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

The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cγ2 through a pathway that is dependent on the Fc receptor γ (FcR γ) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR γ chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin α2β1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR γ chain. This was not true of the other platelet Src-family kinases. The association between the FcR γ chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR γ chain phosphorylation.

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Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽

10.1182/blood.v104.11.3541.3541 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3541-3541

Author(s):

Swaminathan Murugappan ◽

Haripriya Shankar ◽

Satya Kunapuli

Keyword(s):

Tyrosine Kinase ◽

Intracellular Calcium ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Human Platelets ◽

Dependent Manner ◽

Protein Signaling ◽

Glycoprotein Vi ◽

Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

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A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

Blood ◽

10.1182/blood.v106.11.1648.1648 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1648-1648

Author(s):

Ilaria Canobbio ◽

Lucia Stefanini ◽

Gianni F. Guidetti ◽

Cesare Balduini ◽

Mauro Torti

Keyword(s):

Platelet Aggregation ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Lipid Rafts ◽

Platelet Activation ◽

Human Platelets ◽

G Protein Coupled Receptors ◽

Weak Stimulation ◽

High Concentrations ◽

G Protein Coupled

Abstract The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

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Critical role of Src-Syk-PLCγ2 signaling in megakaryocyte migration and thrombopoiesis

Blood ◽

10.1182/blood-2010-03-275990 ◽

2010 ◽

Vol 116 (5) ◽

pp. 793-800 ◽

Cited By ~ 38

Author(s):

Alexandra Mazharian ◽

Steve G. Thomas ◽

Tarvinder S. Dhanjal ◽

Christopher D. Buckley ◽

Steve P. Watson

Keyword(s):

Kinase Inhibitor ◽

Tyrosine Phosphatase ◽

Critical Role ◽

Src Family Kinases ◽

Surface Glycoprotein ◽

Platelet Recovery ◽

Glycoprotein Vi ◽

Syk Kinase ◽

And Migration

Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signaling proteins in regulating MK migration and platelet recovery after immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow–derived MKs on a fibronectin matrix are abolished by the Src family kinases inhibitor PP1, the Syk kinase inhibitor R406 and the integrin αIIbβ3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary phospholipase C γ2 (PLCγ2)–deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the glycoprotein VI–FcRγ-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of platelet endothelial cell adhesion molecule-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCγ2. This study identifies a model in which integrin signaling via Src family kinases and Syk kinase to PLCγ2 is required for MK spreading, migration, and platelet formation.

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Role of Fc receptor γ-chain in platelet glycoprotein Ib–mediated signaling

Blood ◽

10.1182/blood.v97.12.3836 ◽

2001 ◽

Vol 97 (12) ◽

pp. 3836-3845 ◽

Cited By ~ 88

Author(s):

Yi Wu ◽

Katsue Suzuki-Inoue ◽

Kaneo Satoh ◽

Naoki Asazuma ◽

Yutaka Yatomi ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Calcium Release ◽

Human Platelets ◽

Fc Receptor ◽

Platelet Glycoprotein ◽

Wild Type ◽

Glycoprotein Ib ◽

Von Willebrand ◽

Willebrand Factor

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor γ-chain (FcR γ-chain), Syk, linker for activation of T cells (LAT), and phospholipase C γ2 (PLCγ2). Pretreatment of platelets with 10 μM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR γ-chain and Syk, suggesting that Src and Lyn are involved in FcR γ-chain tyrosine phosphorylation and downstream signals. In spite of the PLCγ2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR γ-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR γ-chain–deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCγ2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR γ-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR γ-chain–deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR γ-chain is required for the full activation of platelets mediated by GPIb.

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The Cytoplasmic Tail of GPIbß Modulates Signalling Induced by GPIb/VWF Interactions under High Shear Conditions.

Blood ◽

10.1182/blood.v104.11.1563.1563 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1563-1563

Author(s):

Kenneth Martin ◽

Shuju Feng ◽

Michael H. Kroll ◽

Dermot Kenny

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Calcium Influx ◽

Cytoplasmic Tail ◽

Surface Expression ◽

Inhibitory Effect ◽

Platelet Glycoprotein ◽

Before And After ◽

Induced Aggregation

Abstract The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation. The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above. The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation. RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent. These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.

