Adhesion and Activation of Human Platelets Induced by Convulxin Involve Glycoprotein VI and Integrin α2ॆ1

We analyzed the interaction of convulxin (Cvx), a 72-kDa protein isolated from the venom of Crotalus durissus terrificus, with human platelets. Cvx is a potent platelet agonist that induces an increase in the intracellular Ca2+concentration ([Ca2+] i), granule exocytosis and aggregation. 125I-Labeled Cvx binds specifically and rapidly to platelets at binding sites of high and moderate affinity. Platelets adhere to immobilized Cvx in a time-dependent but cation-independent manner. Platelet exocytosis and aggregation induced by Cvx were inhibited by an anti-integrin α2ॆ1 monoclonal antibody (6F1) and by the Fab fragments of a polyclonal anti-glycoprotein VI (GPVI) antibody. Both the adhesion of platelets to Cvx and the Cvx-induced increase in [Ca2+] i were inhibited by anti-GPVI Fab fragments but not by 6F1. Ligand blotting assay showed that 125I-Cvx binds to a 57-kDa platelet protein with an electrophoretic mobility identical to that of GPVI. In addition, we observed the following: (i)125I-Cvx binds to GPVI immunoprecipitated by the anti-GPVI antibody from a platelet lysate, and (ii) Cvx inhibits the binding of anti-GPVI IgG to GPVI. Taken together, these results demonstrate that GPVI behaves as a Cvx receptor and that the α2ॆ1 integrin appears to be involved in the later stages of Cvx-induced platelet activation,i.e. exocytosis and aggregation. We analyzed the interaction of convulxin (Cvx), a 72-kDa protein isolated from the venom of Crotalus durissus terrificus, with human platelets. Cvx is a potent platelet agonist that induces an increase in the intracellular Ca2+concentration ([Ca2+] i), granule exocytosis and aggregation. 125I-Labeled Cvx binds specifically and rapidly to platelets at binding sites of high and moderate affinity. Platelets adhere to immobilized Cvx in a time-dependent but cation-independent manner. Platelet exocytosis and aggregation induced by Cvx were inhibited by an anti-integrin α2ॆ1 monoclonal antibody (6F1) and by the Fab fragments of a polyclonal anti-glycoprotein VI (GPVI) antibody. Both the adhesion of platelets to Cvx and the Cvx-induced increase in [Ca2+] i were inhibited by anti-GPVI Fab fragments but not by 6F1. Ligand blotting assay showed that 125I-Cvx binds to a 57-kDa platelet protein with an electrophoretic mobility identical to that of GPVI. In addition, we observed the following: (i)125I-Cvx binds to GPVI immunoprecipitated by the anti-GPVI antibody from a platelet lysate, and (ii) Cvx inhibits the binding of anti-GPVI IgG to GPVI. Taken together, these results demonstrate that GPVI behaves as a Cvx receptor and that the α2ॆ1 integrin appears to be involved in the later stages of Cvx-induced platelet activation,i.e. exocytosis and aggregation. Many snake venom proteins are known to interact with platelets. Some behave in vitro as cell agonists, and others are inhibitors of platelet activation induced by physiological agents (1Brinkhous K.M. Smith S.V. Pirkle H. Markland Jr., F.S. Hematology, Hemostasis and Animal Venoms. 7. Marcel Dekker, New York1988: 363-376Google Scholar). Several groups can be distinguished according to their mechanism of action and molecular structure: inhibitory and activating proteases, glycoproteins (aggregoserpentins) and lectins (thrombolectins) that stimulate platelets, or peptides that inhibit platelet aggregation (disintegrins) (2Zingali R.B. Bon C. Ann. Inst. Pasteur. 1991; 4: 267-276Google Scholar). The target of some of these compounds has been identified. Disintegrins interact with the platelet integrin αIIbॆ3 corresponding to glycoprotein (GP) 1The abbreviations used are: GP, glycoprotein; [Ca2+] i, intracellular Ca2+concentration; Cvx, convulxin; PLC, phospholipase C; PRP, platelet-rich plasma; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; ACD-A, acid-citrate-dextrose anticoagulant. IIb-IIIa and inhibit the binding of fibrinogen to this receptor and, hence, platelet aggregation. Another large group of venom proteins interact with membrane GPIb, resulting in platelet agglutination and/or inhibition of the binding of von Willebrand factor to this receptor (3Fujimara Y. Kawasaki T. Titani K. Thromb. Haemostasis. 