Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 186-191 ◽  
Author(s):  
Gillian Stephens ◽  
Yibing Yan ◽  
Martine Jandrot-Perrus ◽  
Jean-Luc Villeval ◽  
Kenneth J. Clemetson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Immune Modulation ◽  
Soluble Fraction ◽  
Platelet Reactivity ◽  
Cleavage Product ◽  
Platelet Surface ◽  
Variable Expression ◽  
Collagen Receptor ◽  
Stimulation Of

Abstract Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI–specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.

Glycoprotein (GP) VI Is Associated with GPIb-IX-V on the Membrane of Resting and Activated Platelets.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1553-1553
Author(s):  
Michael C. Berndt ◽  
Jane F. Arthur ◽  
Elizabeth E. Gardiner ◽  
Yukio Ozaki ◽  
Mark L. Kahn ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Soluble Fraction ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Low Shear Stress ◽  
Pre Treatment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Collagen Receptor ◽  
Low Shear

Abstract The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIbα-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIbα from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association in resting or activated platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIbα and GPVI, since soluble fragments of GPIbα (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

scholarly journals Platelet activation in cystic fibrosis

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4635-4641 ◽  
Author(s):  
Brian P. O'Sullivan ◽  
Matthew D. Linden ◽  
Andrew L. Frelinger ◽  
Marc R. Barnard ◽  
Michele Spencer-Manzon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gene Encoding ◽  
Platelet Surface ◽  
Beneficial Effects ◽  
Plasma Factor

Abstract Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E1 (PGE1). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF. (Blood. 2005;105:4635-4641)

Platelet Activation and Aggregation in Patients with Advanced Adenocarcinoma Undergoing Chemotherapy: Correlation with a Validated Venous Thromboembolism Risk Score

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3445-3445 ◽  
Author(s):  
Arnab Basu ◽  
Rohit Gosain ◽  
Udaya Tantry ◽  
Kenneth Miller ◽  
Paul A Gurbel
Keyword(s):  
Arachidonic Acid ◽  
Risk Factor ◽  
Venous Thromboembolism ◽  
Platelet Aggregation ◽  
Cancer Patients ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Risk Scores ◽  
Platelet Surface ◽  
Thrombotic Risk

Abstract Background: Cancer is known to increase risk of venous thromboembolism (VTE), which is associated with considerable mortality and morbidity. Chemotherapy is an independent risk factor for thrombosis in cancer patients. We examined platelet activation and reactivity based on the Khorana Score, a validated scoring model for VTE risk in patients receiving chemotherapy. Materials and Methods: Patients (n=25) with advanced stage adenocarcinomas (TNM stage III or greater), were enrolled in this study. Patients on antiplatelet therapy or on anticoagulation were excluded. Approximately 10 ml of Citrated blood samples were collected from central access catheters after discarding initial 5 ml to minimize iatrogenic platelet activation. 1μMol, 2μMol, 5μMol of ADP-induced, 2μMol Arachidonic Acid (AA)-induced and 4μMol Collagen-induced platelet aggregation in Platelet Rich plasma (PRP) was assessed with a light transmission aggregometry assay. Maximal aggregation and aggregation velocity were recorded. Concurrent flowcytometry analysis was done to assess the expressions of CD41 (Glycoprotein GpIIb), CD62p (platelet surface p-Selectin), and PAC-1(Activated GP IIb/IIIa). Thromboembolic risk scores (Khorana Scores, KS) were calculated based on a validated scoring system (Khorana AA, et al. Blood. 2008;111:4902-7) as follows: Table 1.Very high risk cancers (pancreatic or gastric)+2 pointsHigh risk cancers (lung, ovarian, or bladder cancer)+1 pointPlatelet count ≥350 x 109/L+1 pointHemoglobin < 10 gm/dL or use of erythropoietin+1 pointLeukocytosis >11 x 109/L+1 pointBMI > 35+1 point Results: Mean values for maximum platelet aggregation were calculated by groups and compared between patient with KS < 3 and KS > = 3 by two sample t-tests. Differences were statistically significant for all concentrations of ADP and collagen, with a positive trend for Arachidonic acid (Table). A significant linear relationship between maximal platelet aggregation and higher KS was observed. However there were no significant differences observed in the expression of platelet surface p-selectin, CD41 or PAC-1 when comparing patients at Khorana scores 0, 3 or more than 3 (not shown). Table 2.Agonist ConcentrationKhorana Score <3Khorana Score >= 3P valueADP 1μMol20.8%28.14%0.02ADP 2μMol38.6%67%0.002ADP 5μMol66.5%79.85%0.006Arachidonic acid 2μMol61.6%73.85%0.12Collagen 4μMol78.6%90.14%0.01 Discussion: The mechanism of cancer-related thromboembolism is not well understood. Chemotherapy is an added risk factor for the development of VTEs. Several new markers such as soluble p-selectin and mean platelet volume have been investigated as adjunct factors to improve predictive ability. Although our study did not find a correlation of some common platelet surface markers for activation with thrombotic risk scores; we find a strong positive correlation of future thrombotic risk derived via Khorana scores with heightened platelet reactivity. Platelet reactivity may mediate a final common pathway for venous thromboembolism in cancer patients and should be validated further as an adjunctive marker in a large scale study. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Antiplatelet Treatment with Aspirin (ASA) on Platelet Reactivity in Patients with Essential Thrombocythemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4253-4253
Author(s):  
Marina Marchetti ◽  
Sara Gamba ◽  
Cinzia Giaccherini ◽  
Cristina Verzeroli ◽  
Laura Russo ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Essential Thrombocythemia ◽  
Platelet Reactivity ◽  
Venous Blood ◽  
Myeloproliferative Neoplasm ◽  
Platelet Surface ◽  
Antithrombotic Prophylaxis ◽  
Mutational Status ◽  
Induced Aggregation

