Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen

Platelet responses to collagen are mediated by the combined actions of the integrin alpha2beta1, which serves as a major collagen-binding receptor, and the GPVI/FcRgamma-chain complex, which transmits collagen-specific activation signals into the cell interior through the action of an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain of the FcRgamma-chain. Despite much progress in identifying components of the signaling pathway responsible for collagen-induced platelet activation, virtually nothing is known about the regulatory elements that modulate this important hemostatic event. PECAM-1, a recently recognized member of the inhibitory receptor family, contains a functional immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain that, when tyrosine phosphorylated, recruits and activates the protein-tyrosine phosphatase, SHP-2. To test the hypothesis that PECAM-1 functions to regulate GPVI/FcRgamma-chain-mediated platelet activation, the responses of wild-type versus PECAM-1-deficient murine platelets to GPVI-specific agonists were compared. Four distinct GPVI/FcRgamma-chain-dependent responses were found to be significantly exaggerated in platelets derived from PECAM-1-deficient mice, including Mg++-independent adhesion to immobilized fibrillar collagen, collagen-induced platelet aggregation, platelet aggregation induced by the GPVI-specific agonist collagen-related peptide, and GPVI/FcRgamma-chain-induced dense granule secretion. Together, these data provide compelling evidence that PECAM-1 modulates platelet responses to collagen, and they implicate this novel member of the inhibitory receptor family in the regulation of primary hemostasis.     

Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents

GPVI is a 62-kDa membrane glycoprotein expressed in noncovalent association with the Fc receptor gamma chain on human and murine platelets and serves as the major activating receptor for collagen. GPVI-specific antibodies have the capacity to specifically deplete GPVI from mouse and human platelets in vivo, rendering them unresponsive to collagen and GPVI-specific agonists. Such antibodies do not remove GPVI from noncirculating platelets in vitro, however, making it difficult to evaluate their antithrombotic potential and mechanism of action, particularly in human platelets. We devised a model system in which human platelets are introduced into the retroorbital plexus of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, allowed to circulate, and evaluated for the effects of GPVI-specific murine monoclonal antibodies (mAbs) on platelet survival and function. GPVI-specific mAbs triggered depletion of GPVI from human, but not murine, platelets. Soluble truncated human GPVI appeared concomitantly in mouse plasma. GPVI-depleted human platelets had markedly diminished responses to GPVI-specific agonists and unexpectedly exhibited somewhat depressed responses to G-protein-coupled agonists. The ability to evaluate in living mice the in vivo function and survival of circulating human platelets may prove valuable for determining mechanisms of antibody-mediated platelet passivation and aid in the development of novel anti-platelet therapeutics.     

Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRgamma-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRgamma-chain complex (FcRgamma(-/-)). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRgamma is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRgamma was associated with a 30% reduction in thrombus growth. Analysis of FcRgamma(-/-) platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRgamma deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRgamma compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRgamma(-/-) mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRgamma deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.     

Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen

The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRgamma. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76(-/-)) or heterozygous (SLP-76(+/-)) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76(+/-) platelets, but not SLP-76(-/-) platelets (P <.01), and failed to stimulate annexin V binding to either SLP-76(+/-) or SLP-76(-/-) platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76(+/-) or SLP-76(-/-) platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76(+/-) and SLP-76(-/-) platelets (P <.001 versus unstimulated platelets). Similar results were obtained with platelets from FcRgamma null mice, for which collagen, but not convulxin, induced procoagulant activity (P <.01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76(+/-) platelets (P <.05), but not in SLP-76(-/-) platelets. SLP-76(-/-) platelets also exhibited less annexin V binding than SLP-76(+/-) platelets after costimulation with thrombin and convulxin (P <.05). These findings demonstrate that an intact GPVI/FcRgamma/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.     