1996; 76: 633-639Crossref PubMed Scopus (63) Google Scholar). Convulxin (Cvx), a 72-kDa glycoprotein, isolated from the venoms ofCrotalus durissus cascavella and Crotalus durissus terrificus (4Prado-Franceschi J. Vital-Brazil O. Toxicon. 1981; 19: 875-887Crossref PubMed Scopus (67) Google Scholar), is an extremely potent platelet activator (5Vargaftig B.B. Prado-Franceschi J. Chignard M. Lefort J. Marlas G. Eur. J. Pharmacol. 1980; 68: 451-464Crossref PubMed Scopus (62) Google Scholar). Platelet aggregation induced by Cvx has been shown to be Ca2+-dependent but fibrinogen-, ADP-, and cyclooxygenase-independent (6Vargaftig B.B Joseph D. Wal F. Marlas G. Chignard M. Chevance L.G. Eur. J. Pharmacol. 1983; 92: 57-68Crossref PubMed Scopus (28) Google Scholar). The transduction pathway activated by Cvx involves the activation of a phospholipase Cγ and activation of tyrosine kinases (7Faili A. Randon J. Francischetti I.M.B. Vargaftig B.B Hatmi M. Biochem. J. 1994; 298: 87-91Crossref PubMed Scopus (26) Google Scholar). 2I. M. B. Francischetti, F. A. Ghazaleh, R. A. M. Reis, C. R. Carlini, and J. A. Guimarães, submitted for publication. Cvx also binds to rabbit platelets with a high affinity (8Francischetti I.M.B. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Most studies performed on Cvx have been conducted on animal, and particularly rabbit, platelets, but platelet membrane components are better known in humans. We have therefore focused our studies on Cvx interaction with human platelets to elucidate its mechanism of action. We show that Cvx is a particularly potent activator of human platelets, inducing an increase in [Ca2+] i, granule exocytosis, and aggregation, and that immobilized Cvx induces a cation-independent platelet adhesion. Cvx binds specifically and rapidly to washed and to fixed platelets with a high affinity. Use of specific antibodies showed that GPIb, GPIV, and GPV do not interact with Cvx but that both the platelet integrin α2ॆ1 (GPIa-IIa) and GPVI are involved in platelet activation induced by Cvx, and that GPVI acts as a platelet receptor for Cvx. Cvx was purified from the venom of C. d. terrificus (Pentapharm, Basel, Switzerland and Syntex Laboratories, Ribero Preto, Brazil) as described previously (8Francischetti I.M.B. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Cvx was labeled with 125I using the IODOGEN procedure (Pierce) and Na125I (Amersham, Les Ulis, France). Specific activity of the labeled compound was 9 MBq/mg, and labeling did not modify the biological properties of Cvx. The monoclonal anti-GPIbα antibody, SZ2, and the anti-GPV antibody, SW16, were purchased from Immunotech (Marseille, France). The anti-α2ॆ1 integrin monoclonal antibodies used were 6F1 (9Coller B.S. Beer J.H. Scudder L.E. Steinberg M.H. Blood. 1989; 74: 182-192Crossref PubMed Google Scholar), a generous gift from Dr. Barry S. Coller (Mount Sinai Medical Center, New York, NY), and Gi9, purchased from Immunotech. The anti-GPIV antibody FA6-152 produced by Dr. Lena Edelman (Institut Pasteur, Paris, France) was kindly provided by Dr. C. Legrand (INSERM U-353, Paris, France). Purification of human polyclonal anti-GPVI IgG, from the serum of a patient with autoimmune thrombocytopenia (10Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar), was performed by chromatography on protein A-Sepharose (Pharmacia Biotech Inc., Uppsala, Sweden). Fab fragments were prepared from purified IgG incubated for 90 min at 37 °C, with papain (Sigma; 1 ॖg/100 ॖg of IgG), in 20 mm sodium phosphate, 150 mm NaCl (PBS), pH 7.4, containing 1 mm EDTA and 1.77 (v/v) ॆ-mercaptoethanol. Iodoacetamide (30 mm) was then added and left for 15 min at 37 °C, and the sample was dialyzed in PBS. Fc fragments were removed on protein A-Sepharose. The purity of Fab fragments and the absence of contaminating IgG, in particular, were controlled by SDS-PAGE. Purified anti-GPVI IgG induced platelet aggregation, whereas Fab fragments inhibited both collagen- and anti-GPVI