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• W2065571808 abstract "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 µg/ml) was inhibitable by an anti-αIIbβ3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to αIIbβ3-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 Cγ2 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. 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 µg/ml) was inhibitable by an anti-αIIbβ3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to αIIbβ3-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 Cγ2 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. glycoprotein von Willebrand factor phospholipase Cγ2 Fc receptor γ chain In both normal hemostasis and thrombotic disease, platelet adhesion and aggregation may be initiated by engagement of specific membrane receptors that leads to platelet activation and αIIbβ3-dependent aggregation (1Weiss H.J. Thromb. Haemostasis. 1995; 74: 117-122Crossref PubMed Scopus (74) Google Scholar, 2Savage B. Salvidar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar, 3Andrews R.K. López J.A Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (181) Google Scholar, 4Kulkarni S. Dopheide S.M. Yap C.L. Ravanat C. Freund M. Mangin P. Heel K.A. Street A. Harper I.S. Lanza F. Jackson S.P. J. Clin. Invest. 2000; 105: 783-791Crossref PubMed Scopus (299) Google Scholar, 5Watson S.P. Thromb. Haemostasis. 1999; 82: 365-376Crossref PubMed Scopus (112) Google Scholar). At high shear stress, platelet adhesion is dependent on binding of the platelet membrane glycoprotein (GP)1Ib-IX-V complex to its ligand, von Willebrand factor (vWF), and is supported by collagen receptors α2β1 integrin (platelet GPIa-IIa) and/or GPVI (3Andrews R.K. López J.A Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (181) Google Scholar, 5Watson S.P. Thromb. Haemostasis. 1999; 82: 365-376Crossref PubMed Scopus (112) Google Scholar). At low shear, α2β1 and/or GPVI support platelet adhesion and activation without the requirement for vWF (5Watson S.P. Thromb. Haemostasis. 1999; 82: 365-376Crossref PubMed Scopus (112) Google Scholar). Viper venom proteins that activate platelets have played a significant role in elucidating mechanisms of platelet activation. Several groups have described a 50-kDa protein of the C-type lectin family, alboaggregin-A, from venom of the white-lipped tree viper,Trimeresurus albolabris, which binds to GPIb-IX-V (6Peng M. Lu W. Kirby E.P. Thromb. Haemostasis. 1992; 67: 702-707Crossref PubMed Scopus (44) Google Scholar, 7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar, 8Kowalska M.A. Tan L. Holt J.C. Peng M. Karczewski J. Calvete J.J. Niewiarowski S. Thromb. Haemostasis. 1998; 79: 609-613Crossref PubMed Scopus (57) Google Scholar, 9Falati S. Edmead C.E. Poole A.W. Blood. 1999; 94: 1648-1656Crossref PubMed Google Scholar) and GPVI (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar, 11Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 2333-2341Crossref PubMed Scopus (65) Google Scholar) and activates platelets by a mechanism that may involve one or both receptors. A related C-type lectin-like protein, aggretin from the Malayan pit viper, Calloselasma rhodostoma, has been reported to activate platelets by a mechanism that involves GPIb and GPIa-IIa but not GPVI (12Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). An analogous, ∼85-kDa C-type lectin-like protein, convulxin, from the venom of the tropical rattlesnake, Crotalus durissus terrificus, activates platelets by a mechanism involving GPVI (5Watson S.P. Thromb. Haemostasis. 1999; 82: 365-376Crossref PubMed Scopus (112) Google Scholar, 13Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Polgar J. Clemetson J.M. Kehrel B.E. Weidemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 15Leduc M. Bon C. Biochem. J. 1998; 333: 389-393Crossref PubMed Scopus (66) Google Scholar). In the present study, we isolated a protein termed alborhagin from T. albolabris venom that is functionally related to convulxin, because it is a potent agonist at GPVI, but that is a member of the metalloproteinase family. Evidence is presented to suggest that alborhagin binds to GPVI at a site distinct from that of convulxin. Alborhagin therefore represents a novel tool to further characterize GPVI. Lyophilized venom from