Glycoprotein VI Expression Research Articles

Platelet collagen receptor glycoprotein VI as a possible novel indicator for the acute coronary syndrome

Platelet collagen receptor glycoprotein VI (GPVI) plays a critical role in acute coronary thrombosis. This prospective study examined the predictive value of GPVI for acute coronary syndromes (ACS) in a large consecutive group of patients with symptomatic coronary artery disease to identify the high-risk cohort with imminent coronary events. We evaluated 1,003 patients with symptomatic coronary artery disease, verified by coronary angiography, and determined the surface expression of GPVI using flow cytometry. In a subgroup of 471 patients, who were treated with aspirin plus clopidogrel for coronary stenting, adenosine disphosphate (20 micromol/L)-induced platelet aggregation was evaluated. Patients with ACS (n = 485) showed a significantly enhanced GPVI expression compared to patients with stable angina pectoris (SAP; n = 518) (mean fluorescence intensity for ACS 19.8 +/- 5.9; SAP 18.7 +/- 8.5, P = .01). Patients with elevated GPVI levels on admission (GPVI cutoff value > or =18.6 mean fluorescence intensity) had a 1.4-fold relative risk for ACS. Logistic regression analysis showed that an elevated platelet GPVI level may indicate ACS independent of biomarkers of myocardial necrosis including troponin, creatine kinase, and creatine kinase-MB. Patients with increased platelet activation (GPVI expression level > or =18.6) showed significant enhanced residual platelet aggregation despite dual antiplatelet therapy compared to patients with low GPVI levels (P = .028). Surface expression of GPVI is enhanced in patients with ACS and indicates an imminent acute coronary event before irreversible myocardial necrosis is evident. High GPVI levels are associated with increased residual platelet aggregation despite antiplatelet therapy. Therefore, GPVI is useful to identify the subgroup of patients with a high risk for coronary stent thrombosis and thromboischemic events.

Characterization of a patient with atypical amegakaryocytic thrombocytopenia

We report a 6-year-old girl with amegakaryocytic thrombocytopenia, the first case of this rare congenital disorder not to have an MPL gene mutation. Although no mutations were identified in MPL, Mpl protein was absent in the platelets and TPO induced phosphorylation of the Janus tyrosine kinase 2 (Jak2) was not detected. In addition to the defect of Mpl, the patient demonstrated markedly reduced expression of glycoprotein VI (GPVI) in contrast to normal expression of other platelet-specific proteins GPIb alpha, GPIb beta, and GPIIb. To explore the causes for the absence of Mpl, the entire coding region of Jak2 and AML1 were sequenced and no mutations were identified. To our knowledge, this is the first report that describes a case of amegakaryocytic thrombocytopenia that is not caused by a mutation in MPL and demonstrates the severe impairment of GPVI expression on platelets.

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

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.

Expression of platelet collagen receptor glycoprotein VI is associated with acute coronary syndrome

Platelet collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial thrombosis. In this observational study, we examined the platelet surface expression of GPVI in patients with symptomatic coronary artery disease (CAD). We evaluated a consecutive cohort of 367 patients with symptomatic CAD, who underwent coronary angiography. The surface expression of platelet activation markers (GPVI, CD62P, and CD42b) was determined by flow cytometry. Patients with acute coronary syndrome (ACS) showed a significantly enhanced GPVI expression on admission when compared with patients with stable angina pectoris (SAP) (ACS: 21.4+/-9.7; SAP: 18.6+/-7.1 mean fluorescence intensity+/-SD; P=0.004). The expression of GPVI correlated with CD62P (r=0.702; P=0.001). Logistic regression analysis demonstrated that on admission, elevated platelet GPVI expression was associated with ACS, independent of markers of myocardial necrosis such as troponin and creatine kinase. Platelet GPVI surface expression is elevated in patients with ACS and is associated with imminent acute coronary events. The determination of the platelet-specific thrombotic marker GPVI may help to identify patients at risk before myocardial ischaemia is evident.

FcRγ-Chain Signals in GPVI-Deficient Platelets.