IgG-induced platelet aggregation as reported previously (10Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar). Control human IgG and Fab fragments used in these studies were prepared from the plasma of healthy donors according to the procedures described above. Anti-Cvx antibody was kindly provided by Dr. M. Leduc (Unité des Venins Institut Pasteur). Calf skin collagen type I, obtained from Stago (Asnières, France), was used according to the manufacturer's instructions. Blood from healthy human volunteers was collected by venipuncture on acid-citrate-dextrose anticoagulant (ACD-A) or on trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation at 110 × g for 15 min. Platelet secretion and adhesion were determined using platelets labeled in PRP (ACD-A) incubated with 0.6 ॖm[14C]5-hydroxytryptamine (Amersham, Les Ulis, France) and 2 ॖCi/ml 51Cr (CIS International, Gif-sur-Yvette, France), for 30 min at 37 °C respectively. Platelets were sedimented at 1100 × g for 15 min after acidification of the PRP to pH 6.5 with ACD-A and addition of 25 ॖg/ml apyrase (Sigma) and 100 nm prostaglandin E1 (Sigma) and resuspended in washing buffer (103 mm NaCl, 5 mm KCl, 1 mmMgCl2, 5 mm glucose, 36 mm citric acid) at pH 6.5 containing 3.5 mg/ml BSA (Sigma), 25 ॖg/ml apyrase, and 100 nm prostaglandin E1 (11Jandrot-Perrus M. Guillin M.C. Nurden A.T. Thromb. Haemostasis. 1987; 58: 915-920Crossref PubMed Scopus (19) Google Scholar). After sedimentation, the platelets were washed twice in this buffer and resuspended at 3 × 108/ml in the reaction buffer composed of 5 mmHepes, 137 mm NaCl, 2 mm KCl, 1 mmMgCl2, 12 mm NaHCO3, 0.3 mm NaH2PO4, 5.5 mmglucose, pH 7.4, containing 3.5 mg/ml BSA. Formaldehyde-fixed platelets were prepared by incubating 1 volume of citrated PRP with 0.98 volume of 10 mm Tris, 150 mm NaCl, pH 7.0, and 0.02 volume of 307 (w/v) formaldehyde for 18 h at room temperature and in the dark. Fixed platelets were washed three times in PBS before resuspension in PBS. PRP or washed platelets were preincubated for 3 min at 37 °C in the presence of buffer or antibodies before aggregation was initiated by Cvx or collagen. Experiments were performed under stirring conditions at 37 °C in a Chrono-Log aggregometer (Chrono-Log Corp, Haverton, PA). Release of [14C]5-HT was measured as described previously (11Jandrot-Perrus M. Guillin M.C. Nurden A.T. Thromb. Haemostasis. 1987; 58: 915-920Crossref PubMed Scopus (19) Google Scholar). Platelet adhesion was measured on microtitration plates. Typical experiments were performed as follows. Collagen (2 ॖg) in 100 ॖl of 20 mm acetic acid, Cvx (1.4 ॖg) in 100 ॖl of PBS, or BSA (5 ॖg) in 100 ॖl of PBS were immobilized on Immulon II plates (Dynatech, St-Cloud, France) for 2 h at room temperature. Plates were then saturated with 2 mg/ml BSA in PBS for 1 h, and washed with PBS and with reaction buffer.51Cr-Labeled platelets (2 × 108/ml in reaction buffer, 100 ॖl) were added to the wells in the presence or absence of 300 ॖm Arg-Gly-Asp-Ser (RGDS) peptide (Bachem Biochimie, Voisins-le-Bretonneux, France) or 2 mm EDTA. A certain number of experiments were performed in the presence of antibodies, as indicated in the text. Wells were emptied and washed three times with reaction buffer after different incubation times at room temperature, One hundred ॖl of 27 SDS (w/v) was subsequently added to each well, and the samples were counted for51Cr. Intracellular free calcium ([Ca2+] i) transients were monitored by fura-2 fluorescence. Platelets were resuspended in washing buffer after centrifugation of the PRP and loaded with 2 ॖm fura 2-acetoxymethylester (fura 2-AM, Sigma) for 60 min at 37 °C, centrifuged again, washed twice, and resuspended in reaction buffer. The platelets (2 × 108/ml) were then preincubated with 2 mm CaCl2 in the absence or presence of different antibodies for 3 min at 37 °C prior to addition of Cvx or collagen. Fluorescence was measured at 37 °C using two excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm on an Hitachi H-2000 spectrofluorimeter (Sciencetec, Les Ulis, France). Maximal and minimal fluorescence were