D G; Quinn, P G; Valeri, C R

1983-08-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

Platelet size does not correlate with platelet age

International Nuclear Information System (INIS)

Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

1983-01-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75 Se-methionine, (2) in vitro labeling with 51 Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75 Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51 Cr and 14 C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization.

Science.gov (United States)

Nisar, Shaista P; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J; Kelly, Eamonn; Mundell, Stuart J

2012-07-13

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

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.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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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.

Presence of intratumoral platelets is associated with tumor vessel structure and metastasis

International Nuclear Information System (INIS)

Li, Rong; Zhang, Xu; Zhang, Zhuo; Zhang, Xiao; Ran, Bing; Wu, Jianbo; Ren, Meiping; Chen, Ni; Luo, Mao; Deng, Xin; Xia, Jiyi; Yu, Guang; Liu, Jinbo; He, Bing

2014-01-01

Platelets play a fundamental role in maintaining hemostasis and have been shown to participate in hematogenous dissemination of tumor cells. Abundant platelets were detected in the tumor microenvironment outside of the blood vessel, thus, platelet -tumor cell interaction outside of the bloodstream may play a role in regulating primary tumor growth and metastasis initiation. However, it is unclear that platelet depletion affects tumor vessel structure and dynamics. Using thrombocytopenia induction in two different tumor-bearing mouse models, tumor tissues were performed by Westernblotting and immunohistochemical staining. Vascular permeability was evaluated by determination of intratumoral Evans blue and Miles vascular permeability assay. Furthermore, microdialysis was used to examining the intratumoral extracellular angiogenic growth factors (VEGF, TGF-β) by ELISA. Platelet depletion showed no change in tumor growth and reduced lung metastasis. Platelet depletion led to reduced tumor hypoxia and Met receptor activation and was associated with a decreased release of MMP-2, 9, PAI-1, VEGF, and TGF-β. Tumor vessels in platelet-depleted mice showed impaired vessel density and maturation. Our findings demonstrate that platelets within the primary tumor microenvironment play a critical role in the induction of vascular permeability and initiation of tumor metastasis

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers.

Science.gov (United States)

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren; Park, Fiona M

2013-07-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.

[3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Marquis, O.; Robaut, C.; Cavero, I.

1988-01-01

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

Decreased mean platelet volume in panic disorder

Directory of Open Access Journals (Sweden)

Göğçegöz Gül I

2014-09-01

Full Text Available Işil Göğçegöz Gül, Gül Eryilmaz, Eylem Özten, Gökben Hizli Sayar Neuropsychiatry Health, Practice, and Research Center, Uskudar University, Istanbul, Turkey Aim: The relationship between psychological stress and platelet activation has been widely studied. It is well known that platelets may reflect certain biochemical changes that occur in the brain when different mental conditions occur. Platelet 5-hydroxytryptamine (5-HT is also extensively studied in psychiatry. The mean platelet volume (MPV, the accurate measure of platelet size, has been considered a marker and determinant of platelet function. The aim of the present study was to search for any probable difference in the MPV of subjects with panic disorder (PD.Methods: A total of 37 drug-free subjects, aged 18 to 65 years, diagnosed with PD, with or without agoraphobia, according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition (DSM-IV criteria and 45 healthy control subjects were included in the study. Platelet count and MPV were measured and recorded for each subject.Results: There were no statistically significant differences between groups in terms of female/male ratio, age, or body mass index between the PD group and control group (P=0.91, P=0.82, and P=0.93, respectively. The MPV was found to be significantly lower in the PD group compared with the control group (8.8±0.9 fL vs 9.2±0.8 fL; P=0.02. All the participants had MPV values in the standard range of 6.9–10.8 fL.Conclusion: We concluded that abnormalities of the 5-HT1A receptor function in the central nervous system of subjects with a diagnosis of PD are also mirrored in as an alteration in platelet activity. Measurements of platelet activity may be used as a tool for neuropsychiatric and psychopharmacological research and for studying how certain mental diseases and medications affect the central nervous system. Keywords: 5-HT, thrombocyte, anxiety 

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

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Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A. H.; Stegner, David; van der Meijden, Paola E. J.; Kuijpers, Marijke J. E.; Varga-Szabo, David; Heemskerk, Johan W. M.; Nieswandt, Bernhard

2010-01-01

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

Blood platelet kinetics and platelet transfusion

OpenAIRE

Aster, Richard H.

2013-01-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 pr...

cDNA for the human β2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor

International Nuclear Information System (INIS)

Kobilka, B.K.; Dixon, R.A.F.; Frielle, T.

1987-01-01

The authors have isolated and sequenced a cDNA encoding the human β 2 -adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster β 2 -adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. They have localized the gene for the β 2 -adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor

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

Science.gov (United States)

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

2015-06-01

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of a tubular flow chamber for studying platelet interaction with biologic and prosthetic materials: deposition of indium 111-labeled platelets on collagen, subendothelium, and expanded polytetrafluoroethylene

International Nuclear Information System (INIS)

Badimon, L.; Turitto, V.; Rosemark, J.A.; Badimon, J.J.; Fuster, V.

1987-01-01

A plastic (Plexiglas) chamber for evaluating platelet deposition under controlled hemodynamic conditions has been developed. The perfusion chamber has been designed to retain the cylindrical shape typical of the vasculature, to be flexible enough to accept a variety of biologic and prosthetic materials, and to simulate a broad range of physiologic flow conditions in either an ex vivo or in vitro perfusion system. Three type of surfaces were exposed to blood flowing directly from the carotid artery of a heparinized pig through the perfusion chamber: de-endothelialized pig aorta, collagen strips from rabbit Achilles tendon, and an expanded polytetrafluoroethylene material (Gore-Tex). Platelets, previously radiolabeled with indium 111 and injected into the animal, were quantified on the material surface, and the total number of deposited platelets determined for a range of blood flow rates (5 to 40 ml/min) and exposure times (0.5 to 20 minutes). The deposition rates were correlated with theory for describing the mass transport of platelets to the test surface. At the wall shear rates investigated (105 to 850 sec-1), the deposition of platelets on subendothelium was strongly dependent on the local flow conditions. Values of deposition on Gore-Tex obtained at similar flow conditions (105 to 425 sec-1) were reduced compared with that observed on subendothelium and showed a markedly weaker dependence on the shear rate. In contrast, deposition of platelets on collagen was more than an order of magnitude greater than on subendothelium and showed a dependence on flow only at the lowest flow rate studied (10 ml/min). The results indicate that collagen is much more reactive than subendothelium and Gore-Tex with respect to the growth and stability of platelet aggregates and moreover suggest that flow mechanisms for depositing platelets on various surface may be substantially different

Characterization of a tubular flow chamber for studying platelet interaction with biologic and prosthetic materials: deposition of indium 111-labeled platelets on collagen, subendothelium, and expanded polytetrafluoroethylene

Energy Technology Data Exchange (ETDEWEB)

Badimon, L.; Turitto, V.; Rosemark, J.A.; Badimon, J.J.; Fuster, V.

