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• W2073590210 abstract "SummaryPlatelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI deficiency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no specific, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a purified system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and FcγRIIA, 9O12.2 Fab fragments completely blocked collagen-induced platelet aggregation and secretion from 5 µg mL−1 and fully prevented CRP-induced activation from 1.5 µg mL−1. 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial flow conditions. We thus describe here for the first time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth. Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI deficiency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no specific, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a purified system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and FcγRIIA, 9O12.2 Fab fragments completely blocked collagen-induced platelet aggregation and secretion from 5 µg mL−1 and fully prevented CRP-induced activation from 1.5 µg mL−1. 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial flow conditions. We thus describe here for the first time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth. Platelet adhesion to collagen and collagen-induced platelet activation are primary key events not only in hemostasis, by initiating plug formation and limiting blood loss, but also in the acute arterial occlusion associated with atherothrombotic disease. Platelet adhesion and aggregation on collagen is a complex process that involves several receptors [1Clemetson K.J. Clemetson J.M. Platelet collagen receptors.Thromb Haemost. 2001; 86: 189-97Crossref PubMed Scopus (238) Google Scholar]. One of these, glycoprotein (GP)VI, plays a critical role as it mediates platelet activation, platelet recruitment and thrombus growth [2Moroi M. Jung S.M. Okuma M. Shinmyozu K. A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.J Clin Invest. 1989; 84: 1440-5Crossref PubMed Google Scholar, 3Kehrel B. Wierwille S. Clemetson K.J. Anders O. Steiner M. Knight C.G. Farndale R.W. Okuma M. Barnes M.J. Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not.Blood. 1998; 91: 491-9Crossref PubMed Google Scholar, 4Heemskerk J.W. Siljander P. Vuist W.M. Breikers G. Reutelingsperger C.P. Barnes M.J. Knight C.G. Lassila R. Farndale R.W. Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.Thromb Haemost. 1999; 81: 782-92Crossref PubMed Scopus (0) Google Scholar, 5Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.EMBO J. 2001; 20: 2120-30Crossref PubMed Scopus (0) Google Scholar]. GPVI is a type I transmembrane protein which belongs to the immunoglobulin superfamily. Its expression is restricted to platelets and megakaryocytes [6Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.Blood. 2000; 96: 1798-807Crossref PubMed Google Scholar]. GPVI is expressed associated with the common Fc receptor γ chain (FcRγ), which acts as the signaling subunit of the complex [7Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.FEBS Lett. 1997; 413: 255-9Crossref PubMed Scopus (257) Google Scholar]. The crucial role of GPVI in the development of vascular lesions has recently been shown in mice displaying immunological depletion [8Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice.J Exp Med. 2001; 193: 459-69Crossref PubMed Scopus (294) Google Scholar] or deficient expression of GPVI [9Konishi H. Katoh Y. Takaya N. Kashiwakura Y. Itoh S. Ra C. Daida H. Platelets activated by collagen through immunoreceptor tyrosine-based activation motif play pivotal role in initiation and generation of neointimal hyperplasia after vascular injury.Circulation. 2002; 105: 912-6Crossref PubMed Scopus (0) Google Scholar]. Impairment of GPVI function results in a long-term antithrombotic effect and prevents neointimal hyperplasia following arterial injury. Interestingly, in these animal models, GPVI deficiency was not associated with increased bleeding, as observed in animals treated with antagonists of the integrin αIIbβ3 or of GPIb. Furthermore, the few patients identified with GPVI deficiency [2Moroi M. Jung S.M. Okuma M. Shinmyozu K. A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.J Clin Invest. 