determined after platelet lysis with 17 (v/v) Triton X-100 and the addition of 2 mm EGTA, respectively. Ca2+ concentrations were calculated using aK d of 224 nm for the interaction between fura 2 and Ca2+ (12Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Washed or formaldehyde-fixed platelets (3 × 108/ml) were incubated at 37 °C in 400 ॖl with various amounts of125I-labeled Cvx. The platelets were transferred to tubes containing 500 ॖl of 207 (w/v) sucrose in PBS after different incubation times, and centrifuged for 5 min at 12,000 ×g. Supernatants were aspirated, and the tips of the tubes were counted for 125I to determine the fraction of Cvx bound to platelets. Nonspecific binding was determined in the presence of a 100–500-fold excess of unlabeled Cvx. Platelet lysates were prepared by solubilization of washed platelets (109/ml) with 27 (w/v) SDS in 20 mm Tris-HCl, 150 mm NaCl, 3 mm EDTA, 5 mm N-ethylmaleimide (Sigma), for 5 min at 100 °C. Proteins (7 ॖg) were separated on 107 acrylamide slab gels (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and transferred to nitrocellulose membranes (Bio-Rad, Ivry-sur-Seine, France). The membranes were then soaked with 57 (w/v) nonfat dry milk in PBS, incubated with 125I-labeled Cvx (50 ng/ml) in PBS, pH 7.4, containing 0.57 (w/v) dry milk and 0.27 (v/v) Tween 20, washed, and exposed to X-AR films (Kodak). Nitrocellulose sheets were incubated alternatively with 9 ॖg/ml anti-GPVI IgG in PBS, pH 8.6, containing 0.17 dry milk and 0.017 Tween 20, followed by125I-labeled protein A or peroxidase-coupled protein A revealed by chemiluminescence (Amersham). Platelet lysates were obtained by solubilization of washed platelets (5 × 109/ml) with 17 (v/v) Nonidet P-40 in 20 mm Tris-HCl, pH 8.0, 150 mm NaCl containing 15 ॖg/ml leupeptin (Sigma), 50 kallikrein-inactivating units of aprotinin, 1 mmphenylmethylsulfonyl fluoride (Sigma), 2 mm benzamidine HCl, and 2 mm EDTA (lysis buffer), at 4 °C for 30 min followed by centrifugation at 13,000 × g at 4 °C for 30 min. Lysates were precleared by incubation with protein A-Sepharose for 30 min at 4 °C and centrifugation to avoid nonspecific precipitation. Cleared lysates were incubated with 200 ॖg/ml anti-GPVI IgG for 30 min at room temperature and then protein A-Sepharose at 4 °C overnight. Samples were centrifuged at 2,000 × g, and the immunoprecipitates were washed three times with the lysis buffer. Immunoprecipitated proteins were eluted by 27 SDS in Laemmli buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes, which were probed using 125I-Cvx or anti-GPVI IgGs, as described above. Washed human platelets aggregated in response to Cvx (Fig.1) as reported previously for rabbit platelets (6Vargaftig B.B Joseph D. Wal F. Marlas G. Chignard M. Chevance L.G. Eur. J. Pharmacol. 1983; 92: 57-68Crossref PubMed Scopus (28) Google Scholar). Aggregation was also observed in PRP and was preceded by a change in cell shape. The effect of Cvx was tested on formaldehyde-fixed platelets to verify that Cvx induced true platelet aggregation rather than passive agglutination. No modification of light transmittance was observed for suspensions of fixed platelets, indicating that Cvx does not agglutinate platelets (data not shown). The threshold concentration of Cvx that typically induced platelet aggregation was between 15 and 35 pm. Important heterogeneity in platelet sensitivity to Cvx was observed with platelets from normal individuals however. Secretion of [14C]5-HT from dense granules paralleled aggregation. The dose-response curve was sigmoid and characterized by a very abrupt slope (Fig. 2 A). Aggregation was prevented by 2 mm EDTA and by 300 ॖm RGDS peptide, which block fibrinogen binding to integrin αIIbॆ3. In contrast, neither cell shape change nor [14C]5-HT release were inhibited by EDTA or RGDS peptide (Fig. 2 B). Along with granule exocytosis and cell aggregation, Cvx induced a dose-dependent increase in [Ca2+] i. Following a lag phase, which could be shortened by increasing the Cvx concentration, [Ca2+] i reached a plateau that remained stable for 3 min (Fig. 3).Figure 2Convulxin-induced platelet secretion.