T. albolabriswas purchased from Sigma or Venom Supplies, Tanunda, South Australia. Bovine α-thrombin was from Sigma. PP1 was purchased from Calbiochem-Novabiochem. Convulxin isolated from the venom of C. durissus terrificus (15Leduc M. Bon C. Biochem. J. 1998; 333: 389-393Crossref PubMed Scopus (66) Google Scholar) was kindly provided by Drs. M. Leduc and C. Bon (Unite des Venens, Institut Pasteur, Paris, France). The cobra venom metalloproteinase mocarhagin was purified from Naja mocambique mocambique venom (Sigma) as previously described (16De Luca M. Dunlop L.C. Andrews R.K. Flannery J.V. Ettling R. Cumming D.A. Veldman G.M. Berndt M.C. J. Biol. Chem. 1995; 270: 26734-26737Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar,17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). The anti-αIIbβ3 monoclonal antibody CRC64 was the gift of Dr. A. Mazurov (Moscow, Russia) and has been described elsewhere (18Khaspekova S.G. Vlasik T.N. Byzova T.V. Vinogradov D.V. Berndt M.C. Mazurov A.V. Br. J. Haematol. 1993; 85: 332-340Crossref PubMed Scopus (23) Google Scholar). An anti-murine GPVI monoclonal antibody, JAQ1, has also been described elsewhere (19Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Rabbit polyclonal antiserum against mocarhagin or convulxin was prepared using standard methods (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar, 20Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (154) Google Scholar). The anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Biotechnology Inc. (Lake Placid, NY). The anti-PLCγ2 and anti-Syk polyclonal antibodies were a gift from Dr. M. G. Tomlinson (DNAX Research Institute, Palo Alto, CA). Throughout the purification procedure, fractions were assayed for their ability to induce platelet aggregation of citrated platelet-rich plasma and analyzed by SDS-polyacrylamide gel electrophoresis as previously described (7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar, 21Andrews R.K. Booth W.J. Gorman J.J. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8317-8326Crossref PubMed Scopus (156) Google Scholar). Lyophilized T. albolabris venom (20 mg) was dissolved in 4 ml of TS buffer (0.01 m Tris-HCl, 0.15 m NaCl, pH 7.4) and loaded at 30 ml/h onto a 1.5 × 20-cm heparin-agarose (Bio-Rad) column and washed with TS buffer. Bound protein was eluted with a linear 0.15–1.0 m NaCl gradient in 0.01m Tris-HCl, pH 7.4. Fractions containing an ∼60-kDa protein (reduced and nonreduced) that eluted from the heparin-agarose column were dialyzed into 5 mmNaH2PO4, pH 6.8 and loaded at 25 ml/h onto a 10 × 1-cm hydroxylapatite column equilibrated in the same buffer. Bound protein was eluted with a linear 5–200 mmNaH2PO4, pH 6.8 gradient. Fractions containing alborhagin were pooled, concentrated using an ultrafiltration device (Amicon, Danvers, MA) fitted with a YM30 membrane, and dialyzed into TS buffer. The concentration of purified protein was estimated using the BCA method with bovine serum albumin as standard according to the manufacturer's instructions (Pierce). N-terminal sequence analysis was performed as previously described (7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar, 17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). For internal sequence analysis, alborhagin (0.58 mg) was dialyzed into distilled water and digested with trypsin (0.1 mg of trypsin/mg of protein) overnight at 37 °C. Tryptic fragments were separated by reverse-phase high pressure liquid chromatography, eluted by a linear 0–60% (v/v) acetonitrile gradient, and sequenced as previously described (17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). Digestion of human fibrinogen (Kabivitrum, Stockholm, Sweden) at a final concentration of ∼100 µg/ml in TS buffer by alborhagin (10 µg/ml, final concentration) for 30 min at 22 °C was carried out in the presence of EDTA (10 mm, final concentration) or CaCl2 (10 mm, final concentration) according to a published method (22Ward C.M. Vinogradov D.V. Andrews R.K. Berndt M.C. Toxicon. 1996; 34: 1203-1206Crossref PubMed Scopus (32) Google Scholar). Platelet aggregation of human platelet-rich plasma (0.32% citrate, final concentration) was performed using a whole blood Lumiaggregometer (Chronolog, Havertown, PA) as previously described (7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar, 17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar, 21Andrews R.K. Booth W.J. Gorman J.J. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8317-8326Crossref PubMed Scopus (156) Google Scholar). Alborhagin was added to platelets at a final concentration of 2.5–25 µg/ml. In some assays, the anti-αIIbβ3 antibody CRC64 at 20 µg/ml or mocarhagin at 10 µg/ml was pre-incubated with the platelets for 5 or 6 min, respectively, at 37 °C prior to the addition of alborhagin. Other assays included EDTA at a final concentration of 10 mm. The effect of PP1 (10 µm) on alborhagin-induced aggregation of washed human platelets, isolated as previously described (23Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Crossref PubMed Scopus (104) Google Scholar), was determined at 37 °C in the presence of indomethacin (10 µm) and apyrase (2 units/ml). Alborhagin-dependent aggregation of mouse platelets (1 × 108/ml) in the absence or presence of the monoclonal antibody JAQ1 at a final concentration of 10 µg/ml was carried out essentially as previously described (19Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The incorporation of carrier-free [32P]orthophosphate (∼8750 Ci/mmol; NEN Life Science Products) into platelet proteins was assessed using previously described methods (7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar). Briefly, washed platelets at 109/ml in 0.01 m HEPES, 0.15 m NaCl, 5 mmEDTA, pH 7.4 (EHS buffer) were labeled with 1 mCi of [32P]orthophosphate for 1 h at 22 °C, washed in the same buffer, and resuspended in a Tyrode's buffer containing 138 mm NaCl, 29 mm KCl, 12 mmNaHCO3, 0.36 mm Na2PO4, 5.5 mm glucose, 1.8 mm CaCl2, 0.49 mm MgCl2, pH 7.4. Stirred32P-labeled platelets (2 × 108/ml) were treated with a final concentration of 10 µg/ml collagen or 10 µg/ml alborhagin for 0.25, 1, 2, or 5 min at 37 °C. Samples were quenched by electrophoresis sample buffer, electrophoresed on SDS-polyacrylamide gels under reducing conditions, and analyzed by autoradiography. For immunoblotting, washed platelets (2 × 108/ml) isolated as previously described (23Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Crossref PubMed Scopus (104) Google Scholar) were treated with 1–15 µg/ml alborhagin or 1 µg/ml convulxin for various times at 37 °C. Reactions were terminated by adding SDS-containing sample buffer, electrophoresed on SDS-polyacrylamide (8–18%) gels under reducing conditions, electrotransferred to polyvinylidene difluoride membranes, and immunoblotted with the anti-phosphotyrosine antibody 4G10. Blots were visualized using a horseradish peroxidase-coupled second antibody (Silenus, Hawthorn, Victoria, Australia) and the ECL detection system (Amersham Pharmacia Biotech). For immunoprecipitation studies, platelets (2 × 108/ml) were pre-incubated with vehicle (0.1% Me2SO) or 10 µm PP1 for 5 min at 37 °C prior to the addition of 3 µg/ml alborhagin or 1 µg/ml convulxin for 90 or 30 s, respectively. Platelets were then lysed by adding an equivalent volume of ice-cold lysis buffer (20 mm Tris-HCl, pH 7.3, 300 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2% (v/v) Nonidet P-40, 2 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A), centrifuged at 15,000 ×g for 10 min to remove detergent-insoluble material, and pre-cleared with 30 µl of a 50% suspension of protein A-Sepharose in TBS-T buffer (20 mm Tris-HCl, 137 mm NaCl, 0.1% (v/v) Tween 20, pH 7.3). Samples were then immunoprecipitated with monoclonal anti-PLCγ2 antibody and 30 µl of protein A-Sepharose for 2 h at 4 °C. The beads were washed once with lysis buffer and three times with TBS-T buffer, then solubilized in electrophoresis sample buffer, and boiled for 10 min. Immunoblotting with the anti-phosphotyrosine antibody 4G10 was carried out as described above. Membranes were then stripped and reprobed with anti-PLCγ2 antibody. K562 human erythroleukemia cells stably cotransfected with empty pRc plasmid and pMG-FcRγ-chain (FcRγ) (pRc/γ-chain) or pRc-GPVI and pMG-FcRγ (GPVI/FcRγ) (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar,23Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Crossref PubMed Scopus (104) Google Scholar) were treated with phorbol 12-myristate 13-acetate for 24 h to increase surface expression of GPVI and then stimulated with alborhagin for 90 