1989; 84: 1440-5Crossref PubMed Google Scholar, 10Arai M. Yamamoto N. Moroi M. Akamatsu N. Fukutake K. Tanoue K. Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency.Br J Haematol. 1995; 89: 124-30Crossref PubMed Google Scholar] present only a mild bleeding tendency, although their platelets display severely impaired responses to collagen. GPVI antagonists may thus have a therapeutic potential in atherothrombosis with the clear-cut advantage of being safe. There is thus a real need for potent, specific and accessible antagonists of human GPVI. To date, only Fab fragments of a polyclonal anti-GPVI antibody raised in one patient have been reported to block human GPVI function [11Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.Blood. 1987; 69: 1712-20Crossref PubMed Google Scholar, 12Goto S. Tamura N. Handa S. Arai M. Kodama K. Takayama H. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions.Circulation. 2002; 106: 266-72Crossref PubMed Scopus (0) Google Scholar]. We have developed several antihuman GPVI monoclonal antibodies (MoAb), obtained by gene immunization of mice with the sequence encoding the extracellular domain of the receptor. All the MoAbs obtained are able to activate platelets. However, one antibody, 9O12.2, blocked the binding of recombinant soluble GPVI to both collagen and convulxin, a specific ligand of GPVI. 9O12.2 was selected to study further its properties and evaluate its utility as a GPVI blocker in humans. 9O12.2 inhibits the adhesion to immobilized collagen and collagen-related peptides (CRP) of cells expressing recombinant GPVI. We show that the Fab fragments of this antibody are potent specific inhibitors of collagen-induced platelet responses, such as aggregation and secretion, and they limit collagen-induced platelet procoagulant activity. 9O12.2 Fabs also prevent GPVI-dependent platelet adhesion to collagen in static conditions. Furthermore, they inhibit thrombus formation under flow at arterial flow conditions. The properties of 9O12.2 are those expected for an accessible blocker of human GPVI and provide a basis for the design of new GPVI antagonists of therapeutic potential. Collagen from equine tendon (Horm collagen) was from Nycomed (Munich, Germany); acid-insoluble fibrillar type I collagen from bovine tendon, prostaglandin E1 (PGE1), grade VII apyrase, the thromboxane analog U46619, RGD peptide, orthophenylenediamine dihydrochloride (OPD) and soluble papain from Sigma (St Louis, MO, USA); H-D-Phe-Arg-pNa (S2238) was from Biogenic (Mauguio, France); bovine serum albumin Cohn fraction V from Research Organics Inc. (Cleveland, OH, USA); immobilized pepsin from Perbio Science (Helsingborg, Sweden); 14C-5-hydroxytryptamine, HiTrap protein A-Sepharose, peroxidase-coupled antimouse antibodies and peroxidase-coupled protein A from Amersham (Les Ulis, France); bovine factor (F)Xa from ERL (South Bend, IN, USA); FITC-coupled annexin V from Beckman-Coulter (Villepinte, France). Human prothrombin was purified in the laboratory [13Bezeaud A. Denninger M.H. Guillin M.C. Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin.Eur J Biochem. 1985; 153: 491-6Crossref PubMed Google Scholar]. Convulxin was purified from the venom of Crotalus durrissus terrificus as described [14Francischetti I.M. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets.Toxicon. 1997; 35: 1217-28Crossref PubMed Scopus (95) Google Scholar]. Collagen-related peptides (CRP-XL) were a generous gift from R. Farndale (Cambridge, UK) [15Morton L.F. Hargreaves P.G. Farndale R.W. Young R.D. Barnes M.J. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.Biochem J. 1995; 306: 337-44Crossref PubMed Scopus (0) Google Scholar]. The anti-FcγRIIA monoclonal antibody IV.3 (sodium azide-free) was from Medarex (Annandale, NJ, USA). Irrelevant mouse Fab fragments, and donkey antihuman Fc antibodies coupled to rhodamine (RedX) were from Jackson ImmunoResearch Labs Inc. (West Grove, PA, USA). Mouse IgG1 and FITC-coupled antimouse F(ab′)2 were from Beckman-Coulter (Villepinte, France). FITC-coupled anticollagen type III antibody was from Research Diagnostics Inc. (Flanders, NJ, USA). The anti-α2β1 monoclonal antibody 6F1 was a kind gift from B. S. Coller (Mount Sinai Medical Center, New York, NY, USA). The human polyclonal anti-GPVI antibodies and Fab fragments were purified from the same batch of a patient's serum [16Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin alpha2beta1.J Biol Chem. 1997; 272: 27035-41Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] kindly provided by M. Okuma (Kyoto University, Japan) [11Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.Blood. 1987; 69: 1712-20Crossref PubMed Google Scholar]. The recombinant soluble human GPVI (rsGPVI-Fc) consists of two extracellular (EC) domains of the receptor, coupled to the Fc fragment of human IgG1. rsGPVI-Fc was produced as already reported [6Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.Blood. 2000; 96: 1798-807Crossref PubMed Google Scholar] and purified by affinity chromatography on HiTrap-Protein A Sepharose. The human cell lines HEL and U937 were engineered to express GPVI as reported [6Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.Blood. 2000; 96: 1798-807Crossref PubMed Google Scholar]. The MoAbs 9O12.2, 3J24.2, 8M14.3 and 1P10.2 were obtained by immunizing Balb/C mice with the cDNA encoding the rsGPVI-Fc fusion protein using the GeneGun technique, as already reported [17Lagrue-Lak-Hal A.H. Debili N. Kingbury G. Lecut C. Le Couedic J.P. Villeval J.L. Jandrot-Perrus M. Vainchenker W. Expression and function of the collagen receptor GPVI during megakaryocyte maturation.J Biol Chem. 2001; 276: 15316-25Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar]. Antihuman GPVI MoAbs were selected by their ability to bind to rsGPVI-Fc in ELISA assays. They were typed as IgG1. 7I20.2 is an isotypic non-relevant IgG. IgGs were purified from cell culture media or from mouse ascites by chromatography on Hi-Trap protein A Sepharose according to the manufacturer's instructions. Purified MoAbs were proteolyzed by soluble papain to obtain Fab fragments, or immobilized pepsin to obtain F(ab′)2 fragments, according to the manufacturers' protocols. The fragments were separated from Fc fragments and non-digested IgG by affinity chromatography on HiTrap Protein A-Sepharose. Purity was checked by SDS–PAGE and blotting with peroxidase-coupled Protein A. Human washed platelets were obtained according to a previously described procedure [16Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin alpha2beta1.J Biol Chem. 1997; 272: 27035-41Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Briefly, blood from healthy volunteers with appropriate consent was collected on acid–citrate–dextrose anticoagulant (ACD-A) and centrifuged at 110 × g for 15 min to obtain platelet-rich plasma (PRP). PRP was centrifuged for 15 min at 1100 × g in the presence of 1/10 v of ACD-A, 100 nm PGE1 and 25 µg mL−1 apyrase. Platelets were washed twice in a modified Tyrode's buffer pH 6.5, containing 100 nm PGE1, 25 µg mL−1 apyrase, 1 mm MgCl2, 2 mm CaCl2 and 3.5 mg mL−1 bovine serum albumin (BSA). Washed platelets were resuspended at the desired concentration in a modified Tyrode-HEPES buffer pH 7.4 containing 1 mm MgCl2, 2 mm CaCl2 and 3.5 mg mL−1 BSA. Platelets (5 × 107 mL−1) were incubated with the purified monoclonal IgGs (5–10 µg mL−1) for 30 min at room temperature. After washing, FITC-coupled antimouse antibodies were added for 30 min and the samples were analyzed in a flow cytometer (FACscalibur; Beckman-Coulter). Alternatively, cells were incubated with FITC-coupled MoAbs, prepared as already described [17Lagrue-Lak-Hal A.H. Debili N. Kingbury G. Lecut C. Le Couedic J.P. Villeval J.L. Jandrot-Perrus M. Vainchenker W. Expression and function of the collagen receptor GPVI during megakaryocyte maturation.J Biol Chem. 2001; 276: 15316-25Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar], before flow cytometry analysis. RsGPVI-Fc binding to collagen Microtitration wells were coated overnight with 20 µg mL−1 collagen type I from equine tendon (Horm) in PBS and blocked with 0.2% BSA in PBS for 2 