1987-12-01

A plastic (Plexiglas) chamber for evaluating platelet deposition under controlled hemodynamic conditions has been developed. The perfusion chamber has been designed to retain the cylindrical shape typical of the vasculature, to be flexible enough to accept a variety of biologic and prosthetic materials, and to simulate a broad range of physiologic flow conditions in either an ex vivo or in vitro perfusion system. Three type of surfaces were exposed to blood flowing directly from the carotid artery of a heparinized pig through the perfusion chamber: de-endothelialized pig aorta, collagen strips from rabbit Achilles tendon, and an expanded polytetrafluoroethylene material (Gore-Tex). Platelets, previously radiolabeled with indium 111 and injected into the animal, were quantified on the material surface, and the total number of deposited platelets determined for a range of blood flow rates (5 to 40 ml/min) and exposure times (0.5 to 20 minutes). The deposition rates were correlated with theory for describing the mass transport of platelets to the test surface. At the wall shear rates investigated (105 to 850 sec-1), the deposition of platelets on subendothelium was strongly dependent on the local flow conditions. Values of deposition on Gore-Tex obtained at similar flow conditions (105 to 425 sec-1) were reduced compared with that observed on subendothelium and showed a markedly weaker dependence on the shear rate. In contrast, deposition of platelets on collagen was more than an order of magnitude greater than on subendothelium and showed a dependence on flow only at the lowest flow rate studied (10 ml/min). The results indicate that collagen is much more reactive than subendothelium and Gore-Tex with respect to the growth and stability of platelet aggregates and moreover suggest that flow mechanisms for depositing platelets on various surface may be substantially different.

Autologous Platelet-Rich Plasma for the Treatment of Pattern Hair Loss.

Science.gov (United States)

Singh, Babu; Goldberg, Lynne J

2016-08-01

Platelet-rich plasma (PRP) is a solution derived from whole blood that is enriched in the platelet fraction. Platelets serve as a reservoir of growth factors and cytokines. When platelets are activated in vivo, signaling molecules are released into the immediate microenvironment and activate receptors for various pathways. Historically, PRP has been applied to wound beds to promote healing of complex wounds. Over the last decade, it has served as a valuable therapeutic tool in various specialties such as maxillofacial surgery, plastic surgery, orthopedics and sports medicine. Only recently has PRP been utilized for dermatologic purposes, more specifically, for the treatment of male and female pattern hair loss. In this review, we discuss molecular and cellular pathways upregulated by PRP important in hair folliculogenesis, and examine clinical evidence from all previously published studies involving the use of PRP for pattern hair loss.

Platelet transfusions can induce transplantation tolerance

International Nuclear Information System (INIS)

Claas, F.H.J.; Blankert, J.J.; Ruigrok, R.; Moerel, L.

1982-01-01

Recently it was shown that the induction of antibodies against the H-2 antigens after multiple platelet transfusions is due to leukocyte contamination of the platelet suspensions. Pure platelets are not able to induce a primary antibody response. The present study shows that the platelets, however, can be recognized by the immune system but they induce a suppression of the response. Mice pretreated with donor platelets will not give a primary antibody response upon a subsequent injection of donor leukocytes and the survival of donor skin grafts will be prolonged. Similar results were obtained by pretreatment of the responder mice with heat-treated donor leukocytes. Furthermore, repeated injections of heat-treated leukocytes of the recipient strain to the donor before bone marrow grafting, will graft-versus-host mortality. The recipient mice were irradiated and received spleen cell injections. These data show that cells which have only class I antigens on their surface and no activating class II antigens, induce a suppression of the response against class I antigens. (Auth.)

Effect of Extreme Wettability on Platelet Adhesion on Metallic Implants: From Superhydrophilicity to Superhydrophobicity.

Science.gov (United States)

Moradi, Sona; Hadjesfandiari, Narges; Toosi, Salma Fallah; Kizhakkedathu, Jayachandran N; Hatzikiriakos, Savvas G

2016-07-13

In order to design antithrombotic implants, the effect of extreme wettability (superhydrophilicity to superhydrophobicity) on the biocompatibility of the metallic substrates (stainless steel and titanium) was investigated. The wettability of the surface was altered by chemical treatments and laser ablation methods. The chemical treatments generated different functionality groups and chemical composition as evident from XPS analysis. The micro/nanopatterning by laser ablation resulted in three different pattern geometry and different surface roughness and consequently wettability. The patterned surface were further modified with chemical treatments to generate a wide range of surface wettability. The influence of chemical functional groups, pattern geometry, and surface wettability on protein adsorption and platelet adhesion was studied. On chemically treated flat surfaces, the type of hydrophilic treatment was shown to be a contributing factor that determines the platelet adhesion, since the hydrophilic oxidized substrates exhibit less platelet adhesion in comparison to the control untreated or acid treated surfaces. Also, the surface morphology, surface roughness, and superhydrophobic character of the surfaces are contributing factors to platelet adhesion on the surface. Our results show that superhydrophobic cauliflower-like patterns are highly resistant to platelet adhesion possibly due to the stability of Cassie-Baxter state for this pattern compared to others. Our results also show that simple surface treatments on metals offer a novel way to improve the hemocompatibility of metallic substrates.

Anti-platelet activity of water dispersible curcuminoids in rat platelets.

Science.gov (United States)

Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

2015-03-01

Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders. Copyright © 2015 John Wiley & Sons, Ltd.

Platelets in thrombosis and hemostasis: old topic with new mechanisms.