s. Cells were lysed and immunoprecipitated with anti-Syk antibody as described above. Proteins were electrophoresed on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted with 4G10. Filters were stripped and reprobed with anti-Syk antibody. The effect of alborhagin (0.1–100 µg/ml, final concentration) on binding of convulxin (1 µg/ml) to GPVI-expressing K562 cells was assessed by flow cytometry using an anti-convulxin IgG and a secondary fluorescein isothiocyanate-labeled anti-rabbit IgG. GPVI-transfected Jurkat cells were transfected with GPVI using methods similar to those described above for K562 cells (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar, 23Pasquet J.M. Bobe R. Gross B. Gratacap M.P. Tomlinson M.G. Payrastre B. Watson S.P. Biochem. J. 1999; 342: 171-177Crossref PubMed Scopus (104) Google Scholar). Jurkat cells are human T lymphocytic cells that do not express endogenous GPVI or FcRγ. Ca2+ flux in response to alborhagin (10 µg/ml) in untransfected or GPVI-transfected cells was measured as previously described (24Berlanga O. Bobe R. Becker M. Murphy G. Leduc M. Bon C. Barry F.A. Gibbins J.M. Garcia P. Frampton J. Watson S.P. Blood. 2000; 96: 2740-2745Crossref PubMed Google Scholar). The effect of alborhagin (0.1–100 µg/ml, final concentration) on binding of convulxin (1 µg/ml) to GPVI-expressing Jurkat cells was assessed using the methods described above for K562 cells. In the course of our purification of the platelet agonist alboaggregin-A from the venom of the white-lipped tree viper,T. albolabris (7Andrews R.K. Kroll M.H. Ward C.M. Rose J.W. Scarborough R.M. Smith A.I. López J.A. Berndt M.C. Biochemistry. 1996; 35: 12629-12639Crossref PubMed Scopus (82) Google Scholar), we identified a structurally distinct protein termed alborhagin that was also a potent platelet agonist. Alborhagin had an apparent molecular mass on SDS-polyacrylamide gels of ∼60 kDa under reducing (Fig.1A) and nonreducing (data not shown) conditions and bound strongly to heparin (eluted at >0.5m sodium chloride). Several lines of evidence suggested that alborhagin was a member of the metalloproteinase-disintegrin family. First, it was immunoreactive toward an antibody against the cobra venom metalloproteinase-disintegrin, mocarhagin (Fig.1 B). This antibody is also cross-reactive with jararhagin (data not shown). Second, like other venom metalloproteinase-disintegrins (22Ward C.M. Vinogradov D.V. Andrews R.K. Berndt M.C. Toxicon. 1996; 34: 1203-1206Crossref PubMed Scopus (32) Google Scholar, 25Kini R.M. Evans H.J. Toxicon. 1992; 30: 265-293Crossref PubMed Scopus (214) Google Scholar), alborhagin showed metalloproteinase activity toward the fibrinogen α-chain in the presence of Ca2+, but there was no significant digestion in the presence of EDTA (Fig. 1 C). Third, amino acid sequences of tryptic fragments corresponding to a total of 36 residues (Fig. 2A) were analogous to sequences within the metalloproteinase domain of the venom metalloproteinase-disintegrins jararhagin (26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar), HR1B (27Takeya H. Oda K. Miyata T. Omori-Satoh T. Iwanaga S. J. Biol. Chem. 1990; 265: 16068-16073Abstract Full Text PDF PubMed Google Scholar), and MT-d (28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar). Fourth, we obtained the sequence from the disintegrin domain by N-terminal sequencing of a 42-kDa autolytic fragment of alborhagin (Fig. 3A). Autolytic digestion of alborhagin was inhibited in the presence of EDTA (Fig.3 B), suggesting that it involved the metalloproteinase activity of alborhagin. The 42-kDa fragment did not bind heparin (data not shown) and was isolated in the flow-through of a heparin-agarose column to allow separation of any undigested alborhagin. The N-terminal sequence of the 42-kDa fragment (Fig. 2 B) was similar to the region at the boundary between the metalloproteinase and disintegrin domains of jararhagin (26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar). This sequence was conserved in MT-d (Fig.2 B), which also undergoes autodigestion (28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar).Figure 2Amino acid sequence analysis of alborhagin. A, amino acid sequences of tryptic peptides of alborhagin compared with sequences from jararhagin (26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar) (residues 