GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

Platelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage.

For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods.

According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.     

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.     

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.     

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking alpha-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor gamma-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C gamma 2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the literature.     

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.     

Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcRgamma-chain complex from the human platelet surface

Platelet glycoprotein (GP) VI is a 62-kDa membrane glycoprotein that exists on both human and murine platelets in a noncovalent complex with the Fc receptor (FcR) gamma chain. The GPVI/FcRgamma-chain complex serves as the major activating receptor for collagen, as evidenced by observations that platelets genetically deficient in GPVI or the FcRgamma chain are highly refractory to collagen-induced platelet activation. Recently, several different rat anti-murine GPVI monoclonal antibodies, termed JAQs 1, 2, and 3, were produced that had the unique property of "immunodepleting" GPVI from the murine platelet surface and rendering it unresponsive to collagen or GPVI-specific agonists like convulxin or collagen-related peptide (CRP). Herein, we describe a patient with a mild bleeding disorder and a moderately reduced platelet count whose platelets fail to become activated in response to collagen or CRP and inefficiently adhere to and form thrombi on immobilized collagen under conditions of arterial shear. Although the amount of GPVI platelet mRNA and the nucleotide sequence of the GPVI gene were found to be normal, both GPVI and the FcRgamma chain were nearly absent from the platelet surface and were markedly reduced in wholeplatelet detergent lysates. Patient plasma contained an autoantibody that bound specifically to GPVI-positive, normal platelets, and cleared soluble GPVI from the plasma, suggesting that the patient suffers from a rare form of idiopathic thrombocytopenic purpura caused by a GPVI-specific autoantibody that mediates clearance of the GPVI/FcRgamma-chain complex from the platelet surface. Since antibody-induced GPVI shedding now has been demonstrated in both humans and mice, these studies may provide a rationale for developing therapeutic reagents that induce temporary depletion of GPVI for the treatment of clinical thrombosis.     

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)     

Identification of human platelet glycoprotein VI-specific IgG autoantibody and its fragments.

Glycoprotein VI (GPVI), as a major receptor for collagen on the platelet surface, mediates the initial platelet contact with collagen, and causes platelet aggregation and thrombosis. Agents of anti-GPVI can inhibit the adhesion, activation and aggregation function of platelets, which can be used for preventing thrombotic diseases. To make humanized monoclonal antibodies by phage surface display technology, we studied plasmas from 44 patients with chronic idiopathic thrombocytopenic purpura using modified monoclonal antibody immobilization of platelet antigen assays. GPVI-specific antibodies were found in six (13.6%) patients. Among these, only one showed significant inhibition of platelet aggregation induced by collagen. The IgG antibody and F(ab')2 fragments of this patient's plasma were further purified and their immunoreactivities and effects on platelet aggregation were reanalyzed. It was found that purified IgG and its F(ab')2 fragments from the patient not only retained the ability to bind to platelet GPVI, but also inhibited collagen-induced platelet aggregation. In this study, therefore, the specific anti-platelet GPVI autoantibody that inhibits collagen-induced platelet aggregation has been successfully screened out, which can be used to develop completely humanized anti-GPVI phage antibody.     

An Acquired Defect Associated with Abnormal Signaling of the Platelet Collagen Receptor Glycoprotein VI

Introduction: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. Aim: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. Methods: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. Results: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ～40-kDa Syk fragment. Conclusions: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.     

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.     

Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily

Injuries to the vessel wall and subsequent exposure of collagen from the subendothelial matrix result in thrombus formation. In physiological conditions, the platelet plug limits blood loss. However, in pathologic conditions, such as rupture of atherosclerotic plaques, platelet-collagen interactions are associated with cardiovascular and cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets, and people who are deficient in GPVI suffer from bleeding disorders. Based on the fact that GPVI is coupled to the Fc receptor (FcR)-gamma chain and thus should share homology with the FcR chains, the genes encoding human and mouse GPVI were identified. They belong to the immunoglobulin (Ig) superfamily and share 64% homology at the protein level. Functional evidence demonstrating the identity of the recombinant protein with GPVI was shown by binding to its natural ligand collagen; binding to convulxin (Cvx), a GPVI-specific ligand from snake venom; binding of anti-GPVI IgG isolated from a patient; and association to the FcR-gamma chain. The study also demonstrated that the soluble protein blocks Cvx and collagen-induced platelet aggregation and that GPVI expression is restricted to megakaryocytes and platelets. Finally, human GPVI was mapped to chromosome 19, long arm, region 1, band 3 (19q13), in the same region as multiple members of the Ig superfamily. This work offers the opportunity to explore the involvement of GPVI in thrombotic disease, to develop alternative antithrombotic compounds, and to characterize the mechanism involved in GPVI genetic deficiencies. (Blood. 2000;96:1798-1807)     

GPIbα-selective activation of platelets induces platelet signaling events comparable to GPVI activation events

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbα (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbα-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbα- and α_IIbβ₃-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbα and anti-α_IIbβ₃ antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbα-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbα engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbα can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.     

Model systems for platelet receptor shedding

Platelet adhesion and aggregation at sites of vascular injury are crucial for hemostasis; however, under pathological conditions, they can cause myocardial infarction and stroke. Platelet adhesion is mediated by binding of the glycoprotein (GP)Ib-V-IX complex to immobilized von Willebrand factor (vWF), thereby enabling the interaction of GPVI with exposed collagen and subsequent platelet activation. This process is reinforced by locally produced thrombin and platelet-derived adenosine diphosphate (ADP) and thromboxane A2 (TxA2), leading to a conformational change of integrins from a low- to a high-affinity state, which allows firm platelet adhesion and aggregation. Stable platelet aggregation requires the formation of fibrin and the coordinated interaction of additional receptors on the adjacent platelets. Platelet surface receptor expression can partially be regulated by proteolytic cleavage, which may represent a mechanism to downregulate platelet reactivity toward extracellular matrix proteins and ligands. It is estimated that approximately 10% of platelet surface receptors are regulated by proteolytic cleavage. In this chapter of the review series we will summarize the platelet receptors that are known to be shed from the cell surface with a focus on the proteins GPIbα, GPV and GPVI, which are cleaved by members of the ‘a disintegrin and metalloproteinase’ (ADAM) family.     

Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito

During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.     

Convulxin-induced platelet adhesion and aggregation: involvement of glycoproteins VI and IaIIa

The interaction of convulxin (Cvx), a 72-kDa glycoprotein isolated from the venom of Crotalus durissus terrificus with human platelets has been studied. Cvx at low concentrations (below 100 pM) induced platelet aggregation, dense body secretion and intracellular calcium mobilization which indicates that Cvx is a potent activator of human platelets. Cvx-induced platelet aggregation and secretion was inhibited by 6Fl an anti-integrin alpha2beta1 monoclonal antibody that was without effect on calcium mobilization. Anti-GPVI Fab fragments inhibited aggregation, secretion and calcium mobilization triggered by Cvx. In addition, immobilized Cvx was found to induce divalent cation-independent platelet adhesion in a static system. Platelet adhesion to Cvx was inhibited by anti-GPVI Fab fragments but not by anti-integrin alpha2beta1 . Cvx was shown to bind to a 57,000 Dalton protein that was identified as GPVI. Altogether, these results indicate that GPVI behaves as a receptor for Cvx, while integrin alpha2beta1 could play a regulatory role in Cvx-induced platelet aggregation. Cvx and collagen interaction with platelets, thus appears to share some characteristics but to also have specific properties.     

Convulxin-induced platelet adhesion and aggregation: involvement of glycoproteins VI and IaIIa

The interaction of convulxin (Cvx), a 72-kDa glycoprotein isolated from the venom of Crotalus durissus terrificus with human platelets has been studied. Cvx at low concentrations (below 100 pM) induced platelet aggregation, dense body secretion and intracellular calcium mobilization which indicates that Cvx is a potent activator of human platelets. Cvx-induced platelet aggregation and secretion was inhibited by 6Fl an anti-integrin alpha2beta1 monoclonal antibody that was without effect on calcium mobilization. Anti-GPVI Fab fragments inhibited aggregation, secretion and calcium mobilization triggered by Cvx. In addition, immobilized Cvx was found to induce divalent cation-independent platelet adhesion in a static system. Platelet adhesion to Cvx was inhibited by anti-GPVI Fab fragments but not by anti-integrin alpha2beta1. Cvx was shown to bind to a 57 000 Dalton protein that was identified as GPVI. Altogether, these results indicate that GPVI behaves as a receptor for Cvx, while integrin alpha2beta1 could play a regulatory role in Cvx-induced platelet aggregation. Cvx and collagen interaction with platelets, thus appears to share some characteristics but to also have specific properties.     

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.     

Glycoprotein VI-mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP

Collagen activates platelets by transducing signals through glycoprotein VI (GPVI). It is not clear whether collagen can directly activate fibrinogen receptors on the adherent platelets without a role for positive feedback agonists. We investigated the contribution of secondary G protein signaling to the mechanism of GPVI-stimulated platelet aggregation using the GPVI-selective agonists, convulxin and collagen-related peptide (CRP) as well as collagen. Adenosine diphosphate (ADP) scavengers or ADP receptor antagonists shifted the concentration-response curve slightly to the right at low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. ADP receptor antagonists shifted the concentration-response curve of collagen- or CRP-induced platelet aggregation to the right at all the concentrations. Protein kinase C inhibitor, Ro 31-8220, or a calcium chelator 5,5'-dimethyl-BAPTA shifted the concentration-response curve of convulxin-induced platelet aggregation to the right. In addition, pretreatment with both Ro 31-8220 and dimethyl-BAPTA resulted in total inhibition of convulxin-mediated aggregation. Blockade of either the calcium- or protein kinase C-regulated pathway leads to inhibition of fibrinogen receptor activation on platelets adherent to collagen, but inhibition of both pathways leads to abolished fibrinogen receptor activation. We conclude that collagen-induced activation of fibrinogen receptor on adherent platelets through GPVI signaling occurs without any significant role for secreted ADP or thromboxane A(2). Furthermore, protein kinase C- and calcium-regulated pathways independently contribute to GPVI-mediated platelet aggregation.     

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.     

A synthetic nonapeptide derived from the sequence of a platelet type I collagen receptor inhibits type I collagen-mediated platelet aggregation

We have cloned the platelet receptor for type I collagen, but the structure-function of the receptor has not been completely established. The purpose of this investigation was to identify a collagen binding site(s) of the platelet receptor. Three peptides were synthesized chemically. Each peptide serves as an inhibitor of type I collagen-induced platelet aggregation, ATP release, platelet protein phosphorylation, and platelet adhesion to artificial matrices and aortic segments. We show that a nonapeptide specifically inhibits type I collagen-induced platelet aggregation and the release of ATP in a dose-dependent fashion. The peptide also inhibits the binding of radiolabeled alpha (I) chain to washed platelets, the adhesion of radiolabeled platelets to type I collagen-coated petri dishes, rabbit aortic segments, and platelet protein phosphorylation. Deletion of this peptide region of the cloned cDNA abolishes the inhibitory effect of the recombinant protein on type I collagen-induced platelet aggregation. These findings support the likelihood that the nonapeptide forms part of the binding site of the platelet receptor for type I collagen.