Role of the recombinant non-integrin platelet collagen receptor P65 on platelet activation induced by convulxin

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom selectively binds with a high affinity to platelets and induces platelet aggregation by a mechanism that resembles that induced by collagen. Taking advantage that P65 has been recently cloned and expressed as a recombinant soluble protein (rec-P65), we examined the role of this non-integrin collagen receptor in platelet activation induced by Cvx. Rec-P65 blocked platelet adhesion to collagen-coated surfaces and inhibited platelet aggregation and ATP secretion induced by type I collagen. On the other hand, rec-P65 did not inhibit platelet aggregation and ATP secretion induced by Cvx, and it did not affect platelet adhesion to Cvx. In addition, ligand-blotting indicated that the Cvx binding to the collagen receptor GPVI was preserved in the presence of rec-P65. These observations indicate that P65 does not play a significant role in platelet activation by Cvx; in contrast, platelet response to collagen involves multiple receptors.     

Constitutive Dimerization of Glycoprotein VI (GPVI) in Resting Platelets Is Essential for Binding to Collagen and Activation in Flowing Blood

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ～29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ～39 and ～44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.     

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.     

Convulxin binding to platelet receptor GPVI: competition with collagen related peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.     

Evidence for two distinct epitopes within collagen for activation of murine platelets

It has recently been shown that the monoclonal antibody JAQ1 to murine glycoprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody cross-linking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem. 275, 23998-24002). In the present study, we have shown that cross-linking of GPVI by JAQ1 results in tyrosine phosphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) gamma-chain, Syk, LAT, SLP-76, and phospholipase C gamma 2. In contrast, platelet aggregation and tyrosine phosphorylation of these proteins were inhibited when mouse platelets were preincubated with JAQ1 in the absence of cross-linking and were subsequently stimulated with a collagen-related peptide (CRP) that is specific for GPVI and low concentrations of collagen. However, at higher concentrations of collagen, but not CRP, aggregation of platelets and tyrosine phosphorylation of the above proteins (except for the adapter LAT) is re-established despite the presence of JAQ1. These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, is occupied in the presence of high concentrations of collagen. Although this could be a second site on GPVI that is activated by a novel motif within the collagen molecule, the absence of LAT phosphorylation in response to collagen in the presence of JAQ1 suggests that this is more likely to be caused by activation of a second receptor that is also coupled to the FcR gamma-chain. The possibility that this response is mediated by a receptor that is not coupled to FcR gamma-chain is excluded on the grounds that aggregation is absent in platelets from FcR gamma-chain-deficient mice.     

Phosphatidylinositol 3'-kinase and tyrosine-phosphatase activation positively modulate Convulxin-induced platelet activation. Comparison with collagen

In this report we have studied the role of phosphatidylinositol 3'-kinase (PI3-K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3-K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx-induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca2+]i. However, PAO does not inhibit Cvx-induced tyrosine phosphorylation of platelet proteins, including Syk and PLCgamma2, but blocked collagen-induced platelet aggregation as well as tyrosine phosphorylation of PLCgamma2. In contrast, Cvx-induced PLCgamma2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCgamma2 activity; and (iii) besides protein tyrosine kinases, PI3-K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI-Fc receptor gamma chain signal to result in full activation and tyrosine phosphorylation of PLCgamma2 in Cvx-stimulated platelets.     

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.     

Selective blockade of glycoprotein VI clustering on collagen helices

Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.     

Novel synthetic collagen fibers, poly(PHG), stimulate platelet aggregation through glycoprotein VI

Novel synthetic collagen fibers, poly(PHG) made by polycondensation of Pro-Hyp-Gly, spontaneously assume polymeric structure with molecular weights greater than 10⁵. Its application for biomaterials has been explored, but that for a platelet agonist has not been investigated. Poly(PHG)-induced platelet aggregation independently of thromboxane A₂ and integrin α2β1. Poly(PHG)-induced tyrosine phosphorylation of glycoprotein VI (GPVI)-related molecules and failed to activate GPVI/FcRγ-deficient platelets. Binding of GPVI to poly(PHG) was confirmed by a surface plasmon resonance spectroscopy, suggesting that poly(PHG) activates platelets through GPVI. Poly(PHG) is an useful research tool to investigate GPVI-mediated signals and a substitute for collagen in platelet functional assays.     

Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcRgamma chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcRgamma chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcRgamma chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcRgamma chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcRgamma chain-deficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcRgamma chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcRgamma chain is critical for its expression and function.     

Fc Rgamma -independent signaling by the platelet collagen receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) is structurally homologous to multisubunit immune receptors and signals through the immune receptor adaptor Fc Rgamma. Multisubunit receptors are composed of specialized subunits thought to be dedicated exclusively to ligand binding or signal transduction. However, recent studies of the intracellular region of GPVI, a ligand-binding subunit, have suggested the existence of protein-protein interactions that could regulate receptor signaling. In the present study we have investigated the signaling role of the GPVI intracellular domain by stably expressing GPVI mutants in RBL-2H3 cells, a model system that accurately reproduces the GPVI signaling events observed in platelets. Studies of mutant GPVI receptor protein-protein interaction and calcium signaling reveal the existence of discrete domains within the receptor's intracellular tail that mediate interaction with Fc Rgamma, calmodulin, and Src family tyrosine kinases. These receptor interactions are modular and mediated by non-overlapping regions of the receptor transmembrane and intracellular domains. GPVI signaling requires all three of these domains as receptor mutants able to couple to only two interacting proteins exhibited severe signaling defects despite normal surface expression. Our results demonstrate that the ligand-binding subunit of the GPVI-Fc Rgamma receptor participates directly in receptor signaling by interacting with downstream signaling molecules other than Fc Rgamma through an adaptor-like mechanism.     

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors.

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.     

Expression of the platelet receptor GPVI confers signaling via the Fc receptor gamma -chain in response to the snake venom convulxin but not to collagen

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fcepsilon receptor gamma-chain (Fc Rgamma), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagen-related peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc Rgamma partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc Rgamma and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the alpha(2)beta(1) integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.     

Identification of residues within human glycoprotein VI involved in the binding to collagen: evidence for the existence of distinct binding sites

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.     

Convulxin binds to native,human glycoprotein Ib alpha

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.     

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.     

Chimeric Fc receptors identify ligand binding regions in human glycoprotein VI

Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcalphaRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcalphaRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.     

Analysis of protein networks in resting and collagen receptor (GPVI)-stimulated platelet sub-proteomes

Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.     

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.