4–14, 50–61, and 67–80), HR1B (27Takeya H. Oda K. Miyata T. Omori-Satoh T. Iwanaga S. J. Biol. Chem. 1990; 265: 16068-16073Abstract Full Text PDF PubMed Google Scholar) (residues 3–12, 48–59, and 65–78), and MT-d (28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar) (residues 193–202, 239–250, and 256–269).B, N-terminal sequence of the 42-kDa fragment of alborhagin compared with jararhagin residues 206–223 (26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar) and MT-d residues 393–410 (28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar). The boundary between the metalloproteinase (metalloprot.) and disintegrin (Disint.) domains of jararhagin is indicated. Identical residues or conservative substitutions are boxed.View Large Image Figure ViewerDownload (PPT)Figure 3Autodigestion of alborhagin. A, time course for autodigestion of alborhagin (Albo) at 4 °C. Samples at each time point were electrophoresed under reducing conditions on SDS-polyacrylamide (5–20%) gels and stained with Coomassie Blue. B, alborhagin treated under the same conditions as in A, except in the presence of 10 mm EDTA.View Large Image Figure ViewerDownload (PPT) Alborhagin induced aggregation in platelet-rich plasma, with maximal activity at ∼7.5 µg/ml (Fig.4A). Aggregation was strongly inhibited by the anti-αIIbβ3 antibody CRC64, which blocks ligand binding to αIIbβ3 (18Khaspekova S.G. Vlasik T.N. Byzova T.V. Vinogradov D.V. Berndt M.C. Mazurov A.V. Br. J. Haematol. 1993; 85: 332-340Crossref PubMed Scopus (23) Google Scholar), and by EDTA (Fig. 4 A), suggesting that aggregation was αIIbβ3-dependent and involved alborhagin-induced platelet activation. In the presence of EDTA, alborhagin still induced a platelet shape change (Fig.4 A), suggesting that alborhagin could induce platelet activation independently of its metalloproteinase function. In contrast to intact alborhagin, however, the ∼42-kDa alborhagin fragment corresponding to the disintegrin and C-terminal regions minus the metalloproteinase domain (see above) did not induce platelet aggregation at a final concentration of 10 µg/ml in platelet-rich plasma (data not shown). Together, these results suggested that an intact metalloproteinase domain, but not proteolysis, is necessary for alborhagin-induced platelet activation. Pre-treating platelets with the cobra venom metalloproteinase mocarhagin had no effect on alborhagin-dependent platelet aggregation (data not shown). Mocarhagin has previously been shown to cleave the GPIbα chain between Glu-282 and Asp-283 and abolish binding of vWF to the GPIb-IX-V complex on platelets and vWF-dependent platelet aggregation (17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). The lack of effect of mocarhagin on alborhagin activity therefore suggests that alborhagin does not act through GPIb-IX-V on platelets. However, alborhagin-induced aggregation of washed human platelets was strongly inhibited by PP1 (Fig. 4 B). PP1 is an Src family kinase inhibitor previously shown to abolish GPVI-dependent platelet aggregation (29Briddon S.J. Watson S.P. Biochem. J. 1999; 337: 203-209Crossref Scopus (82) Google Scholar). Treating [32P]orthophosphate-loaded platelets with alborhagin at 10 µg/ml resulted in phosphorylation of the previously characterized protein kinase C substrates p47/pleckstrin (30Kroll M.H. Hellums J.D. Guo Z. Durante W. Razdan K. Hroblich J.K. Schafer A.I. J. Biol. Chem. 1993; 268: 3520-3524Abstract Full Text PDF PubMed Google Scholar) and myosin light chain (31Scholey J.M. Taylor K.A. Kendrick-Jones J. Nature. 1980; 287: 233-235Crossref PubMed Scopus (224) Google Scholar) on a time scale comparable with that induced by collagen (data not shown). Additionally, alborhagin stimulated the tyrosine phosphorylation of a range of proteins, measured using an anti-phosphotyrosine antibody (Fig.5A). This platelet phosphorylation profile resembled that induced by collagen or convulxin (Fig. 5 B), a venom protein of the C-type lectin family previously shown to activate platelets by an interaction with GPVI (13Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar,14Polgar J. Clemetson J.M. Kehrel B.E. Weidemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). For both alborhagin and convulxin stimulation, prominent phosphorylated