Characterization of Three Alboaggregins Purified from Trimeresurus albolabris Venom

Summary

Alboaggregins (AL-A, AL-B, AL-C) isolated from Trimeresurus albolabris snake venom represent a new family of proteins which bind to platelet glycoprotein lb (GPIb). These alboaggregins were purified to homogeneity with ion exchange HPLC (Mono-Q column) and hydrophobic HPLC (TSK Phenyl-5PW column). On SDS-polyacrylamide gel electrophoresis, apparent molecular weights of AL-A, AL-B and AL-C were 52 kDa, 26 kDa, and 121 kDa respectively, under nonreducing conditions. Upon reduction, each alboaggregin showed two types of chains with apparent molecular weights in the range of 15-20 kDa. All three alboaggregins agglutinated formalin-fixed platelets. Agglutination activities and binding of labeled alboaggregins to GPIb were specifically inhibited by the monoclonal antibody AK2 which is directed against the 45 kDa N-terminal region on GPIb, but not by monoclonal antibodies against other epitopes on GPIb. ¹²⁵I-alboaggregin binding to platelets was not altered by the presence of thrombin. Alboaggregins did not bind to GPIIb/IIIa. Alboaggregins were competitive inhibitors for ¹²⁵I-bovine vWF binding to platelets. Mutual competition studies between AL-A, AL-B and AL-C for the binding of labeled bovine vWF and AL-B to platelets demonstrated that AL-B and AL-C had a significantly higher affinity than AL-A.

• References

• 1 Weiss HJ, Baumgartner HR, Tschopp TB, Turito VT, Cohen D. Correction by factor VIII of the impaired platelet adhesion to subendothelium in von Willebrand disease. Blood 1978; 51: 267-279
• 2 Howard MA, Firkin BG. Ristocetin - a new tool in the investigation of platelet aggregation. Thromb Diath Haemorrh 1971; 26: 362-369
• 3 Read MS, Smith SV, Lamb MA, Brinkhous KM. Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor. Blood 1989; 74: 1031-1035
• 4 Kirby EP, Mills DCB. The interaction of bovine factor VIII with human platelets. J Clin Invest 1975; 56: 491-502
• 5 Forbes CD, Prentice CRM. Aggregation of human platelets by purified porcine and bovine antihaemophilic factor. Nature - New Biol 1973; 241: 149-150
• 6 Tobelem G, Levy-Toledano S, Bredoux R, Michel H, Nurden A, Caen J. New approaches to determination of specific functions of platelet membrane sites. Nature (London) 1976; 263: 427-429
• 7 Suzuki K, Nishioka J, Hashimoto S. Identical binding site on human platelets for von Willebrand factor and bovine platelet aggregating factor. Thromb Res 1980; 17: 215-223
• 8 Schmaier AH, Colman RW. Crotalocytin: Characterization of the timber rattlesnake platelet activating protein. Blood 1980; 56: 1020-1028
• 9 Davey MG, Luscher EF. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 1967; 216: 857-858
• 10 Read MS, Shermer RW, Brinkhous KM. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc Natl Acad Sci 1980; 75: 4514-4518
• 11 Huang TF, Holt JC, Lukasiewicz H, Niewiarowski S. Trigramin, a low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. J Biol Chem 1987; 262: 16157-16163
• 12 Williams J, Rucinski B, Holt JC, Niewiarowski S. Elegantin and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor. Biochem Biophys Acta 1990; 1039: 81-89
• 13 Peng M, Lu W, Kirby EP. Alboaggregin-B: A new platelet agonist that binds to platelet membrane glycoprotein lb. Biochemistry 1991; 30: 11529-11536
• 14 Ogilvie ML, Byl JW, Gartner TK. Platelet-aggregation is stimulated by lactose-inhibitable snake venom lectins. Thromb Haemostas 1989; 62: 704-707
• 15 Ganguly P, Fossett NG. Interaction of lentil lectin with human platelets. Evidence against glycoprotein II as aggregation mediator. Biochem Biophys Res Commun 1980; 92: 499-504
• 16 Ganguly P, Fossett NG. Evidence for multiple mechanisms of interaction between wheat germ agglutinin and human platelets. Biochem Biophys Acta 1980; 627: 256-261
• 17 Bakhshi MR, Kirby EP. Proteolytic and immunologic comparison of human and bovine von Willebrand factor. Thromb Haemostas 1990; 63: 517-523
• 18 Mascelli MA, Edgington TS, Kirby EP. Characterization of a fragment of bovine von Willebrand factor that binds to platelets. Biochemistry 1986; 25: 6325-6335
• 19 Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range 1 to 100 kDa. Anal Biochem 1987; 166: 368-379
• 20 Kirby EP. The agglutination of human platelets by bovine factor VIII :R. J Lab Clin Med 1982; 100: 963-976
• 21 Fling SP, Gregerson DS. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem 1986; 155: 83-88
• 22 Berndt MC, Du XP, Booth WJ. Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex. Biochemistry 1988; 27: 633-640
• 23 Harmon JT, Jamieson GA. The glycocalicin portion of platelet glycoprotein lb expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets. J Biol Chem 1986; 261: 13224-13229
• 24 George JN, Torres MM. Thrombin decreases von Willebrand factor binding to platelet glycoprotein lb. Blood 1988; 71: 1253-1259