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LAT Is Required for Tyrosine Phosphorylation of Phospholipase Cγ2 and Platelet Activation by the Collagen Receptor GPVI

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8326 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8326-8334 ◽

Cited By ~ 136

Author(s):

Jean-Max Pasquet ◽

Barbara Gross ◽

Lynn Quek ◽

Naoki Asazuma ◽

Weiguo Zhang ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Human Platelets ◽

Major Pathway ◽

Glycoprotein Vi ◽

Fibrinogen Receptor ◽

Protein Receptor ◽

Thrombin Activation ◽

Granule Secretion ◽

A Minor ◽

Collagen Receptor

ABSTRACT In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cγ2 (PLCγ2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcγRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCγ2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCγ2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin αIIbβ3 in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCγ2, leading to downstream responses such as α-granule secretion and activation of integrin αIIbβ3. The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCγ2. We propose a model in which LAT and SLP-76 are required for PLCγ2 phosphorylation but are regulated through independent pathways downstream of Syk.

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Genetic and Pharmacological Analyses of Involvement of Src-family, Syk and Btk Tyrosine Kinases in Platelet Shape Change

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615689 ◽

2001 ◽

Vol 85 (02) ◽

pp. 331-340 ◽

Cited By ~ 20

Author(s):

Markus Bauer ◽

Petra Maschberger ◽

Lynn Quek ◽

Stephen Briddon ◽

Debabrata Dash ◽

...

Keyword(s):

Tyrosine Kinases ◽

Shape Change ◽

Thromboxane A2 ◽

Human Platelets ◽

Src Family Kinases ◽

G Protein Coupled Receptors ◽

Protein Tyrosine Phosphorylation ◽

G Protein Coupled ◽

Platelet Shape ◽

Stimulation Of

SummaryPlatelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADPand thromboxane A2-G-protein coupled receptors in human platelets and thromboxane A2 receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin αIIb β3 complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin αIIb β3 exposure during shape change.

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Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins

Blood ◽

10.1182/blood-2003-07-2392 ◽

2004 ◽

Vol 103 (6) ◽

pp. 2088-2095 ◽

Cited By ~ 138

Author(s):

Angel García ◽

Sripadi Prabhakar ◽

Sascha Hughan ◽

Tom W. Anderson ◽

Chris J. Brock ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Proteome Analysis ◽

Human Platelets ◽

Rgs Proteins ◽

Protein Signaling ◽

Glycoprotein Vi ◽

Activated Platelets ◽

Tyrosine Kinase 2 ◽

Proteomics Approach ◽

Thrombotic Diseases

Abstract We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, αIIbβ3. These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic diseases.

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Human Atheromatous Plaques Stimulate Thrombus Formation by Activating Platelet Glycoprotein VI.

Blood ◽

10.1182/blood.v104.11.2623.2623 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2623-2623

Author(s):

Armin J. Reininger ◽

Richard Brandl ◽

Sandra Penz ◽

Pankaj Goyal ◽

Tamer Rabie ◽

...

Keyword(s):

Tissue Factor ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Initial Step ◽

Dense Granule ◽

Platelet Glycoprotein ◽

Atherosclerotic Plaques ◽

Glycoprotein Vi ◽

Atheromatous Plaques ◽

Dense Granule Secretion

Abstract Lipid-rich atherosclerotic plaques are vulnerable, and upon disruption trigger intraarterial thrombus formation. Tissue factor activating blood coagulation is viewed as the major prothrombotic stimulus within the plaque. We isolated lipid-rich atheromatous plaques from 50 patients with carotid artery stenosis and identified morphologically diverse collagenous structures within in the plaques. They stimulated platelet adhesion, dense granule secretion and aggregation, and triggered thrombus formation in hirudin-anticoagulated blood under arterial flow conditions. Even in fully anticoagulated flowing blood, i.e. in the absence of tissue factor-mediated coagulation, plaques were able to activate platelets. Thrombus formation was more rapid and stable when blood was anticoagulated with a low concentration of heparin, but, although fibrin was detectable within the thrombus, the initial step was always single platelet adhesion and not fibrin formation. In contrast, absence or inhibition of the platelet collagen receptor glycoprotein VI prevented platelet adhesion to atheromatous plaques and thrombus formation. We thus identified platelet glycoprotein VI as being essential and sufficient to mediate plaque-induced thrombus formation. Our study suggests a novel anti-thrombotic strategy to prevent and treat atherothrombosis in patients with vulnerable atherosclerotic plaques. Figure Figure

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Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C

Blood ◽

10.1182/blood.v100.10.3561 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3561-3569 ◽

Cited By ~ 39

Author(s):

Andrew C. Pearce ◽

Jonathan I. Wilde ◽

Gina M. Doody ◽

Denise Best ◽

Osamu Inoue ◽

...

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

G Protein ◽

Cell Types ◽

Human Platelets ◽

Glycoprotein Vi ◽

Activation Motif ◽

G Protein Coupled ◽

Collagen Receptor

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)–coupled collagen receptor GPVI and by the G protein–coupled receptor agonist thrombin. The glycoprotein VI (GPVI)–specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

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