The receptor glycoprotein VI (GPVI) is required for hemostasis and collagen-induced platelet aggregation. GPVI and FcRγ-chain form a noncovalent signaling complex. Genetic ablation of murine FcRγ-chain prevents expression of GPVI, and immunodepletion of GPVI removes FcRγ chain from platelet membranes. The GPVI/FcRγ-chain relationship has confounded elucidation of the individual role(s) of GPVI and FcRγ-chain in signal transduction. For example, botrocetin (bt)/von Willebrand factor (vWf) and γ-thrombin induced aggregation of washed platelets each appear to be FcRγ-chain-dependent. However, since FcRγ-chain−/− platelets also lack GPVI, the independent role(s), if any, of these signaling molecules in this aggregation could not be established. But, genetic ablation of murine GPVI does not exclude expression of the FcRγ-chain. Therefore, based on analyses of genetically deficient GPVI platelets that express FcRγ-chain, we demonstrate here that bt/vWf and thrombin-induced aggregation of washed platelets are each dependent on FcRγ-chain thereby demonstrating that FcRγ-chain can function in the absence of GPVI. Presumably, another receptor or receptors is/are able to activate FcRγ-chain in GPVI −/− and GPVI +/+ platelets. GPIb may not directly activate FcRγ-chain because GPIb elicited activation of αIIbβ3 is not FcRγ-chain dependent. The integrin αIIbβ3 appears to be common to the signaling described here because both the non-agglutination elicited bt/vWf-induced, and the γ-thrombin-induced TxA2 production and ATP secretion were β3-dependent. Therefore, αIIbβ3 may have activated this FcRγ-chain-dependent signaling. This hypothesis was tested by investigating the role of FcRγ-chain in αIIbβ3-mediated outside-in signaling induced by a palmitoylated derivative of the peptide KVGFFKR, a peptide which directly activates αIIbβ3 causing β3-dependent signaling that elicits TxA2 production, ATP secretion and platelet aggregation. As with γ-thrombin-treated FcRγ-chain−/− platelets, pKVGFFKR-treated FcRγ-chain−/− platelets did not produce TxA2, secrete ATP or aggregate. But, pKVGFFKR-treated GPVI−/− platelets produced the wild type level of TxA2, secreted the wild type level of ATP, and aggregated normally demonstrating that pKVGFFKR-induced αIIbβ3-mediated outside-in signaling is FcRγ-chain dependent. These results support the hypothesis that activation of FcRγ-chain is αIIbβ3-dependent in the signaling described here. The results of ELISA studies support this conclusion.

The influence of N-linked glycosylation on the function of platelet glycoprotein VI

AbstractUsing recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). In COS-7 cells transiently transfected with GPVI, deglycosylation with peptide-N-glycosidase F (PNGase F; specific for complex N-linked glycans) or tunicamycin decreases the molecular weight of GPVI and reduces transfected COS-7 cell binding to both CRP and CVX. In stably transfected Dami cells, the substitutions N92A or S94A, but not L95H, resulted in a 30% to 40% decrease in adhesion to CVX, but a 90% or greater decrease in adhesion to CRP and a 65% to 70% decrease in adhesion to type I collagen. Treatment with PNGase F, but not Endoglycosidase H (Endo H) (specific for high-mannose N-linked glycans), produced an equivalent decrease in molecular weight. Neither N92A nor S94A affected the expression of GPVI, based on the direct binding of murine anti–human GPVI monoclonal antibody 204-11 to transfected Dami cells. These findings indicate that N-linked glycosylation at N92 in human GPVI is not required for surface expression, but contributes to maximal adhesion to type I collagen, CRP and, to a lesser extent, CVX.

Selective impairment of platelet activation to collagen in the absence of GATA1

AbstractDefects in the X-linked DNA-binding megakaryocyte transcription factor GATA1 cause thrombocytopenia and abnormal platelet function. However, detailed studies of GATA1 function in platelet activation are lacking. Here, we studied platelets from GATA1-deficient mice and from a male patient (S14) with a bleeding diathesis attributed to a single amino acid substitution (R216Q) in the N-terminal GATA1 zinc finger that alters binding to DNA. In both cases there was inhibition of aggregation to collagen and decreased tyrosine phosphorylation of glycoprotein VI (GPVI)–signaling proteins. This effect was more marked in GATA1-deficient murine platelets, where it was associated with a significant reduction in surface GPVI expression. Moreover, both human and murine GATA1-mutant platelets showed reduced adhesion and aggregate formation on a collagen matrix at an intermediate rate of shear, although this could not be accounted solely by the thrombocytopenia and altered GPVI expression, indicating that GATA1 regulates additional factors important for platelet activation under shear. In contrast, there was no inhibition of responses to G protein–coupled receptor agonists in GATA1-perturbed platelets. Our results are consistent with GATA1 regulating some but not all pathways of platelet activation, leading to an impairment of aggregate formation under flow, which cannot be attributed solely to the thrombocytopenia.

Thrombopoietin Initiates Demethylation-Based Transcription of GP6 during Megakaryocyte Differentiation.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-1, Sp1 and Fli-1. However, the mere presence of these is not sufficient to initiate transcription during megakaryocyte differentiation, and other mechanisms are suggested to be involved in the regulation of megakaryocyte-specific expression of GPVI. In this study, we show that GPVI expression during megakaryocytic differentiation is dependent on CpG demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI non-expressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human c-mpl, were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation which can be directly initiated by TPO. Our results establish, for the first time, a role for TPO in dynamic changes in CpG methylation status that are involved in the epigenetic regulation of megakaryocyte-specific gene expression.

Surface expression of collagen receptor Fc receptor-gamma/glycoprotein VI is enhanced on platelets in type 2 diabetes and mediates release of CD40 ligand and activation of endothelial cells.