bands were observed at ∼135, ∼72, ∼36, and ∼12 kDa. The time course for convulxin-induced phosphorylation peaked at 15 s before declining. In contrast, alborhagin stimulated a slower, sustained increase in tyrosine phosphorylation reminiscent of responses to collagen and collagen-related peptide. The transient nature of the increase in tyrosine phosphorylation induced by convulxin is thought to be related to its more powerful stimulatory action (32Asazuma N. Wilde J.I. Berlanga O. Leduc M. Leo A. Schweighoffer E. Tybulewicz V.L.J. Bon C. Liu S.K. McGlade C.J. Schraven B. Watson S.P. J. Biol. Chem. 2000; 275: 33427-33434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). PLCγ2 was identified as being specifically phosphorylated in response to both alborhagin and convulxin, a response that was blocked by PP1 (Fig. 6) along with the increase in whole cell phosphorylation (data not shown). Of the other phosphorylated bands, it is probable that the 12, 36, and 72 kDa bands phosphorylated both by alborhagin and convulxin correspond to the FcRγ, LAT, and Syk/SLP76, respectively, based on previous studies (13Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Polgar J. Clemetson J.M. Kehrel B.E. Weidemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar,32Asazuma N. Wilde J.I. Berlanga O. Leduc M. Leo A. Schweighoffer E. Tybulewicz V.L.J. Bon C. Liu S.K. McGlade C.J. Schraven B. Watson S.P. J. Biol. Chem. 2000; 275: 33427-33434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Figure 6Effect of PP1 on alborhagin- or convulxin-induced PLCγ2 phosphorylation.Washed platelets (2 × 108/ml) were pre-incubated with vehicle (0.1% Me2SO) or 10 µm PP1 for 5 min and then treated with 3 µg/ml alborhagin for 90 s or 1 µg/ml convulxin for 30 s. Lysates were immunoprecipitated with anti-PLCγ2 monoclonal antibody and analyzed by immunoblotting with either the anti-phosphotyrosine antibody 4G10 (top panel), or following stripping of the membrane, the anti-PLCγ2 antibody (lower panel). IP, immunoprecipitation.View Large Image Figure ViewerDownload (PPT) The similar pattern of tyrosine phosphorylation and sensitivity to PP1 in platelets treated with alborhagin implicated GPVI in the mechanism of alborhagin activity. We therefore examined the activity of alborhagin on two cell lines transfected with GPVI. In FcRγ-expressing K562 cells transfected with GPVI (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar), alborhagin induced concentration-dependent GPVI-dependent phosphorylation of Syk, whereas there was no response in mock-transfected cells (Fig.7A). K562 cells contain no endogenous GPIb-IX-V or GPVI expression, as assessed by flow cytometry (data not shown), suggesting that GPVI was specifically required for alborhagin-induced Syk phosphorylation in this system. Alborhagin also induced elevation of cytosolic Ca2+, specifically in GPVI-transfected Jurkat cells but not in untransfected cells (Fig.7 B). Jurkat cells do not express GPVI or FcRγ, although the role of the latter is taken by the T cell receptor ζ chain. 2D. Tulasne, T. Bori, and S. P. Watson, unpublished observation. These results provide additional evidence that alborhagin targets GPVI. Binding of convulxin (1 µg/ml) to GPVI-expressing K562 cells was partially inhibited by alborhagin in a dose-dependent manner. There was ∼40% inhibition at a final concentration of 10 µg/ml alborhagin, with no increased inhibition up to 100 µg/ml (Fig.8A). Similar experiments using GPVI-expressing Jurkat cells showed that there was only minimal displacement of convulxin binding by 10 µg/ml alborhagin, with no further inhibition at 100 µg/ml (Fig. 8 A). Together, these data suggest that although alborhagin and convulxin may both bind GPVI, they may recognize separate binding sites. This is supported by studies on mouse platelets using a rat monoclonal antibody that binds GPVI, JAQ1. JAQ1 has previously been shown to specifically inhibit aggregation of mouse platelets by collagen-related peptide and low concentrations of collagen but not by convulxin or high concentrations of collagen (19Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 33Schulte V. Snell D. Bermeier W. Zirngibl H. Watson S.P. Nieswandt B. J. Biol. Chem. 2001; 276: 364-368Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 34Nieswandt B. Schulte