Science.gov (United States)

Wang, Yiming; Andrews, Marc; Yang, Yan; Lang, Sean; Jin, Joseph W; Cameron-Vendrig, Alison; Zhu, Guangheng; Reheman, Adili; Ni, Heyu

2012-12-01

Platelets are small anucleate cells generated from megakaryocytes in the bone marrow. After being released into the circulation, platelets play key roles in the surveillance of vascular injury, and can quickly adhere and aggregate at the site of injury, which are critical events for vascular repair and hemostasis. However, the same biological processes of platelet adhesion and aggregation may also cause thrombotic disorders. The formation of a platelet plug at sites of atherosclerotic lesion rupture is the most common mechanism leading to myocardial or cerebral infarction. Platelet-related deep vein thrombosis is also one of the leading causes of mortality worldwide. The contribution of several platelet receptors and their ligands has been highlighted in these processes. In platelet adhesion, particularly at high shear stress, GPIbα-von Willebrand factor (VWF) interaction may initiate this event, which is followed by GPVI signalling and firm platelet adhesion mediated by members of the integrin family, such as β3 (αIIbβ3) and β1 (α2β1, α5β1) integrins. In platelet aggregation, although GPIbα-VWF, P selectin-sulfatides, and other molecules, may be involved, the process is mainly mediated by β3 (αIIbβ3) integrin and its ligands, such as fibrinogen and VWF. It is intriguing that platelet adhesion and aggregation still occur in mice lacking both fibrinogen and VWF, suggesting that other unforeseen molecule(s) may also be important in these processes. Identification and characterization of these molecules will enrich our knowledge in the basic science of hemostasis and thrombosis, and may lead to the development of new therapies against bleeding disorders and thrombotic diseases.

Platelets subvert T cell immunity against cancer via GARP-TGFβ axis.

Science.gov (United States)

Rachidi, Saleh; Metelli, Alessandra; Riesenberg, Brian; Wu, Bill X; Nelson, Michelle H; Wallace, Caroline; Paulos, Chrystal M; Rubinstein, Mark P; Garrett-Mayer, Elizabeth; Hennig, Mirko; Bearden, Daniel W; Yang, Yi; Liu, Bei; Li, Zihai

2017-05-05

Cancer-associated thrombocytosis has long been linked to poor clinical outcome, but the underlying mechanism is enigmatic. We hypothesized that platelets promote malignancy and resistance to therapy by dampening host immunity. We show that genetic targeting of platelets enhances adoptive T cell therapy of cancer. An unbiased biochemical and structural biology approach established transforming growth factor β (TGFβ) and lactate as major platelet-derived soluble factors to obliterate CD4 + and CD8 + T cell functions. Moreover, we found that platelets are the dominant source of functional TGFβ systemically as well as in the tumor microenvironment through constitutive expression of the TGFβ-docking receptor glycoprotein A repetitions predominant (GARP) rather than secretion of TGFβ per se. Platelet-specific deletion of the GARP-encoding gene Lrrc32 blunted TGFβ activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. Last, this study shows that T cell therapy of cancer can be substantially improved by concurrent treatment with readily available antiplatelet agents. We conclude that platelets constrain T cell immunity through a GARP-TGFβ axis and suggest a combination of immunotherapy and platelet inhibitors as a therapeutic strategy against cancer. Copyright © 2017, American Association for the Advancement of Science.

Potential fluid mechanic pathways of platelet activation.

Science.gov (United States)

Shadden, Shawn C; Hendabadi, Sahar

2013-06-01

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.

Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy

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Enko, Dietmar; Mangge, Harald; Münch, Andreas; Niedrist, Tobias; Mahla, Elisabeth; Metzler, Helfried; Prüller, Florian

2017-01-01

Introduction The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%. Conclusions Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced. PMID:28392742

Combined aspirin and cilostazol treatment is associated with reduced platelet aggregation and prevention of exercise-induced platelet activation.

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Cleanthis, M; Bhattacharya, V; Smout, J; Ashour, H; Stansby, G

2009-05-01

Cilostazol has proven efficacy in increasing walking distance in claudicants, but it has not been demonstrated to be more effective than placebo in secondary cardiovascular prevention. The direct effect of exercise on platelet function remains less well defined. We have investigated the effect of combination treatment with aspirin and cilostazol on platelet activity in claudicants subjected to repeated treadmill exercise. Nineteen claudicants completed a double-blind, randomised, controlled, cross-over trial. Each subject received a 2-week course of aspirin (75mg) and placebo and aspirin and cilostazol (100mg twice daily). Following each 2-week treatment period, patients participated in a standardised treadmill test (3.2kmh(-1), 10 degrees incline) walking to maximal claudication distance. The exercise was repeated thrice in total, and blood was sampled before and after exercise. Platelet activation was measured using free platelet counting aggregation, flow cytometry for surface markers of platelet activation and soluble P-selectin assay. Compared to aspirin and placebo, combination treatment with aspirin and cilostazol was associated with reduced arachidonic-acid-induced platelet aggregation (pWilcoxon signed-rank test). Aspirin and placebo treatment were associated with elevated P-selectin expression, platelet-monocyte aggregation and reduced CD42b expression (pWilcoxon signed-rank test) post-exercise. No difference was seen in spontaneous platelet aggregation whilst soluble P-selectin was reduced post-exercise with combination treatment with aspirin and cilostazol (pWilcoxon signed-rank test). Combination treatment with aspirin and cilostazol results in suppression of platelet activation and reduces the effect of exercise on platelets. The benefit seen may be a result of cilostazol enhancing the inhibitory effect of aspirin on the cyclo-oxygenase pathway.

Platelet apoptosis by cld-induced glycoportein Ibα clustering

DEFF Research Database (Denmark)

van der Wal, Dianne E; Du, V X; Lo, KS

2010-01-01

Summary Background:  Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα-distribution indicative of receptor clustering and initiates thromboxane A2-formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells...

Platelet survival in idiopathic thrombocytopenic purpura and response to splenectomy

International Nuclear Information System (INIS)

Monteiro, M.E.; Verhaeghe, R.; Devos, P.

Platelet survival combined with surface counting was performed in 9 patients with idiopathic thrombocytopenic purpura, resistent to steroid therapy. All patients had a markedly enhanced platelet turnover, five of them showed an augmented trapping of radioactivity over the spleen compared to liver and heart. These five patients underwent splenectomy: the platelet count increased in all of them but this increase was not always sustained. (Author) [pt

An investigation on platelet transport during thrombus formation at micro-scale stenosis.