[14C]5-HT-labeled washed platelets (3 × 108/ml) were incubated with various concentrations of Cvx in the aggregometer cuvette. After 2 min of incubation, samples were transferred to EDTA and centrifuged and the secretion of [14C]5-HT was measured as described under 舠Experimental Procedures.舡 The release is expressed as the percentage of the total platelet content in [14C]5-HT. In A, experiments were performed in the presence of 2 mmCaCl2 and results are the mean ± S.D. of five experiments. In B, platelets were suspended in reaction buffer containing: 2 mm CaCl2 (open bar), 2 mm EDTA (dotted bar), or 300 ॖm RGDS (hatched bar). Cvx concentration was 200 pm. Results are from one representative experiment out of three.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Convulxin-induced increase in intracellular Ca2+. Platelets (2 × 108/ml) that had been loaded with Fura 2-AM were incubated at 37 °C with various concentrations of Cvx: a, 91 pm; b, 61 pm; c, 46 pm. Fluorescence changes were monitored as described under 舠Experimental Procedures.舡 The Ca2+ mobilization curves that are illustrated here are representative of three similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 51Cr-Labeled platelets were found to bind to microtitration plates coated with Cvx (Fig.4). Platelets also bound to immobilized collagen under similar static conditions. Platelet adhesion was a function of the coating concentration in both cases and reached a maximum for Cvx and collagen concentrations ≥ 10 ॖg/ml (data not shown). A total of 43 ± 27 of the platelets were bound to immobilized Cvx after 1 h of incubation when the proportion of platelets bound to immobilized collagen was 2-fold lower and less than 0.57 were bound to immobilized BSA. The number of platelets bound to Cvx and collagen decreased to 12.7 ± 0.57 and 8.6 ± 0.87, respectively, due to inhibition of platelet aggregation when experiments were performed in the presence of 300 ॖm RGDS (Fig. 4). These results indicate that platelet aggregation is more important in Cvx-coated wells than collagen-coated wells and confirms that Cvx has a high potency for inducing platelet activation. The proportion of platelets bound to Cvx remained unchanged in the presence of EDTA (2 mm) as in the presence of RGDS, but platelet adhesion to collagen was prevented by EDTA (Fig. 4), as reported previously (14Santoro S.A. Cell. 1986; 46: 913-920Abstract Full Text PDF PubMed Scopus (294) Google Scholar). 125I-Cvx bound to washed platelets in a time- and concentration-dependent manner (Fig.5). Nonspecific binding measured in the presence of a 100-fold excess of unlabeled Cvx represented less than 107 of the total radioactivity added. Association of125I-Cvx with human platelets at 37 °C was rapid and reached a steady state at 15 min (Fig. 5 A). Addition of a 500-fold excess of unlabeled Cvx 15 min after the addition of125I-Cvx to platelets showed, in contrast, that dissociation was very slow, with less than 17 of the bound125I-Cvx being displaced per minute in the presence of a 100-fold excess of unlabeled Cvx (data not shown). Scatchard plot analysis of specific binding of 125I-Cvx to human platelets indicated two classes of binding sites, one of high affinity and low capacity and one of moderate affinity and capacity (TableI). Comparable results were obtained when formaldehyde-fixed platelets were used. Affinities were slightly lower in this case, but receptor number was increased for both classes of sites (Table I). Additional experiments were performed in the presence of sugars such as 45 mm galactose or mannose or in the presence of 10 ॖg/ml WGA, suspected previously to interact with the Cvx binding site on platelets (15Sano-Martins S.J. Daimon T. Toxicon. 