h. rsGPVI-Fc was then added at different concentrations, or for different times, in the presence or absence of anti-GPVI MoAbs. After washing, bound rsGPVI-Fc was detected using peroxidase-coupled Protein A and subsequent hydrolysis of OPD. Binding of convulxin to rsGPVI-Fc Microtitration wells were coated with a non-blocking anti-GPVI MoAb, 1P10.2 (5 µg mL−1) and saturated with BSA. rsGPVI-Fc (0.5 nm) was added for 2 h at room temperature. After washing and preincubation of the wells with anti-GPVI MoAbs or buffer, 125I-Cvx was added and the incubation was prolonged for 10 min. Wells were washed and counted for 125I-Convolxin (Cvx) in a γ-counter. Mouse aortas were fixed in formalin and paraffin-embedded. Cryostat sections were fixed in paraformaldehyde–lysine–periodate. rsGPVI-Fc (0.1 µg mL−1) was preincubated for 15 min with either 10 µg mL−1 9O12.2 or the same concentration of an isotype-matched control antibody (8M14), and then allowed to interact with aorta sections. Bound rsGPVI-Fc was detected using donkey antihuman Fc antibodies coupled to rhodamine (RedX, 2 µg mL−1). Otherwise, sections were incubated with FITC-coupled anticollagen type III antibodies to visualize vascular collagen in the media. Experiments were performed mainly as previously described [6Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.Blood. 2000; 96: 1798-807Crossref PubMed Google Scholar, 17Lagrue-Lak-Hal A.H. Debili N. Kingbury G. Lecut C. Le Couedic J.P. Villeval J.L. Jandrot-Perrus M. Vainchenker W. Expression and function of the collagen receptor GPVI during megakaryocyte maturation.J Biol Chem. 2001; 276: 15316-25Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar]. Briefly, microtitration wells were coated either with collagen type I from equine tendon (Horm), convulxin or CRP (20 µg mL−1 each in PBS). After blocking with BSA, HEL cells (5 × 106 mL−1) in Tyrode–HEPES buffer were added to the wells and allowed to adhere for 45 min under gentle agitation. Adherent cells were quantified by measuring alkaline phosphatase activity using para-nitrophenylphosphate (PNP). Aggregation of human washed platelets (3 × 108 mL−1 in Tyrode–HEPES buffer) was performed at 37 °C under stirring according to standard procedures, in a Chronolog aggregometer (Chrono Log Corp., Havertown, PA, USA). Secretion of dense granule content was measured using 14C-5-HT [16Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin alpha2beta1.J Biol Chem. 1997; 272: 27035-41Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Platelet adhesion to immobilized collagen was performed in static conditions as described above for cell adhesion, using washed platelets at 2 × 108 mL−1. Platelet adhesion to collagen under flow was measured as described elsewhere [18Moog S. Mangin P. Lenain N. Strassel C. Ravanat C. Schuhler S. Freund M. Santer M. Kahn M. Nieswandt B. Gachet C. Cazenave J.P. Lanza F. Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.Blood. 2001; 98: 1038-46Crossref PubMed Scopus (0) Google Scholar]. Briefly, citrate-anticoagulated whole blood was labeled with 1 µm DiOC6 for 10 min and then perfused with a syringe pump at 1500 s−1, through glass capillaries coated with insoluble type I bovine collagen. Platelet interaction with immobilized collagen was monitored in real time under a fluorescence microscope and recorded for off-line analysis. The number of individual adhered platelets was analyzed frame by frame (24 frames s−1) over the first 30 s (6400 µm2 field). Human washed platelets (108 mL−1 in Tyrode–HEPES buffer) were preincubated in the presence of increasing concentrations of 9O12.2 Fabs for 5 min at 37 °C, and activated by 10 µg mL−1 Horm collagen for 10 min at 37 °C. FXa (250 pm) and Ca2+ (5 mm) were added to the activated cells and the incubation was prolonged for 2 min, before addition of 2 µm prothrombin. After 3 min of incubation at 37 °C, the reaction was stopped by addition of 1 mm EDTA. The amount of thrombin formed was quantified by measuring the rate of S2238 hydrolysis at 405 nm. Data are expressed as mean ± SEM. Student's t-test or a one-factor anova followed by the Bonferroni test were used to evaluate statistical significance. A value of P < 0.05 was considered to be statistically significant. 