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Francisco Javier Tovar-Lopez

Full Text Available This paper reports on an investigation of mass transport of blood cells at micro-scale stenosis where local strain-rate micro-gradients trigger platelet aggregation. Using a microfluidic flow focusing platform we investigate the blood flow streams that principally contribute to platelet aggregation under shear micro-gradient conditions. We demonstrate that relatively thin surface streams located at the channel wall are the primary contributor of platelets to the developing aggregate under shear gradient conditions. Furthermore we delineate a role for red blood cell hydrodynamic lift forces in driving enhanced advection of platelets to the stenosis wall and surface of developing aggregates. We show that this novel microfluidic platform can be effectively used to study the role of mass transport phenomena driving platelet recruitment and aggregate formation and believe that this approach will lead to a greater understanding of the mechanisms underlying shear-gradient dependent discoid platelet aggregation in the context of cardiovascular diseases such as acute coronary syndromes and ischemic stroke.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

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Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B; Svalgaard, Jesper D; Borup, Rehannah; Talman, Maj-Lis M; Elberg, Jens J; Kølle, Stig-Frederik T; Drzewiecki, Krzysztof T; Fischer-Nielsen, Anne

2017-02-01

Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

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Taisuke Watanabe

2017-01-01

Full Text Available Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF and concentrated growth factors (CGF according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

VacA, the vacuolating cytotoxin of Helicobacter pylori, binds to multimerin 1 on human platelets

OpenAIRE

Satoh, Kaneo; Hirayama, Toshiya; Takano, Katsuhiro; Suzuki-Inoue, Katsue; Sato, Tadashi; Ohta, Masato; Nakagomi, Junko; Ozaki, Yukio

2013-01-01

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive exper...

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

Science.gov (United States)

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

Influence of caffeine on blood pressure and platelet aggregation

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José Wilson S. Cavalcante

2000-08-01

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

Heterogeneity of rabbit platelets

International Nuclear Information System (INIS)

Karpatkin, S.

1978-01-01

Rabbits were injected intravenously with a cohort platelet label, 75 Se-selenomethionine. Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with larger platelets. The lightest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the lightest platelet fraction. The mean platelet survival of the lightest two fractions was significantly shorter than that of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction had six absent to markedly diminished platelet proteins. The data are compatible with two models, (1) heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membranes and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope that lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets. The light-smaller platelets which are released later into the circulation have a shorter survival. (author)

Platelet function in brown bear (Ursus arctos compared to man

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Särndahl Eva

2010-06-01

Full Text Available Abstract Background Information on hemostasis and platelet function in brown bear (Ursus arctos is of importance for understanding the physiological, protective changes during hibernation. Objective The study objective was to document platelet activity values in brown bears shortly after leaving the den and compare them to platelet function in healthy humans. Methods Blood was drawn from immobilized wild brown bears 7-10 days after leaving the den in mid April. Blood samples from healthy human adults before and after clopidogrel and acetylsalicylic acid administration served as control. We analyzed blood samples by standard blood testing and platelet aggregation was quantified after stimulation with various agonists using multiple electrode aggregometry within 3 hours of sampling. Results Blood samples were collected from 6 bears (3 females between 1 and 16 years old and from 10 healthy humans. Results of adenosine diphosphate, aspirin, and thrombin receptor activating peptide tests in bears were all half or less of those in humans. Platelet and white blood cell counts did not differ between species but brown bears had more and smaller red blood cells compared with humans. Conclusion Using three different tests, we conclude that platelet function is lower in brown bears compared to humans. Our findings represent the first descriptive study on platelet function in brown bears and may contribute to explain how bears can endure denning without obvious thrombus building. However, the possibility that our findings reflect test-dependent and not true biological variations in platelet reactivity needs further studies.

Concomitant nitrates enhance clopidogrel response during dual anti-platelet therapy.

Science.gov (United States)

Lee, Dong Hyun; Kim, Moo Hyun; Guo, Long Zhe; De Jin, Cai; Cho, Young Rak; Park, Kyungil; Park, Jong Sung; Park, Tae-Ho; Serebruany, Victor

2016-01-15

Despite advances in modern anti-platelet strategies, clopidogrel still remains the cornerstone of dual anti-platelet therapy (DAPT) in patients undergoing percutaneous coronary interventions (PCI). There is some inconclusive evidence that response after clopidogrel may be impacted by concomitant medications, potentially affecting clinical outcomes. Sustained released nitrates (SRN) are commonly used together with clopidogrel in post-PCI setting for mild vasodilatation and nitric oxide-induced platelet inhibition. We prospectively enrolled 458 patients (64.5 ± 9.6 years old, and 73.4% males) following PCI undergoing DAPT with clopidogrel and aspirin. Platelet reactivity was assessed by the VerifyNow™ P2Y12 assay at the maintenance outpatient setting. Concomitant SRN (n=266) significantly (p=0.008) enhanced platelet inhibition after DAPT (251.6 ± 80.9PRU) when compared (232.1 ± 73.5PRU) to the SRN-free (n=192) patients. Multivariate logistic regression analysis with the cut-off value of 253 PRU for defining heightened platelet reactivity confirmed independent correlation of more potent platelet inhibition during DAPT and use of SRN (Relative risk=1.675; Odds ratio [1.059-2.648]; p=0.027). In contrast, statins, calcium-channel blockers, beta blockers, angiotensin receptor blockers, ACE-inhibitors, diuretics, and anti-diabetic agents did not significantly impact platelet inhibition following DAPT. The synergic ability of SRN to enhance response during DAPT may have important clinical implications with regard to better cardiovascular protection, but extra bleeding risks, requiring further confirmation in a large randomized study. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mapuche herbal medicine inhibits blood platelet aggregation.

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Falkenberg, Susan Skanderup; Tarnow, Inge; Guzman, Alfonso; Mølgaard, Per; Simonsen, Henrik Toft

2012-01-01

12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM) and collagen- (2.0 μg/mL) induced aggregations in human blood. These four species in respective extracts (in brackets) were Blechnum chilense (MeOH), Luma apiculata (H(2)O), Amomyrtus luma (DCM : MeOH 1 : 1) and Cestrum parqui (DCM : MeOH 1 : 1). The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1), and L. apiculata (H(2)O) were substantial and confirmed by inhibition of platelet surface activation markers.

The life cycle of platelet granules [version 1; referees: 2 approved

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Anish Sharda

2018-02-01

Full Text Available Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types—dense granules, α-granules, and lysosomes—although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans-Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

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

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Ibtissam Youlyouz

2002-01-01

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

Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

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Zainab S Al-Hosni

2016-11-01

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.