V.V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (303) Google Scholar). JAQ1 inhibited alborhagin-induced shape change and aggregation of mouse platelets (Fig. 8 B), suggesting that it binds to a site that is the same as, or located close to, that used by collagen-related peptide but that is distinct from that used by convulxin. Convulxin binding is not blocked by JAQ1 (34Nieswandt B. Schulte V.V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (303) Google Scholar), implying that alborhagin binds to a distinct epitope. In this regard, the effect of JAQ1 is consistent with alborhagin binding to the collagen-related peptide binding site, whereas convulxin may bind to the site used by the second binding site in collagen (33Schulte V. Snell D. Bermeier W. Zirngibl H. Watson S.P. Nieswandt B. J. Biol. Chem. 2001; 276: 364-368Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In the present study, we isolated a novel protein termed alborhagin from T. albolabris venom that is functionally related to alboaggregin-A and convulxin, because it potently activated platelets by an interaction with GPVI; but in contrast to these C-type lectin-like proteins, alborhagin was structurally different. Several lines of evidence suggested that alborhagin may be a member of the metalloproteinase-disintegrin family. First, the molecular mass of alborhagin on SDS-polyacrylamide gels (∼60 kDa; nonreduced and reduced) and its metalloproteinase activity (EDTA-inhibitable) toward fibrinogen α-chain were consistent with other metalloproteinase-disintegrins (17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar, 22Ward C.M. Vinogradov D.V. Andrews R.K. Berndt M.C. Toxicon. 1996; 34: 1203-1206Crossref PubMed Scopus (32) Google Scholar, 25Kini R.M. Evans H.J. Toxicon. 1992; 30: 265-293Crossref PubMed Scopus (214) Google Scholar, 26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar, 35Andrews R.K. Berndt M.C. Toxicon. 2000; 38: 775-791Crossref PubMed Scopus (70) Google Scholar). Second, alborhagin was immunoreactive toward an antibody against the cobra venom metalloproteinase mocarhagin (17Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). Third, the sequences of four peptides throughout the length of alborhagin revealed similarity to jararhagin, HR1B, and MT-d of the metalloproteinase-disintegrin family (26Paine M.J.I. Desmond H.P. Theakston R.D.G. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar, 27Takeya H. Oda K. Miyata T. Omori-Satoh T. Iwanaga S. J. Biol. Chem. 1990; 265: 16068-16073Abstract Full Text PDF PubMed Google Scholar, 28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar). This combined evidence suggests that alborhagin is very likely to be a metalloproteinase-disintegrin, although in the absence of the full sequence, we cannot exclude the possibility that it is a closely related protein. Like MT-d, a venom metalloproteinase-disintegrin from Agkistrodon halys brevicaudus (28Jeon O.H. Kim D.S. Eur. J. Biochem. 1999; 263: 526-533Crossref PubMed Scopus (39) Google Scholar), alborhagin underwent autolysis, yielding an ∼42-kDa fragment. Autolysis was prevented by the presence of EDTA, suggesting that it involved the metalloproteinase activity of alborhagin. The N-terminal sequence of the 42-kDa fragment corresponded to sequences at the N terminus of two naturally occurring jararhagin-derived fragments, one-chain botrocetin (36Fujimura Y. Titani K. Usami Y. Suzuki M. Oyama R. Matsui T. Fukui H. Sugimoto M. Ruggeri Z.M. Biochemistry. 1991; 30: 1957-1964Crossref PubMed Scopus (117) Google Scholar) and jaracetin (37De Luca M. Ward C.M. Ohmori K. Andrews R.K. Berndt M.C. Biochem. Biophys. Res. Commun. 1995; 206: 570-576Crossref PubMed Scopus (90) Google Scholar). This result supports the possibility that autodigestion might be a general mechanism for processing venom metalloproteinases. Several lines of evidence demonstrated that alborhagin was a potent platelet agonist and that, like convulxin, it appeared to target GPVI. First, alborhagin induced platelet aggregation that was αIIbβ3-dependent, because it was inhibitable by a blocking anti-αIIbβ3 monoclonal antibody (CRC64). Second, alborhagin activated protein kinase C, as shown by phosphorylation of p47/pleckstrin and myosin light chain (30Kroll M.H. Hellums J.D. Guo Z. Durante W. Razdan K. Hroblich J.K. Schafer A.I. J. Biol. Chem. 1993; 268: 3520-3524Abstract Full Text PDF PubMed Google Scholar, 31Scholey J.M. Taylor K.A. Kendrick-Jones J. Nature. 