9O12.2 was obtained by immunizing mice with the cDNA encoding the soluble domain of GPVI, expressed as a fusion protein [17Lagrue-Lak-Hal A.H. Debili N. Kingbury G. Lecut C. Le Couedic J.P. Villeval J.L. Jandrot-Perrus M. Vainchenker W. Expression and function of the collagen receptor GPVI during megakaryocyte maturation.J Biol Chem. 2001; 276: 15316-25Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar], and was selected for its unique properties. 9O12.2 bound to human platelets as shown by FACS analysis (Fig. 1A). It also bound to GPVI-transduced U937 cells but not to mock-transduced cells. The specificity of 9O12.2 was restricted to primates as no binding was observed on rodent platelets (mice, rat and rabbit) (data not shown). In Western blot analysis of human platelet proteins under non-reducing conditions, 9O12.2 labeled a 58-kDa band corresponding to GPVI. It did not recognize GPVI after reduction, suggesting that its epitope is structural rather than linear (Fig. 1B). 9O12.2 IgGs were also able to immunoprecipitate GPVI from human platelet lysate (not shown). Purified IgGs induced platelet aggregation after a prolonged lag phase, with a threshold concentration of 2.5 µg mL−1. Aggregation was prevented by anti-GPVI polyclonal IgG Fab fragments from a patient suffering from autoimmune thrombocytopenia [11Sugiyama T. Okuma M. Ushikubi F. Sensaki S. Kanaji K. Uchino H. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.Blood. 1987; 69: 1712-20Crossref PubMed Google Scholar], and also by the anti-FcγRIIA MoAb IV.3 (Fig. 1C). F(ab′)2 fragments derived from 9O12.2 (= 25 µg mL−1) enhanced platelet responses induced by suboptimal doses of collagen. At higher concentrations (= 75 µg mL−1), they induced full aggregation in the absence of any other agonist (Fig. 1D). To evaluate the effect of 9O12.2 on GPVI interactions with its ligands, binding assays were performed using a recombinant soluble form of the receptor. 9O12.2 IgGs inhibited the binding of rsGPVI-Fc to collagen with an IC50 of approximately 1.5 µg mL−1, 90% inhibition being reached for a concentration of 10 µg mL−1 (Fig. 2A). 9O12.2 also prevented the binding of convulxin to immobilized rsGPVI-Fc (Fig. 2B): the IC50 was 3 µg mL−1 and the inhibition reached 80% for concentrations above 25 µg mL−1. In addition, 9O12.2 IgGs (10 µg mL−1) inhibited the binding of rsGPVI-Fc to immobilized CRP by about 95%. In contrast, an isotype-identical control antibody, 7I20, had no effect on the binding of rsGPVI-Fc to collagen, convulxin or CRP. The ability of 9O12.2 IgGs to prevent the binding of GPVI to vascular collagen was then evaluated using an immunofluorescence-based assay. Results are presented in Fig. 2C. rsGPVI-Fc, detected using a rhodamine-coupled anti-Fc antibody, bound to the media of mouse aortic sections (upper left panel) at sites where type III collagen fibers were also found, as visualized using an anticollagen III antibody (lower left panel). The binding of rsGPVI-Fc to vascular collagen was completely prevented in the presence of 10 µg mL−1 9O12.2 IgGs (upper right panel), whereas it was unaffected by the presence of an isotype-identical IgG (10 µg mL−1) (lower right panel), demonstrating that 9O12.2 specifically inhibits GPVI interaction with native type III collagen. As 9O12.2 inhibited the binding of purified soluble GPVI to its ligands, we next addressed the question of its effect on cell-expressed GPVI. HEL cells overexpressing recombinant human GPVI (rGPVI-HEL) at their surface [6Jandrot-Perrus M. Busfield S. Lagrue A.H. Xiong X. Debili N. Chickering T. Le Couedic J.P. Goodearl A. Dussault B. Fraser C. Vainchenker W. Villeval J.L. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.Blood. 