Tyrosine phosphorylation of platelet derived growth factor β receptors in coronary artery lesions: implications for vascular remodelling after directional coronary atherectomy and unstable angina pectoris

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Abe, J; Deguchi, J; Takuwa, Y; Hara, K; Ikari, Y; Tamura, T; Ohno, M; Kurokawa, K

1998-01-01

Background—Growth factors such as platelet derived growth factor (PDGF) have been postulated to be important mediators of neointimal proliferation observed in atherosclerotic plaques and restenotic lesions following coronary interventions. Binding of PDGF to its receptor results in intrinsic receptor tyrosine kinase activation and subsequent cellular migration, proliferation, and vascular contraction.
Aims—To investigate whether the concentration of PDGF β receptor tyrosine phosphorylation obtained from directional coronary atherectomy (DCA) samples correlate with atherosclerotic plaque burden, the ability of diseased vessels to remodel, coronary risk factors, and clinical events.
Methods—DCA samples from 59 patients and 15 non-atherosclerotic left internal thoracic arteries (LITA) were analysed for PDGF β receptor tyrosine phosphorylation content by receptor immunoprecipitation and antiphosphotyrosine western blot. The amount of PDGF β receptor phosphorylation was analysed in relation to angiographic follow up data and clinical variables.
Results—PDGF β receptor tyrosine phosphorylation in the 59 DCA samples was greater than in the 15 non-atherosclerotic LITA (mean (SD) 0.84 (0.67) v 0.17 (0.08) over a control standard, p atherectomy;  restenosis PMID:9616351

In vivo detection of activated platelets allows characterizing rupture of atherosclerotic plaques with molecular magnetic resonance imaging in mice.

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Dominik von Elverfeldt

Full Text Available BACKGROUND: Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture. METHODS: An antibody targeting ligand-induced binding sites (LIBS on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE(-/- mice (60 weeks-old were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO. RESULTS: LIBS-MPIO injected animals showed a significant signal extinction (p<0.05 in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥ 2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01. CONCLUSION: in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE(-/- mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions.

Modified method for labeling human platelets with indium-111 oxine using albumin density-gradient separation

International Nuclear Information System (INIS)

Bunting, R.W.; Callahan, R.J.; Finkelstein, S.; Lees, R.S.; Strauss, H.W.

1982-01-01

When labeling platelets with indium-111 oxine, albumin density-gradient separation minimizes the time spent to resuspend those platelets that have been centrifuged against a hard surface. Labeling efficiency or platelet viability, as measured by platelet survival or aggregation with adenosine diphosphate, are not adversely affected

Mean platelet volume and mean platelet volume/platelet count ratio

African Journals Online (AJOL)

Amira M. Elsayed

2016-03-30

Mar 30, 2016 ... The aim of this study was to compare the MPV and mean platelet volume/platelet count ... brain stroke, both in the acute phase and long after disease.17 ... males, while the healthy controls comprised 12 females and 8.

Procoagulant expression in platelets and defects leading to clinical disorders.

Science.gov (United States)

Solum, N O

1999-12-01

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant

Detection of activated platelets using activation-specific monoclonal antibody (SZ-51) in clinical disorders

International Nuclear Information System (INIS)

Wu Guoxin; Li Fugang; Li Jianyong; Ruan Changgeng

1991-10-01

A direct test for activated platelets in whole blood was developed by radioimmunoassay with 125 I labeled SZ-51, an antibody specific for an α-granule membrane protein (GMP-140) that associates with the platelet surface during secretion. The assay had sufficient sensitivity to detect as few as 2% activated platelets. In 50 normal subjects, minimal GMP-140 molecules per platelet were expressed on the surface of circulating platelets. Ten patients undergoing cardiopulmonary bypass had transiently increased expression of GMP-140 molecules during the bypass procedure, especially at the end of bypass. Evaluation of 18 patients with epidemic hemorrhagic fever (EHF) has shown that the number of GMP-140 molecules on the platelet surface was closely related to the four different phases of EHF. In six patients suffered from acute myocardial infarction (AMI), the number of GMP-140 molecules changed with the procession of AMI and the highest occurred 48 h after AMI. The GMP-140 molecules were also increased in patients with asthma attack (n = 14), but not in patients with idiopathic thrombocytopenic purpura (n = 11) and diabetic mellitus (n = 48). Taken together, these studies suggest that activated platelet can be reliably measured in whole blood using radiolabeled SZ-51 antibody and the detection of activated platelets is potentially useful in identifying patients with certain thrombotic disorders and others

Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

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Singh Ravindra

2009-01-01

Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

Cloning and expression of a human kidney cDNA for an α2-adrenergic receptor subtype

International Nuclear Information System (INIS)

Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

1988-01-01

An α 2 -adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet α 2 -adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet α 2 -adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the α 2 -adrenergic ligand [ 3 H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the α 2 B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet α 2 -adrenergic receptor (α 2 A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective α-adrenergic ligands

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

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Mundell, S J; Rabbolini, D; Gabrielli, S; Chen, Q; Aungraheeta, R; Hutchinson, J L; Kilo, T; Mackay, J; Ward, C M; Stevenson, W; Morel-Kopp, M-C

2018-01-01

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is

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

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

2005-01-01

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

Mapuche Herbal Medicine Inhibits Blood Platelet Aggregation

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Susan Skanderup Falkenberg

2012-01-01

Full Text Available 12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM and collagen- (2.0 μg/mL induced aggregations in human blood. These four species in respective extracts (in brackets were Blechnum chilense (MeOH, Luma apiculata (H2O, Amomyrtus luma (DCM : MeOH 1 : 1 and Cestrum parqui (DCM : MeOH 1 : 1. The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1, and L. apiculata (H2O were substantial and confirmed by inhibition of platelet surface activation markers.

The Dynamics of Platelet Activation during the Progression of Streptococcal Sepsis.

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Sinead M Hurley

Full Text Available Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.

Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

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Adam Z. Blatt

2017-11-01

Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

The effect of the substituted amphetamines, 2.4-methylenedioxymethamphetamine (MDMA) and P-methoxyamphetamine (PMA), on platelet aggregation

International Nuclear Information System (INIS)

Sluggett, A.J.; Irvine, R.J.; Bochner, F.; Rodgers, S.; Lloyd, J.V.