1980; 287: 233-235Crossref PubMed Scopus (224) Google Scholar). Third, alborhagin induced a tyrosine phosphorylation profile similar to that induced by convulxin (this study; 12Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Polgar J. Clemetson J.M. Kehrel B.E. Weidemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 38Ichinohe T. Takayama H. Ezumi Y. Arai M. Yamamoto N. Takahashi H. Okuma M. J. Biol. Chem. 1997; 272: 63-68Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 39Ezumi Y. Shindo K. Tsuji M. Takayama H. J. Exp. Med. 1998; 188: 267-276Crossref PubMed Scopus (186) Google Scholar, 40Hers I. Berlanga O. Tiekstra M.J. Kamiguti A.S. Theakston R.D. Watson S.P. Eur. J. Biochem. 2000; 267: 2088-2097Crossref PubMed Scopus (36) Google Scholar). One of these tyrosine-phosphorylated proteins, PLCγ2, was identified as being phosphorylated in response to both convulxin and alborhagin. PLCγ2 phosphorylation induced by either agonist was inhibitable by the Src kinase family inhibitor PP1. Importantly, PP1 also blocked both alborhagin- and convulxin-dependent platelet aggregation. Additional evidence that alborhagin could target GPVI was provided by studies showing that alborhagin, like convulxin, induced GPVI-dependent phosphorylation of Syk in FcRγ-expressing K562 cells cotransfected with GPVI but not in untransfected cells. Alborhagin also induced elevation of cytosolic Ca2+ in Jurkat cells expressing recombinant GPVI but had no effect in untransfected cells. Interestingly, although both alborhagin and convulxin appeared to target GPVI, alborhagin only partially inhibited (by ∼40%) convulxin binding to recombinant GPVI expressed on K562 cells and had a negligible effect on binding to Jurkat cells, suggesting that the proteins may recognize separate sites. This was supported by the observation that alborhagin-dependent aggregation of mouse platelets was inhibited by an anti-mouse GPVI monoclonal antibody, JAQ1. This antibody also blocks aggregation to collagen-related peptide and low concentrations of collagen but not convulxin (34Nieswandt B. Schulte V.V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (303) Google Scholar). Finally, the observation that structurally distinct viper venom proteins, convulxin and alboaggregin-A of the C-type lectin family (10Asazuma N. Marshall S. Berlanga O. Snell D. Poole A. Berndt M.C. Andrews R.K. Watson S.P. Blood. 2001; 97: 3989-3991Crossref PubMed Scopus (27) Google Scholar,11Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 2333-2341Crossref PubMed Scopus (65) Google Scholar, 13Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Polgar J. Clemetson J.M. Kehrel B.E. Weidemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) and alborhagin (this study), may target distinct sites at the same receptor (GPVI) parallels the observation that other viper venom proteins target distinct sites on vWF. Two-chain botrocetin of the C-type lectin family and jararhagin of the metalloproteinase-disintegrin family interact with vWF at distinct sites, enabling it to bind GPIb-IX-V (35Andrews R.K. Berndt M.C. Toxicon. 2000; 38: 775-791Crossref PubMed Scopus (70) Google Scholar). These latter observations may be related to recent evidence suggesting that C-type lectin-like proteins and metalloproteinase-disintegrins might be derived from a common precursor (41Kini R.M. Toxicon. 1996; 34: 1287-1294Crossref PubMed Scopus (20) Google Scholar). In conclusion, we have identified a novel viper venom protein, alborhagin, that is a potent platelet agonist. Alborhagin is functionally related to convulxin, because it appears to target GPVI, but is structurally different and appears to target the collagen receptor at a distinct site, thereby making it a novel probe for analysis of GPVI-dependent platelet activation. We gratefully acknowledge the excellent technical assistance of Carmen Llerena and Mary Matthew." @default.
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• W2065571808 title "A Novel Viper Venom Metalloproteinase, Alborhagin, Is an Agonist at the Platelet Collagen Receptor GPVI" @default.
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