Abstract INTRODUCTION: Essential Thrombocythemia (ET) is a myeloproliferative neoplasm characterized by an increased rate of thrombotic complications. Antithrombotic prophylaxis with aspirin (ASA), alone or in combination with cytoreduction with hydroxyurea (HU), is widely utilized in ET patients. However, thrombosis occurrence/recurrence in spite of antithrombotic prophylaxis remains a relevant issue. Growing data support the possible contribution to this failure of the inter-individual variability of pharmacological ASA response. AIM: Aim of this study was to characterize, in a group of ET patients receiving 100 mg/d ASA, the platelet reactivity in terms of platelet aggregation and activation properties. MATERIALS AND METHODS: Venous blood samples were obtained from 77 ET patients (26M/51F), and two control groups, i.e., one including 72 non-ET patients receiving chronic ASA prophylaxis, and the other including 111 healthy control subjects (57M/54F). The mutational status of ET was: 35 patients were JAK2V617F⁺, 22 CALR⁺, 3 MPL⁺, and 17 triple negative. Thirty-three ET patients were on ASA+HU, 23 on ASA alone, 5 on HU alone, and 16 were not receiving any of these drugs. Platelet aggregation was assessed in whole blood by the Multiplate® analyzer (Roche). The platelet response to the thrombin receptor activating peptide (TRAP) trigger was the measure of the overall platelet aggregation capacity, while the response to the arachidonic acid (AA) trigger was the measure of ASA effect on platelet aggregation. A normalized AA-induced aggregation (r-AA-agg), defined as AA/TRAP ratio, was calculated for each sample to reflect the individual variation of platelet inhibition by ASA. The platelet activation status was evaluated before and after aggregation by measuring the surface expression of CD62P (P-selectin) by flow cytometry (Accuri™ C6, BD Bioscience). RESULTS: The analysis of subgroups according to treatments shows that AA-induced platelet aggregation in ASA- and ASA+HU-treated ET patients was significantly lower compared to non-ASA ET subjects (p<0.001), and was significantly greater compared to ASA-treated non-ET patients (145±85 AU; p<0.001). The same results were observed with TRAP-induced platelet aggregation. Accordingly, the r-AA-agg. was greater in ET subjects on ASA (=53%) or ASA+HU (=50%) as compared to non-ET ASA-treated individuals (=19%). Furthermore, among ET patients on ASA±HU, those with platelet >450x109/L showed AA-induced aggregation significantly greater than subjects with platelet <450x109/L. The increment of platelet surface CD62P expression after AA stimulation (as a marker of platelet activation) was not influenced by anti-platelet therapy, but was significantly associated with JAK2V617F mutation. CONCLUSIONS: Our data show that in more than 70% of ET patients, in spite of ASA intake, the platelet reactivity remains higher than in non-ET patients receiving the same drug regimen. This phenomenon, together with the so-called "turnover" resistance, i.e. increased platelet turnover associated to short aspirin half-life, may contribute to aspirin failure in ET. Studies are necessary to evaluate the efficacy and safety of a different dose or timing of ASA administration in these patients. Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.). Disclosures Falanga: Pfizer: Speakers Bureau; Aspen: Speakers Bureau; Janssen: Speakers Bureau.

The Effect of Heparin vs. Citrate on the Interaction of Platelets with Vascular Graft Materials

1985 ◽  
Vol 54 (04) ◽  
pp. 842-848 ◽  
Author(s):  
Kandice Kottke-Marchant ◽  
James M Anderson ◽  
Albert Rabinovitch ◽  
Richard A Huskey ◽  
Roger Herzig
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Vascular Graft ◽  
Time Course ◽  
Platelet Reactivity ◽  
Polymer Surfaces ◽  
In Vitro Perfusion ◽  
Citrate Anticoagulation ◽  
Platelet Release

SummaryHeparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

scholarly journals Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713 ◽  
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

scholarly journals Investigation of the mechanisms of monoclonal antibody-induced platelet activation

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1019-1026 ◽  
Author(s):  
P Horsewood ◽  
CP Hayward ◽  
TE Warkentin ◽  
JG Kelton
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Binding Sites ◽  
Fc Receptors ◽  
Secondary Antibody ◽  
Igg Antibodies ◽  
Platelet Surface ◽  
Platelet Protein ◽  
Platelet Binding ◽  
Protein Components

Abstract Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).

A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen

2003 ◽  
Vol 89 (06) ◽  
pp. 996-1003 ◽  
Author(s):  
Jun Mizuguchi ◽  
Sachiko Kawashima ◽  
Michiko Nagamatsu ◽  
Yoshiki Miura ◽  
Tomohiro Nakagaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Collagen Binding ◽  
Glycoprotein Vi ◽  
Phosphorylation Of Proteins ◽  
Dimeric Form ◽  
Collagen Receptor

SummaryThe newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient’s auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.

Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 111-111 ◽  
Author(s):  
Meghna Ulhas Naik ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
Ulhas Pandurang Naik
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Adhesion Molecule ◽  
Resting State ◽  
Human Platelets ◽  
Artery Occlusion ◽  
In Vivo Model ◽  
Platelet Surface ◽  
Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

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