2001-01-01

Full text: Illicit substituted amphetamines such as 3,4-methylenedioxymethamphetamine (MDMA) and p-methoxyamphetamine (PMA) can cause severe toxicity. Disruption of normal coagulation mechanisms have been observed in most fatal cases. However, the precise mechanisms underlying these events are not clearly understood. MDMA and PMA are known to inhibit serotonin transporter function in the central nervous system (Daws et al 2000) and platelet serotonin transporter sites (Rudnick and Wall 1992). Serotonin is in high concentrations in platelets and activation of 5HT 2 receptors on the platelet surface potentiates aggregation of platelets. Therefore, we postulated that MDMA and PMA may have effects on coagulation via inhibition of normal platelet function. Human citrated platelets were incubated in the presence of MDMA (43- 435μM) or PMA (49-498μM) and their aggregator y response to a critical dose of adenosine diphosphate (ADP) determined. These responses were compared to the serotonin reuptake inhibitor fluoxetine (13-130μM). All 3 compounds were found to inhibit platelet aggregation. The IC50s for % aggregation at 5 minutes were MDMA 197μM ± 63μM PMA 344μM ±76μM and fluoxetine 24μM ±1 1μM (n=4). The effect of these drugs on the uptake of 14 C-5HT (0.9 μM /ml) into platelets was also determined and the IC50s observed were MDMA 62.3 μM ±11μM , PMA 24μM ±6μM and fluoxetine 2.5μM ± 0.6μM (n=4). The in vitro effects of MDMA and PMA on aggregation and uptake observed here are close to concentrations reported to have occurred in human fatalities. Therefore it is possible that direct effects of these drugs on coagulation mechanisms may contribute to the toxicity of these compounds. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

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

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

2017-06-01

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 platelet count and IPC (both p-values Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 platelet turnover and COX-1 expression (all p-values platelet turnover and COX-1 and COX-2 expressions (all p-values platelet turnover in ITP patients (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

Further studies on the relationship between platelet buoyant density and platelet age

International Nuclear Information System (INIS)

Boneu, B.; Vigoni, F.; Boneu, A.; Caranobe, C.; Sie, P.

1982-01-01

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using eight discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3-4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

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Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The creation of an antithrombotic surface by apyrase immobilization

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Nilsson, Per H; Engberg, Anna E; Bäck, Jennie; Faxälv, Lars; Lindahl, Tomas L.; Nilsson, Bo; Ekdahl, Kristina Nilsson

2010-01-01

Blood incompatibility reactions caused by surfaces often involve platelet activation and subsequent platelet-initiated activation of the coagulation and complement cascades. The goal of this study was to immobilize apyrase on a biomaterial surface in order to develop an enzymatically active surface that would have the capacity to inhibit platelet activation by degrading ADP. We were able to immobilize apyrase on a polystyrene surface with preservation of the enzymatic activity. We then analyzed the hemocompatibility of the apyrase surface and of control surfaces by incubation with platelet-rich plasma (PRP) or whole blood. Monitoring of markers of platelet, coagulation, and complement activation and staining of the surfaces revealed decreased levels of platelet and coagulation activation parameters on the apyrase surface. The formation of antithrombin-thrombin and antithrombin-factor XIa complexes and the extent of platelet consumption were significantly lower on the apyrase surface than on any of the control surfaces. No significant differences were seen in complement activation (C3a levels). Staining of the apyrase surface revealed low platelet adherence and no formation of granulocyte-platelet complexes. These results demonstrate that it is possible to create an anti-thrombotic surface targeting the ADP amplification of platelet activation by immobilizing apyrase. PMID:20211488

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

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A. A. Lomonosova

2012-01-01

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

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

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Langan, Stacey A; Navarro-Núñez, Leyre; Watson, Steve P; Nash, Gerard B

2017-07-20

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Platelet biomechanics, platelet bioenergetics, and applications to clinical practice and translational research.

Science.gov (United States)

George, Mitchell J; Bynum, James; Nair, Prajeeda; Cap, Andrew P; Wade, Charles E; Cox, Charles S; Gill, Brijesh S

2018-07-01

The purpose of this review is to explore the relationship between platelet bioenergetics and biomechanics and how this relationship affects the clinical interpretation of platelet function devices. Recent experimental and technological advances highlight platelet bioenergetics and biomechanics as alternative avenues for collecting clinically relevant data. Platelet bioenergetics drive energy production for key biomechanical processes like adhesion, spreading, aggregation, and contraction. Platelet function devices like thromboelastography, thromboelastometry, and aggregometry measure these biomechanical processes. Platelet storage, stroke, sepsis, trauma, or the activity of antiplatelet drugs alters measures of platelet function. However, the specific mechanisms governing these alterations in platelet function and how they relate to platelet bioenergetics are still under investigation.

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

DEFF Research Database (Denmark)

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

2001-01-01

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

Platelet Function Tests

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... 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 ...

Development of Antifouling and Bactericidal Coatings for Platelet Storage Bags Using Dopamine Chemistry.

Science.gov (United States)

Hadjesfandiari, Narges; Weinhart, Marie; Kizhakkedathu, Jayachandran N; Haag, Rainer; Brooks, Donald E

2018-03-01

Platelets have a limited shelf life, due to the risk of bacterial contamination and platelet quality loss. Most platelet storage bags are made of a mixture of polyvinyl chloride with a plasticizer, denoted as pPVC. To improve biocompatibility of pPVC with platelets and to inhibit bacterial biofilm formation, an antifouling polymer coating is developed using mussel-inspired chemistry. A copolymer of N,N-dimethylacrylamide and N-(3-aminopropyl)methacrylamide hydrochloride is synthesized and coupled with catechol groups, named DA51-cat. Under mild aqueous conditions, pPVC is first equilibrated with an anchoring polydopamine layer, followed by a DA51-cat layer. Measurements show this coating decreases fibrinogen adsorption to 5% of the control surfaces. One-step coating with DA51-cat does not coat pPVC efficiently although it is sufficient for coating silicon wafers and gold substrates. The dual layer coating on platelet bags resists bacterial biofilm formation and considerably decreases platelet adhesion. A cationic antimicrobial peptide, E6, is conjugated to DA51-cat then coated on silicon wafers and introduces bactericidal activity to these surfaces. Time-of-flight second ion-mass spectroscopy is successfully applied to characterize these surfaces. pPVC is widely used in medical devices; this method provides an approach to controlling biofouling and bacterial growth on it without elaborate surface modification procedures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study

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Perumal, Ramesh; Rajendran, Maheashwari; Krishnamurthy, Malathi; Ganji, Kiran Kumar; Pendor, Sunil Dattuji