1992; 30: 141-150Crossref PubMed Scopus (5) Google Scholar), to rule out the possibility that Cvx binding to platelets might be due to a lectin-like activity. None of the above compounds modified Cvx binding to platelets (data not shown).Table IBinding of 125I-labeled Cvx to human plateletsSitesWashed plateletsFixed plateletsK dB maxK dB maxnmmol/plateletnmmol/plateletHigh affinity sites0.08 ± 0.02130 ± 300.22 ± 0.09300 ± 160Moderate affinity sites1.20 ± 0.141700 ± 4004.42 ± 1.02900 ± 450Washed or formaldehyde-fixed platelets (3 × 108/ml) were incubated at 37 °C with various concentrations of 125I-Cvx. Specific binding of Cvx to platelets was determined as described in the Methods section. Results from the Scatchard analysis are the means ± S.E. of three independent experiments. K d is expressed in nm and B max in molecules/platelet. Open table in a new tab Washed or formaldehyde-fixed platelets (3 × 108/ml) were incubated at 37 °C with various concentrations of 125I-Cvx. Specific binding of Cvx to platelets was determined as described in the Methods section. Results from the Scatchard analysis are the means ± S.E. of three independent experiments. K d is expressed in nm and B max in molecules/platelet. Specific antibodies to known membrane glycoproteins were tested for their effect on Cvx-induced platelet activation to discover whether these glycoproteins were involved in Cvx interaction with platelets. We tested antibodies to glycoproteins already identified as receptors for different cell agonists or ligands, such as the GPIb-V-IX complex (receptor for von Willebrand factor), the integrin α2ॆ1 and GPVI (receptors for collagen), and GPIV (CD 36), which has a less clearly defined function and may act as a collagen and/or thrombospondin receptor (16Clemetson K.J. Thromb. Haemostasis. 1995; 74: 111-116Crossref PubMed Scopus (73) Google Scholar). Monoclonal antibodies with inhibitory activity against GPIbα (SZ2), GPIV (FA6-152), and GPV (SW16) had no effect on Cvx-induced platelet aggregation, platelet adhesion to Cvx, or Cvx binding to platelets (data not shown). Cvx-induced platelet aggregation and secretion were both inhibited, in contrast by 6F1, a monoclonal antibody against integrin α2ॆ1and by polyclonal anti-GPVI Fab fragments (Fig.6, A and B). Inhibition by 6F1 was concentration-dependent and reached a maximum after preincubation of the platelets with 1 ॖg/ml antibody for 3 min at 37 °C. Inhibition was total for low concentrations of Cvx (<50 pm) but could be overcome by increasing the concentration of Cvx above 0.1 nm. Another monoclonal anti-α2ॆ1 antibody, Gi9, also inhibited Cvx-induced platelet aggregation but was less potent than 6F1, inhibition being 307 and 807 with 1 ॖg/ml and 5 ॖg/ml of Gi9, respectively. The anti-GPVI Fab fragments, at 0.2 mg/ml, produced a ∼507 inhibition of platelet aggregation and secretion induced by 30–40 pm Cvx. As with 6F1, inhibition of platelet aggregation and secretion by the anti-GPVI Fab fragments was overcome by increasing the concentration of Cvx. Control experiments showed that nonimmune human Fab had no effect on Cvx-induced platelet aggregation and secretion (data not shown). The concentrations of 6F1, Gi9, and anti-GPVI Fab fragments that inhibited Cvx-induced platelet aggregation also inhibited collagen-induced platelet aggregation (data not shown and Refs. 9Coller B.S. Beer J.H. Scudder L.E. Steinberg M.H. Blood. 1989; 74: 182-192Crossref PubMed Google Scholar and 10Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar). The increase in [Ca2+] i concentration induced by Cvx was prevented by preincubation of platelets with the anti-GPVI Fab fragments (Fig. 6 C). 6F1 (1 ॖg/ml) did not decrease the signal induced by Cvx in contrast but completely inhibited Ca2+ mobilization induced by collagen (Fig. 6 C). The effects of 6F1 and anti-GPVI Fab-fragments on platelet adhesion to immobilized Cvx and collagen were also analyzed. All experiments were performed in the presence of 300 ॖm RGDS to avoid platelet aggregation. None of the antibodies modified the number of51Cr-labeled platelets bound to immobilized Cvx after a 60-min incubation, whereas 6F1 inhibited platelet adhesion to collagen by 907 (data not shown), as shown by others (9Coller B.S. Beer J.H. Scudder L.E. Steinberg M.H. Blood. 