A familial platelet function disorder associated with abnormal signalling through the glycoprotein VI pathway

The platelet collagen receptor, glycoprotein (GP)VI, of the immunoreceptor family forms a complex with the von Willebrand factor (VWF) receptor, GPIb-IX-V, critical for initiating thrombus formation. GPVI is co-associated with Fc receptor gamma-chain (FcRgamma), which contains a cytoplasmic immunoreceptor tyrosine-based activation motif domain, involved in activation of Syk, and a signalling cascade leading to (i) activation of alpha(IIb)beta(3), which binds VWF and fibrinogen and mediates platelet aggregation, and (ii) metalloproteinase-mediated shedding of the GPVI ectodomain (blocked by Syk inhibitors), a key mechanism for regulating GPVI surface expression. In this study, we report a familial case of abnormal platelet aggregation with dysfunctional signalling through GPVI that uniquely demonstrates divergent alpha(IIb)beta(3)-activating and GPVI-shedding pathways. The patient is a 60-year-old female with a history of immune disorders, excessive bleeding from childhood and a life-threatening haemorrhage post-trauma. Platelet aggregation to ADP, thrombin receptor-agonist peptide or ristocetin/VWF was normal (indicating normal expression and function of alpha(IIb)beta(3)), but platelet aggregation to GPVI agonists, collagen, collagen-related peptide, or convulxin, was defective. Both GPVI/FcRgamma expression and ligand-induced GPVI ectodomain shedding were normal, confirming expression of functional GPVI/FcRgamma, but suggesting a signalling defect downstream of Syk. A genetic defect in GPVI/Fcgamma signalling compromising platelet function is hypothesised in this family.     

Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation

Collagen-induced platelet aggregation is a complex process and involves synergistic action of integrins, immunoglobulin (Ig)-like receptors, G-protein-coupled receptors and their ligands, most importantly collagen itself, thromboxane A(2) (TXA(2)), and adenosine diphosphate (ADP). The precise role of each of these receptor systems in the overall processes of activation and aggregation, however, is still poorly defined. Among the collagen receptors expressed on platelets, glycoprotein (GP) VI has been identified to play a crucial role in collagen-induced activation. GPVI is associated with the FcRgamma chain, which serves as the signal transducing unit of the receptor complex. It is well known that clustering of GPVI by highly specific agonists results in platelet activation and irreversible aggregation, but it is unclear whether collagen has the same effect on the receptor. This study shows that platelets from Galphaq-deficient mice, despite their severely impaired response to collagen, normally aggregate on clustering of GPVI, suggesting this not to be the principal mechanism by which collagen activates platelets. On the other hand, dimerization of GPVI by a monoclonal antibody (JAQ1), which by itself did not induce aggregation, provided a sufficient stimulus to potentiate platelet responses to Gi-coupled, but not Gq-coupled, agonists. The combination of JAQ1 and adrenaline or ADP, but not serotonin, resulted in alpha(IIb)beta(3)-dependent aggregation that occurred without intracellular calcium mobilization and shape change in the absence of Galphaq or the P2Y(1) receptor. Together, these results provide evidence for a cross-talk between (dimerized) GPVI and Gi-coupled receptors during collagen-induced platelet aggregation. (Blood. 2001;97:3829-3835)     

Tec regulates platelet activation by GPVI in the absence of Btk

The Tec family kinase Btk plays an important role in the regulation of phospholipase C gamma 2 (PLC gamma 2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLC gamma 2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLC gamma 2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLC gamma 2 in the absence of Btk.     

Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase

Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 microM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.     

GPVI levels in platelets: relationship to platelet function at high shear

We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and alpha 2 beta 1 on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of alpha 2 beta 1, which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and alpha 2 beta 1 levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor gamma-chain (FcR gamma-chain) complex or a functional FcR gamma-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s-1 and 1500 s-1. These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear.     

Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.     

Role of calmodulin in platelet receptor function

Platelet glycoprotein (GP)Ib-IX-V and GPVI are unique platelet receptors that bind von Willebrand factor or collagen, respectively, and control the initial interaction of circulating platelets with the blood vessel wall in physiology (hemostasis) or pathology (heart attack or stroke). Engagement of GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by von Willebrand factor or GPVI by collagen, leads to mobilization of cytosolic Ca2+, secretion of platelet agonists such as ADP, cytoskeletal changes, and activation of the platelet integrin alphaIIbbeta3 that mediates von Willebrand factor- or fibrinogen-dependent platelet aggregation. Recent evidence suggests the cytosolic regulatory protein, calmodulin, plays a central role in regulating GPVI or GPIb-IX-V: first, calmodulin directly binds to conserved, juxtamembrane motifs within cytoplasmic domains of both GPVI and GPIb-IX-V (GPIbbeta and GPV subunits) on resting platelets, interactions that dissociate upon platelet activation; second, an intact calmodulin-binding site within GPVI in transfected cells is required for CaCa2+ signaling, but not for GPVI-dependent pathways involving Src family kinases or co-associated FcRgamma-chain; and third, calmodulin regulates metalloproteinase-dependent ectodomain shedding of GPVI and GPV from human platelets. Other vascular cell adhesion receptors, i.e. leukocyte L-selectin, or PECAM-1 (platelet-endothelial cell adhesion molecule-1), also bind calmodulin within the juxtamembrane region of their cytoplasmic tails, an interaction involved in their proteolytic regulation. Further studies should define the precise functional role of calmodulin in thrombus formation initiated by GPIb-IX-V or GPVI.     

Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation

Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling.     

Platelets activated by collagen through the immunoreceptor tyrosine-based activation motif in the Fc receptor gamma-chain play a pivotal role in the development of myocardial ischemia-reperfusion injury

Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor gamma-chain (FcR gamma) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRgamma. To examine the functional significance of platelet FcR gamma/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRgamma-deficient (FcRgamma(-/-)) mice that lack GPVI. The infarct size was significantly smaller in FcRgamma(-/-) mice subjected to occlusion and reperfusion of the coronary artery than in control FcR gamma(+/+) mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR gamma(-/-) mice. Platelet Syk was scarcely activated in the FcR gamma(-/-) mice after myocardial ischemia and reperfusion, but significantly activated in the FcR gamma(+/+) mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRgamma(-/-) mice than in the FcRgamma(+/+) mice. These results suggest that the collagen-induced activation of platelets through the FcR gamma plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRgamma and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury.     

Murine GPVI stimulates weak integrin activation in PLCgamma2-/- platelets: involvement of PLCgamma1 and PI3-kinase

Collagen stimulates platelet activation through a tyrosine kinase-based pathway downstream of the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. Genetic ablation of FcR gamma-chain results in a complete inhibition of aggregation to collagen. In contrast, a steady increase in light transmission is induced by collagen in phospholipase Cgamma2-deficient (PLCgamma2-/-) platelets in a Born aggregometer, indicating a weak level of activation. This increase is inhibited partially in the presence of an alpha2beta1-blocking antibody or an alphaIIbbeta3 antagonist and completely by a combination of the 2 inhibitors. It is also abolished by the Src kinase inhibitor PP1 and reduced in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. The GPVI-specific agonists convulxin and collagen-related peptide (CRP) also stimulate weak aggregation in PLCgamma2-/- platelets, which is inhibited by wortmannin and PP1. Collagen and CRP stimulate tyrosine phosphorylation of PLCgamma1 at its regulatory site, Tyr 783, in murine but not in human platelets through a Src kinase-dependent pathway. Adhesion of PLCgamma2-/- platelets to a collagen monolayer is severely reduced at a shear rate of 800 s-1, relative to controls, whereas it is abolished in FcR gamma-chain-/- platelets. These results provide strong evidence that engagement of GPVI stimulates limited integrin activation in PLCgamma2-/- platelets via PLCgamma1 and PI3-kinase.     

Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: de-ITAM-izing FcgammaRIIa

Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcRgamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcgammaRIIa, also down-regulates GPVI expression. Agonists that signal through FcgammaRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcgammaRIIa also underwent rapid proteolysis in platelets treated with agonists for FcgammaRIIa (VM58/14A2) or GPVI/FcRgamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcgammaRIIa also bound calmodulin and W7 induced FcgammaRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcgammaRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, micro-calpain cleaved an FcgammaRIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRgamma and FcgammaRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcgammaRIIa after immunologic insult.     

Antibody against platelet membrane glycoprotein VI in a patient with systemic lupus erythematosus.

Platelet-collagen interaction is important in primary hemostasis and collagen receptors on the platelet surface include membrane glycoprotein (GP) Ia/IIa and VI. Platelets from a 47-year-old woman with systemic lupus erythematosus (SLE) and a mild bleeding symptom showed a defective collagen-induced aggregation and an impaired adhesion to collagen surface. The patient's platelets had a markedly decreased content of GPVI. The patient had an antibody against GPVI in serum and the patient's plasma induced aggregation and release reaction of normal platelets. These findings indicate that GPVI is an important receptor for collagen on the platelet surface, and that anti-GPVI antibody activates the platelets, resulting in aggregation. This is the first documented case of SLE who acquired a platelet-aggregating anti-GPVI antibody.     

Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.