2000; 96: 1798-807Crossref PubMed Google Scholar] were allowed to adhere to immobilized collagen, convulxin or CRP, in the presence or absence of 9O12.2 IgGs. 9O12.2 dose-dependently inhibited cell adhesion to collagen and CRP with a similar IC50 of 1 µg mL−1 (Fig. 3A,B) and a plateau reaching 70% and 90% inhibition, respectively. The adhesion of rGPVI-HEL to convulxin was hardly affected by 9O12.2 (<10% inhibition at 10 µg mL−1, Fig. 3C), in apparent contradiction with the results obtained in the binding assay using soluble GPVI. Previous results indicated that 9O12.2 competes with collagen for the binding to GPVI, and should therefore inhibit platelet activation by collagen. To circumvent the activating effect of 9O12.2 IgGs and F(ab′)2, monovalent Fab fragments were used in studies measuring platelet activation induced by collagen and GPVI ligands. Indeed, 9O12.2 Fabs, at concentrations up to 200 µg mL−1, did not induce platelet aggregation or secretion as measured by 14C-5-HT release (data not shown). 9O12.2 Fabs dose-dependently inhibited collagen-induced platelet aggregation (Fig. 4A), full inhibition being attained from 5 to 20 µg mL−1 Fabs, depending on individual blood donors. The Fabs also inhibited collagen-induced aggregation in PRP in the same concentration range (data not shown). Dense granule secretion induced by collagen (1 µg mL−1) decreased from 35% to 4% in the presence of 20 µg mL−1 Fabs. α-Granule exocytosis was inhibited as well, as indicated by a decrease in P-selectin expression (not shown). In contrast, irrelevant mouse Fab fragments or isotype-matched Fab fragments from another anti-GPVI MoAb (3J24.2) did not significantly impair aggregation triggered by collagen (Fig. 4A) and had no effect on granule secretion. The release of 14C-5-HT reached 32% for irrelevant mouse Fabs (50 µg mL−1) and 29.5% for 3J24 Fabs (20 µg mL−1), compared with 35% for the control. 9O12.2 Fabs also behaved as potent inhibitors of CRP-induced platelet aggregation, full inhibition being attained for concentrations of 1–1.5 µg mL−1 (Fig. 4B). 9O12.2 Fabs had little effect on platelet aggregation and secretion induced by convulxin (Fig. 4C), in agreement with the results obtained on adhesion of rGPVI-HEL to immobilized convulxin (Fig. 3C). Only a slight delay in the aggregation could be observed when using a threshold concentration of convulxin (200 pm). Fabs 50 µg mL−1 moderately increased the lag time and had no effect on the amplitude of the response (Fig. 4C). 9O12.2 Fabs at concentrations up to 50 µg mL−1 had no effect on platelet aggregation or secretion induced by thrombin or the TxA2 analog U46619 (Fig. 4D), but the Fabs (20 µg mL−1) completely prevented platelet aggregation induced by 10 µg mL−1 9O12.2 IgGs, while irrelevant or 3J24 isotype-matched Fab fragments did not (Fig. 4E). Taken together, these results demonstrate that 9O12.2 Fab fragments behave as a specific antagonist of GPVI-coupled platelet responses induced by collagen. Collagen-activated platelets expose procoagulant phospholipids at their surface allowing the assembly of the tenase and prothrombinase complexes. We investigated the ability of 9O12.2 Fabs to inhibit the prothrombinase activity of collagen-stimulated washed platelets. Platelets were incubated with increasing doses of 9O12.2 Fabs prior to stimulation by 10 µg mL−1 Horm collagen. Thrombin generation, initiated by the addition of FXa, calcium and prothrombin, was almost completely inhibited by 50 µg mL−1 of 9O12.2 Fabs, reaching a level comparable to that obtained with unstimulated platelets (Fig. 5). Recent studies have suggested that GPVI plays a key role in platelet adhesion to collagen [5Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen.EMBO J. 2001; 20: 2120-30Crossref Pub" @default.
• W2073590210 created "2016-06-24" @default.
• W2073590210 creator A5005081144 @default.
• W2073590210 creator A5017603464 @default.
• W2073590210 creator A5018452732 @default.
• W2073590210 creator A5049243856 @default.
• W2073590210 creator A5061171100 @default.
• W2073590210 creator A5076513030 @default.
• W2073590210 creator A5080727918 @default.
• W2073590210 creator A5089889286 @default.
• W2073590210 date "2003-12-01" @default.
• W2073590210 modified "2023-10-14" @default.
• W2073590210 title "Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments" @default.
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• W2073590210 cites W1604870150 @default.
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