Diabetes is associated with an enhanced collagen-mediated platelet activation that contributes significantly to thromboischemic complications. In this study, the platelet collagen receptor glycoprotein VI (GPVI) was studied in 385 patients with type 2 diabetes. Surface expression of the platelet Fc receptor that forms a functional complex with GPVI was significantly increased in patients with diabetes compared with those without diabetes (P = 0.02). Fc receptor expression correlated with GPVI expression and was found to be independently associated with diabetes (r = 0.529, P < 0.001). Stimulation of GPVI through a specific anti-GPVI monoclonal antibody significantly enhanced surface expression of CD40L (P = 0.006). Because CD40L is a potent platelet-derived cytokine that is involved in thrombosis and atherosclerosis, we evaluated the effect of GPVI-mediated release of CD40L on activation of endothelial cells. Coincubation of GPVI-stimulated platelets resulted in substantial enhanced endothelial surface expression of CD62P, alphavbeta3, and intercellular adhesion molecule 1 (P < 0.05) and secretion of monocyte chemoattractant protein 1 of cultured human umbilical vein endothelial cells (P < 0.01). These results suggest that the function of collagen receptor GPVI is altered in type 2 diabetes and may play an important role in atherothrombotic complications. Inhibition of GPVI may be a promising pharmacological target in the treatment of high-risk diabetic patients.

Expression of glycoprotein VI in vascular endothelial cells

Glycoprotein (GP) VI, a collagen receptor, plays an important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a ˜6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuno-precipitation in both HUVEC and platelet lysates. In addition, a ˜70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRγ chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

The Fc receptor gamma-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin.

There is extensive evidence that FcR gamma-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR gamma-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR gamma-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR gamma-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR gamma-chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR gamma-chain. These results demonstrate that reconstitution of the GPVI-FcR gamma-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide.

The Platelet Receptor GPVI Mediates Both Adhesion and Signaling Responses to Collagen in a Receptor Density-dependent Fashion

The platelet response to collagen is a primary event in hemostasis and thrombosis, but the precise roles of the numerous identified platelet collagen receptors remain incompletely defined. Attention has recently focused on glycoprotein VI (GPVI), a receptor that is expressed on platelets in association with a signaling adapter, the Fc receptor gamma chain (Fc Rgamma). Genetic and pharmacologic loss of GPVI function results in loss of collagen signaling in platelets, but studies to date have failed to demonstrate that GPVI-Fc Rgamma expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc Rgamma may require the collagen-binding integrin alpha2beta1 as a co-receptor, but this model has not been supported by a recent study of mouse platelets lacking alpha2beta1. In the present study we have used a novel anti-GPVI monoclonal antibody to measure the level of GPVI on human platelets and to guide the development of GPVI-expressing cell lines to assess the role of GPVI in mediating platelet collagen responses. GPVI receptor density on human platelets appears tightly regulated, is independent from the level of alpha2beta1 expression, and significantly exceeds that on previously characterized GPVI-expressing RBL-2H3 cells. Using newly generated GPVI-expressing RBL-2H3 cells with receptor densities equivalent to that on human platelets, we demonstrate that GPVI expression confers both adhesive and signaling responses to collagen in a graded fashion that is proportional to the GPVI receptor density. These results resolve some of the conflicting data regarding GPVI-collagen interactions and demonstrate that 1) GPVI-Fc Rgamma expression is sufficient to confer both adhesion and signaling responses to collagen, and 2) GPVI-mediated collagen responses are receptor density-dependent at the receptor levels expressed on human platelets.

Expression and Function of the Collagen Receptor GPVI during Megakaryocyte Maturation

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34(+) cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41(+) cells including some CD41(+)/CD34(+) cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcription-polymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41(high)). As in platelets, megakaryocyte GPVI associates with the Fc receptor gamma chain (FcRgamma). The FcR gamma chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, alpha(2)beta(1) integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8-10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by alpha(2)beta(1) integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcRgamma, and PLC(gamma)2. Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor γ-chain (FcR γ-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca++ elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase Cγ2 (PLCγ2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca++]i, during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR γ-chain during differentiation of megakaryocytes.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

AbstractThis study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor γ-chain (FcR γ-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca++ elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase Cγ2 (PLCγ2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca++]i, during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR γ-chain during differentiation of megakaryocytes.

Molecular Cloning, Genomic Structure, Chromosomal Localization, and Alternative Splice Forms of the Platelet Collagen Receptor Glycoprotein VI

Glycoprotein VI (GPVI) is the major collagen receptor underlying platelet activation. We cloned the full-length cDNA for GPVI (GPVI-1) and its two isoforms (GPVI-2 and -3) from phorbol-ester-stimulated CMK cells. The GPVI-1 cDNA was identical in the coding region with the cDNA that has recently been reported to belong to the immunoglobulin superfamily. The GPVI gene consisted of 8 exons spanning over 23 kbp and was mapped on the chromosome 19q13.4. The promoter of GPVI gene lacked TATA and CAAT boxes and had multiple transcription start sites like other megakaryocytic genes. When COS-7 cells were cotransfected with the GPVI isoforms and Fc receptor gamma chain, Fc receptor gamma chain was associated with GPVI-1 and -2 but did not affect the GPVI expression levels. GPVI-1 and -2 could bind the collagen-related peptide, which exhibits triple-helical and polymeric structure of collagen to activate platelets via 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)-γ 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-γ 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.

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

AbstractInjuries 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)-γ 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-γ 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.