2014-01-01

Background: The primary etiologic factor of periodontitis is the subgingival infection with a group of Gram negative pathogens. Transient bacteremia in periodontitis patients underlie chronic production and systemic increases of various proinflammatory mediators, including Interleukin (IL)-1α, IL-6, C-reactive protein and Tumor necrosis factor (TNF)-α. P- selectin is a member of selectin family of cell surface receptor which is located in the membrane of the secretory granules (alpha granules) of platelets and in the membrane of the Weibel-Palade bodies of the vascular endothelial cells. P selectin redistributes from the membrane of the granules to the plasma membrane when platelets and endothelial cells are activated and thus degranulated. Aim: To compare the level of platelet activation, soluble P Selectin level and morphological changes and aggregation of platelets in patients in periodontitis patients compared to healthy controls. Materials and Methods: 80 patients were included in the study with the age group of 35-60. The patients were divided into 2 groups, 40 subjects with generalized chronic periodontitis and 40 healthy subjects taken as control. Periodontal Examination using clinical parameters namely, Bleeding Index, Plaque Index, Probing Pocket Depth and Clinical Attachment Level were recorded. Collection of blood samples for estimation of serum soluble P- selectin level by ELISA method. Evaluation of Platelet morphology and grading the platelet aggregation. Results: P-selectin expression shows that the mean value for control group is 4.97 ± 16.56 ng/mL and study group 13.05 ± 29.94 ng/mL which was significantly higher than control group with P value 0.001. Platelet morphological changes shows small form – mean value for control group is 75.83% ± 14.24% while for study group is 39.08%. ± 21.59; Big form – mean value for control group 0.80% ± 0.35% while for study group 0.48% ± 1.3%and Spider form- mean value for control group 23.88% ± 14

Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

DEFF Research Database (Denmark)

Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

2010-01-01

-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

Equid herpesvirus type 1 activates platelets.

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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

Difluorothromboxane A2 and stereoisomers: Stable derivatives of thromboxane A2 with differential effects on platelets and blood vessels

International Nuclear Information System (INIS)

Morinelli, T.A.; Okwu, A.K.; Mais, D.E.; Halushka, P.V.; John, V.; Chen, Chienkuang; Fried, J.

1989-01-01

The present study reports on the selective effects on human platelets and canine saphenous veins of four stable difluorinated analogues and thromboxane A 2 (TXA 2 ), in which the characteristic 2,6-dioxa[3.1.1]bicycloheptane structure of TXA 2 has been retained. The four compounds differ in their stereochemistry of the 5,6 double bond and/or the 15-hydroxyl group. Only 10,10-difluoro-TXA 2 (compound I) with the natural stereochemistry of TXA 2 was an agonist in both platelets and canine saphenous veins. (15R)-10,10-Difluoro-TXA 2 (compound II), (5E)-10,10-difluoro-TXA 2 (compound III), and (5E,15R)-10,10-difluoro-TXA 2 (compound IV) were antagonists of platelet aggregation stimulated by compound I. However, compounds II, III, and IV stimulated contraction of canine saphenous veins. All four compounds could displace the TXA 2 /prostaglandin H 2 antagonist 9,11-dimethylmethano-11,12-methano-16-(3- 125 I-4-hydroxyphenyl)-13,14-dihydro-13-aza-15αβ-ω-tetranor-TXA 2 from its platelet receptor. These results support the existence of two subtypes of TXA 2 /prostaglandin H 2 receptors and emphasize the importance of the stereochemical requirements of these TXA 2 analogues for interaction with these receptors. These stable fluorinated TXA 2 analogues should prove useful tools for the further characterization of these and other TXA 2 /prostaglandin H 2 receptors

Analysis of angiotensin II binding to human platelets: Differences in young and old subjects

International Nuclear Information System (INIS)

Siebers, M.J.; Goodfriend, T.L.; Ball, D.; Elliott, M.E.

1990-01-01

We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125 I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125 I-AII alone, with 125 I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125 I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects

Evaluation of amotosalem treated platelets over 7 days of storage with an automated cytometry assay panel.

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Diquattro, M; De Francisci, G; Bonaccorso, R; Tagliavia, A M; Marcatti, M; Palma, B; Agliastro, R

2013-12-01

Pathogen Inactivation allows to overcome microbial contamination and growth related to storage of platelets concentrates (PC) at room temperature. The aim of our study was to evaluate the platelet storage lesion extending the storage period of pathogen inactivated platelet concentrates over 7 days using an automated cytometry assay panel. We analyzed 43 concentrates subjected to pathogen inactivation (CPPI) at 3, 5 and 7 days evaluating: platelet count, mean platelet volume, platelets at low optical density, platelets at high density, GPIIb-IIIa glycoprotein, platelet microparticles, lactate dehydrogenase. The collection bags (Fenwal) and the IBS kit made in PL2410/PL2411 are approved for the conservation of PC up to 7 days. Data analysis was performed with anova test. All the parameters except small platelets and PMP were statistically different among day 7 vs. 3 and day 7 vs. 5. Our study showed a progressive modification of pathogen inactivated platelet concentrates observed up to 7 days. The persistence of the secretory pool and the presence of the platelet membrane fibrinogen receptor suggest the persistence of a potential hemostatic efficacy. Clinical studies are necessary to directly correlate this type of analysis to 24 h recovery or survival of transfused platelets in humans. © 2013 John Wiley & Sons Ltd.

Nanodiamonds activate blood platelets and induce thromboembolism.

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Kumari, Sharda; Singh, Manoj K; Singh, Sunil K; Grácio, José J A; Dash, Debabrata

2014-03-01

Nanodiamonds (NDs) have been evaluated for a wide range of biomedical applications. Thus, thorough investigation of the biocompatibility of NDs has become a research priority. Platelets are highly sensitive and are one of the most abundant cell types found in blood. They have a central role in hemostasis and arterial thrombosis. In this study, we aim to investigate the direct and acute effects of carboxylated NDs on platelet function. In this study, pro-coagulant parameters such as platelet aggregability, intracellular Ca(2+) flux, mitochondrial transmembrane potential (ΔΨm), generation of reactive oxygen species, surface exposure of phosphatidylserine, electron microscopy, cell viability assay and in vivo thromboembolism were analyzed in great detail. Carboxylated NDs evoked significant activation of human platelets. When administered intravenously in mice, NDs were found to induce widespread pulmonary thromboembolism, indicating the remarkable thrombogenic potential of this nanomaterial. Our findings raise concerns regarding the putative biomedical applications of NDs pertaining to diagnostics and therapeutics, and their toxicity and prothrombotic properties should be critically evaluated.

