[Show abstract][Hide abstract] ABSTRACT: Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.
Journal of Thrombosis and Haemostasis 08/2007; 5 Suppl 1(s1):220-9. DOI:10.1111/j.1538-7836.2007.02521.x · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
[Show abstract][Hide abstract] ABSTRACT: Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.
[Show abstract][Hide abstract] ABSTRACT: The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s(-1)), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb alpha (12G1 Fab(2)) and alpha(2)beta(1) (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca(2+) signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting alpha(2)beta(1) revealed its involvement in regulating Ca(2+) signaling, PS exposure, and aggregate size. Both GPIb alpha and alpha(2)beta(1) contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb alpha or alpha(2)beta(1). In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with alpha(2)beta(1) mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.
[Show abstract][Hide abstract] ABSTRACT: Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.
[Show abstract][Hide abstract] ABSTRACT: The collagen-platelet interaction is central to haemostasis and may be a critical determinant of arterial thrombosis, where subendothelium is exposed after rupture of atherosclerotic plaque. Recent research has capitalized on the cloning of an important signalling receptor for collagen, glycoprotein VI, which is expressed only on platelets, and on the use of collagen-mimetic peptides as specific tools for both glycoprotein VI and integrin alpha 2 beta 1. We have identified sequences, GPO and GFOGER (where O denotes hydroxyproline), within collagen that are recognized by the collagen receptors glycoprotein VI and integrin alpha 2 beta 1 respectively, allowing their signalling properties and specific functional roles to be examined. Triple-helical peptides containing these sequences were used to show the signalling potential of integrin alpha 2 beta 1, and to confirm its important contribution to platelet adhesion. Glycoprotein VI appears to operate functionally on the platelet surface as a dimer, which recognizes GPO motifs that are separated by four triplets of collagen sequence. These advances will allow the relationship between the structure of collagen and its haemostatic activity to be established.
Biochemical Society Symposium 02/2003; 70(70):81-94. DOI:10.1042/bss0700081 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine if the results obtained in platelet function tests and whole blood perfusions are associated with those in platelet function analyser (PFA)-100. We used collagen type I monomers and fibrils to analyse the distinct roles of glycoprotein (GP) Ia/IIa and other collagen receptors in flowing blood under a high shear rate (1600/s) and in aggregation studies. Also, anticoagulation [citrate vs. D-phenylalanyl-1-prolyl-1 arginine chloromethyl ketone (PPACK)] was varied to enhance the functions of GP Ia/IIa, since it has been shown that the cation-poor environment of citrated blood impairs GP Ia/IIa-dependent platelet recruitment. Large interindividual variability (45-fold) was detected in deposition of platelets in whole blood perfusions over collagen monomers, whereas this variation was only fourfold in fibrils. In PFA, this variation was reduced to 2.5-fold. However, platelet deposition on monomers is associated with epinephrine-enhanced PFA (r=-.49, P<.03), whereas platelet deposition on fibrils is correlated with adenosine diphosphate (ADP)-enhanced PFA (r=-.47, P<.05), suggesting a distinct synergism between epinephrine and monomers (GP Ia/IIa) as well as ADP with fibrils (other collagen receptors). Donors with 807 C/C polymorphism of GP Ia (n=14) had longer lag phase in aggregation experiments compared with C/T (n=7) both by monomers and fibrils (P<.04), but these polymorphisms with their mild impact on GP Ia/IIa activity did not markedly differ in other tests. In conclusion, the results obtained in perfusion studies and PFA experiments correlated, but PFA fails to reveal the large-scale variability related to collagen-induced platelet responses.
Thrombosis Research 08/2001; 103(2):123-33. DOI:10.1016/S0049-3848(01)00283-3 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.
[Show abstract][Hide abstract] ABSTRACT: Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-m-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.
[Show abstract][Hide abstract] ABSTRACT: In a new 2-stage assay of platelet procoagulant activity (PCA), we first subjected gel-filtered platelets to adhesion on collagen (as a model of primary hemostasis) or plasma clots (as a model of preformed thrombus) for 30 minutes, and then the adherent platelets were supplemented with pooled, reptilase-treated, diluted plasma. Defibrinated plasma provided coagulation factors for assembly on platelet membranes without uncontrolled binding of thrombin to fibrin(ogen). Platelet adhesion to both surfaces showed modest individual variation, which increased at platelet densities that allowed aggregation. However, adhesion-induced PCA varied individually and surface-independently >3-fold, suggesting a uniform platelet procoagulant mechanism. Permanently adhered platelets showed markedly enhanced PCA when compared with the platelet pool in suspension, even after strong activation. The rate of thrombin generation induced by clot-adherent platelets was markedly faster than on collagen-adherent platelets during the initial phase of coagulation, whereas collagen-induced PCA proceeded slowly, strongly promoted by tissue thromboplastin. Therefore at 10 minutes, after adjustment for adhered platelets, collagen supported soluble thrombin formation as much as 5 times that of the thrombin-retaining clots. Activation of platelets by their firm adhesion was accompanied by formation of microparticles, representing about one third of the total soluble PCA. Collagen-adhered platelets provide soluble thrombin and microparticles, whereas the preformed clot serves to localize and accelerate hemostasis at the injury site, with the contribution of retained thrombin and microparticles.
[Show abstract][Hide abstract] ABSTRACT: The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s(-1), whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg(2+) concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg(2+) during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV.
[Show abstract][Hide abstract] ABSTRACT: Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.
Thrombosis and Haemostasis 06/1999; 81(5):782-92. · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P-selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P-selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.
[Show abstract][Hide abstract] ABSTRACT: Abstract—In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca,1]i. Platelets