1989; 74: 182-192Crossref PubMed Google Scholar). We used shorter incubation times (5 min for Cvx and 15 min for collagen) to increase the chances of observing an inhibition since binding of Cvx to platelets is rapid, whereas dissociation is very slow. Approximately the same percentage of platelets adhered to the two proteins under these conditions. Platelet adhesion to Cvx at 5 min was not significantly reduced by the presence of 6F1 (even at concentrations up to 10 ॖg/ml), but platelet adhesion to collagen at 15 min was clearly inhibited by 6F1 at a concentration as low as 0.5 ॖg/ml (Fig.7). Gi9 also failed to inhibit platelet adhesion to Cvx, but it inhibited platelet adhesion to collagen (data not shown). The anti-GPVI Fab fragments (0.2 mg/ml) inhibited platelet adhesion to Cvx significantly in contrast, and also inhibited platelet adhesion to collagen at 15 min (Fig. 7). The possibility that Cvx recognizes a specific platelet protein was tested using a ligand blotting assay, since Cvx has a very high affinity for platelets. Binding of125I-Cvx was observed to occur on a single band ofM r = 57,000 ± 1000 (Fig.8 A) after separation of proteins from whole platelet lysates by SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes. Labeling of this band was not observed when the incubation was performed in the presence of a 100-fold excess of unlabeled Cvx, or 50 ॖg/ml anti-Cvx antibody, indicating that binding was specific. No binding of labeled Cvx was observed when the platelet proteins were reduced with 57 ॆ-mercaptoethanol before electrophoresis. Binding was not inhibited in the presence of 5 ॖg/ml WGA, and was still observed after pretreatment of platelets with 5 nm thrombin, or 3 ॖg/ml protease of Serratia marcescens or 1 ॖmα-chymotrypsin, respectively (data not shown). The latter findings indicated that this 57-kDa protein was neither a binding site for WGA nor degraded to any great extent by these proteases. We also observed a protein band migrating at the same position in membrane extracts obtained after cellular fractionation (data not shown). It appeared likely that this 57-kDa platelet protein corresponded to GPVI since the size of the protein recognized by 125I-Cvx was similar to that reported for GPVI (10Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. Blood. 1987; 69: 1712-1720Crossref PubMed Google Scholar, 15Sano-Martins S.J. Daimon T. Toxicon. 1992; 30: 141-150Crossref PubMed Scopus (5) Google Scholar, 16Clemetson K.J. Thromb. Haemostasis. 1995; 74: 111-116Crossref PubMed Scopus (73) Google Scholar) and platelet activation by Cvx was inhibited by the anti-GPVI Fab fragments. Immunoprecipitates of platelet lysates obtained with the anti-GPVI IgG were probed with125I-Cvx to test this possibility. Fig. 8 B shows that the immunoprecipitated platelet protein interacted with125I-Cvx. Competition experiments using nitrocellulose membranes incubated with the anti-GPVI IgG in the presence of cold Cvx were therefore performed. The intensity of the band labeled by the anti-GPVI IgG decreased as the concentration of Cvx increased, thus confirming that Cvx and the anti-GPVI IgG bind to the same protein,i.e. GPVI (Fig. 8 C). The present study demonstrates that Cvx is a very potent activator of human platelets and that the membrane glycoproteins GPVI and integrin α2ॆ1 are both involved in the activation pathway. Cvx at picomolar concentrations induces extensive activation of stirred platelets, resulting in exocytosis of dense granules and aggregation. Although some authors (6Vargaftig B.B Joseph D. Wal F. Marlas G. Chignard M. Chevance L.G. Eur. J. Pharmacol. 1983; 92: 57-68Crossref PubMed Scopus (28) Google Scholar) have reported that Cvx-induced activation of rabbit platelets is calcium-dependent, our results show that Cvx-induced platelet activation of human platelets occurs in the absence of external Ca2+ and is in agreement with the findings of Sano-Martins and Daimon (15Sano-Martins S.J. Daimon T. Toxicon. 1992; 30: 141-150Crossref PubMed Scopus (5) Google Scholar). However, Cvx induces an increase in [Ca2+] i. The kinetic of Cvx-induced Ca2+ mobilization is slow when compared with the very rapid response induced by other agonists such as thrombin (19Kroll M.H. Schafer A.I. Blood. 1989; 74: 1181-1195Crossref PubMed Google Scholar), and t