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

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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.

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor

International Nuclear Information System (INIS)

Thorsen, L.I.; Brosstad, F.; Gogstad, G.; Sletten, K.; Solum, N.O.

1986-01-01

Direct binding of 125 -I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule

Influence of nitriding atmosphere on the modification of surface titanium with focus on the behavior of blood platelets adhesion; Influencia da atmosfera nitretante na modificacao de superficies de titanio com enfase no comportamento de adesao de plaquetas sanguineas

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Vitoriano, J.O.; Alves, C. [Universidade Federal Rural do Semi-Arido (UFERSA), RN (Brazil); Braz, D.C.; Camara, R.B.G.; Rocha, H.A.O., E-mail: clodomiro.jr@hotmail.com [Universidade Federal do Rio Grande do Norte (UFRN), RN (Brazil)

2014-07-01

The present study aimed to analyze the influence of surface modification of titanium on the adhesion of blood platelets, through techniques of adhesion and morphological analyzes. Discs of titanium grade II received different surface treatments with plasma of Ar + N{sub 2} + H{sub 2} and Ar + H{sub 2}, forming two experimental groups including only polished samples used as standard. Before and after treatment the samples were characterized according to topography, crystalline structure and wettability, using atomic force microscopy, X-ray diffraction, Raman spectroscopy and testing of sessile drop, respectively. Platelet rich plasma (PRP) was applied on the modified surfaces in a culture plates. Images obtained by electron microscopy of adhered platelets were analyzed to verify the behavior of platelets in the different experimental conditions. (author)

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

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He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

Effects of epinephrine on ADP-induced changes in platelet inositol phosphates

International Nuclear Information System (INIS)

Vickers, J.D.; Keraly, C.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

1986-01-01

Epinephrine (EPI) does not aggregate rabbit platelets, but it does increase the labelling of inositol phosphate (IP) at 60s (21%, p + , in platelets prelabelled with [ 3 H] inositol. In contrast, 0.5 μM ADP which causes aggregation, increases the labelling of inositol bisphosphate (IP 2 ) by 30% (p 2 by 154% (p 2 stimulated by ADP + EPI was greater than the increase caused by ADP (p 2 due to 0.2 μM ADP + 0.6 μM EPI by 70% (p 2 by 108% (0 2 metabolism stimulated via the α-adrenergic receptor

[Platelet function in acute myeloid leukemia. II. Aggregation of isolated platelets].

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Zawilska, K; Komarnicki, M; Mańka, B

1978-01-01

In 22 patients with acute myeloid leukaemia (17 cases of myeloblastic leukaemia, 4 cases of myelomonocytic leukaemia and 1 case of undifferentiated-cell leukaemia) platelets were isolated from the plasma by the method of Nicholls and Hampton as modified by Levy-Toledano by centrifugation in albumin gradient. The aim of platelet isolation was their "concentration" in cases of thrombocytopenia to values making possible aggregation tests, and platelet separation from the influence of plasma factors. Then aggregation of isolated platelets caused by ADP was studied. In 16 out of 22 patients a fall of aggregation was observed, with the mean values of aggregation rate and intensity were significantly lower. Parallelly done determinations of aggregating activity released from the platelets by thrombin showed lower values as compared with platelets from healthy subjects. In might be thought, in this connection, that the demonstrated reduction of isolated platelets is associated with a diminution of the nucleotide pool or disturbances of the platelet release reaction. The disturbances of the platelet release reaction. The disturbances of aggregation of isolated platelets and reduction of the aggregating activity were most pronounced in acute myelomonocytic leukaemia.

Platelet-derived growth factor receptor-β, carrying the activating mutation D849N, accelerates the establishment of B16 melanoma

International Nuclear Information System (INIS)

Suzuki, Shioto; Heldin, Carl-Henrik; Heuchel, Rainer Lothar

2007-01-01

Platelet-derived growth factor (PDGF)-BB and PDGF receptor (PDGFR)-β are mainly expressed in the developing vasculature, where PDGF-BB is produced by endothelial cells and PDGFR-β is expressed by mural cells, including pericytes. PDGF-BB is produced by most types of solid tumors, and PDGF receptor signaling participates in various processes, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. Furthermore, PDGF-BB-producing tumors are characterized by increased pericyte abundance and accelerated tumor growth. Thus, there is a growing interest in the development of tumor treatment strategies by blocking PDGF/PDGFR function. We have recently generated a mouse model carrying an activated PDGFR-β by replacing the highly conserved aspartic acid residue (D) 849 in the activating loop with asparagine (N). This allowed us to investigate, in an orthotopic tumor model, the role of increased stromal PDGFR-β signaling in tumor-stroma interactions. B16 melanoma cells lacking PDGFR-β expression and either mock-transfected or engineered to express PDGF-BB, were injected alone or in combination with matrigel into mice carrying the activated PDGFR-β (D849N) and into wild type mice. The tumor growth rate was followed and the vessel status of tumors, i.e. total vessel area/tumor, average vessel surface and pericyte density of vessels, was analyzed after resection. Tumors grown in mice carrying an activated PDGFR-β were established earlier than those in wild-type mice. In this early phase, the total vessel area and the average vessel surface were higher in tumors grown in mice carrying the activated PDGFR-β (D849N) compared to wild-type mice, whereas we did not find a significant difference in the number of tumor vessels and the pericyte abundance around tumor vessels between wild type and mutant mice. At later phases of tumor progression, no significant difference in tumor growth rate was

Involvement of nuclear factor κB in platelet CD40 signaling

International Nuclear Information System (INIS)

Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

2012-01-01

Highlights: ► sCD40L induces TRAF2 association to CD40 and NF-κB activation in platelets. ► IκBα phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. ► IκBα is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

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Alexey Navdaev

Full Text Available von Willebrand factor/ristocetin (vWF/R induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

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Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

2014-01-01

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

Calcium-binding proteins from human platelets

International Nuclear Information System (INIS)

Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

1983-01-01

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

NARCIS (Netherlands)

Fijnheer, R.; Pietersz, R. N.; de Korte, D.; Gouwerok, C. W.; Dekker, W. J.; Reesink, H. W.; Roos, D.

1990-01-01

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from

Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

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Kissopoulou, Antheia; Jonasson, Jon; Lindahl, Tomas L.; Osman, Abdimajid

2013-01-01

Background Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. Results Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. Conclusion The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and

Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

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Antheia Kissopoulou

Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

Overview of platelet physiology and laboratory evaluation of platelet function.