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.     

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.     

Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI,but not the monomeric form,shows affinity to fibrous collagen

Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019-29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc(2)). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc(2), and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc(2) bound to the surface of immobilized collagen with a dissociation constant (K(D)) of 5.76 x 10(-7) m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.     

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.     

Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.     

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.     

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.     

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.     

Ligand binding rapidly induces disulfide-dependent dimerization of glycoprotein VI on the platelet plasma membrane

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.     

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.     

Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL^-1 and 10 μg.mL^-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy.

Trial Registration

ClinicalTrials.gov NCT00763893     

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.     

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

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.     

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.     

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.     

Nanoparticles that display short collagen-related peptides. Potent stimulation of human platelet aggregation by triple helical motifs

Collagen plays a key role in the activation and adhesion of blood platelets via their cell-surface receptors. Normally, collagen-related peptides (CRPs), even one as long as a 30-mer (10 Gly-Pro-Hyp (GPO) repeats), are unable to effectively express collagen's platelet-activating behavior. We attached two short CRPs, AcHN-(Gly-Pro-Hyp)nGly-OH with n = 5 (1) and n = 10 (2), via the C-terminus to amino-functionalized latex nanoparticles to create a multimeric display of triple helical motifs. These nanomaterials were characterized by dynamic light scattering and environmental scanning electron microscopy. The nanoparticles bearing the 31-mer CRP sequence, 2, but not the 16-mer sequence, 1, effectively induced the aggregation of human platelets, with a potency level approaching that of native type I collagen. Our results highlight the importance of presenting triple helical CRP motifs of sufficient length on a suitable scaffold in order to stimulate 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.     

Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets

Activation of platelets by exposed collagen after vessel wall injury is a primary event in the pathogenesis of stroke and myocardial infarction. Two collagen receptors, integrin alpha2beta1 and glycoprotein VI (GPVI), are expressed at similar levels on human and mouse platelets, but their individual roles during collagen activation remain poorly defined. Recent genetic and pharmacologic experiments have revealed an essential role for GPVI but have failed to define the role of alpha2beta1 or explain how two structurally distinct collagen receptors might function together to mediate platelet collagen responses. Discriminating the roles of these two collagen receptors is complicated by evidence suggesting that GPVI and platelet integrins may activate a common intracellular signaling pathway. To determine how alpha2beta1 and GPVI activate platelets in response to collagen, we have (i) examined collagen signaling conferred by expression of these receptors in hematopoietic cell lines; (ii) determined the effect of blocking each receptor on the activation of human platelets by collagen; (iii) generated low-GPVI mice in which the alpha2beta1/GPVI receptor ratio has been altered from 1:1 to 50:1 to expose alpha2beta1 function; (iv) studied the collagen responses of mouse platelets lacking LAT, an adaptor protein critical for GPVI but not integrin signaling; and (v) addressed the mechanism by which soluble collagens activate wild-type platelets. These studies demonstrate that alpha2beta1 requires inside-out signals to participate in collagen signaling and that alpha2beta1 is required for collagen activation of platelets when GPVI signals are reduced by blocking anti-GPVI antibody, low receptor number, specific disruption of the GPVI signaling pathway, or forms of collagen that bind weakly to GPVI relative to alpha2beta1. We propose a reciprocal two-receptor model of collagen signaling in platelets in which the nonintegrin receptor GPVI provides the primary collagen signal that activates and recruits the integrin receptor alpha2beta1 to further amplify collagen signals and fully activate platelets through a common intracellular signaling pathway. This model explains many of the genetic and pharmacologic observations regarding collagen signaling in platelets and demonstrates a novel mechanism by which hematopoietic cells integrate signaling by structurally distinct receptors that share a common ligand.     

The specific recognition of a cell binding sequence derived from type I collagen by Hep3B and L929 cells

In this study, the affinity of two different cell types toward a specific cell binding sequence (Gly-Phe-Hyp-Gly-Glu-Arg or GFOGER) derived from type I collagen using peptide template (PT)-assembled collagen peptides of different triple helicity as a model for natural collagen is examined. A series of biophysical studies, including melting curve analysis and circular dichroism spectroscopy, demonstrated the presence of stable triple-helical conformation in the PT-assembled (GPO)3-GFOGER-(GPO)3, (GPO)-GFOGER-(GPO), and (Pro-Hyp-Gly)5 solution. Conversely, non-templated peptides, except (GPO)3-GFOGER-(GPO)3, showed no evidence of assembly into triple-helical structure. Biological assays, including cell adhesion, competitive inhibition, and immunofluorescence staining, revealed a correlation of triple-helical conformation with the cellular recognition of GFOGER in an integrin-specific manner. The triple helix was shown to be important, but not crucial for cell adhesion to native collagen. Hep3B and L929 cells displayed significant differences in the recognition of GFOGER, mainly because of the differences in their expression of specific integrin receptors for collagen. For example, PT-assembled (GPO)3-GFOGER-(GPO)3 was shown to perform comparably to collagen for L929, but not Hep3B, cell adhesion. The result showed that a specific cell binding motif may not fully mimic the extracellular matrix (ECM) microenvironment, suggesting the need to use a combination of two or more cell binding sequences for targeting a wide range of integrin receptors expressed by a specific cell type to better mimic the ECM.