Isolation and Characterization of jararaca GPIb-BP, a Snake Venom Antagonist Specific to Platelet Glycoprotein lb^[*]

Summary

A platelet glycoprotein lb-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with M_r of 30,000, and two distinct bands with M_r. of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 ± 2,521 molecules per normal platelet, with Kd 39.1 ± 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or botrocetin-induccd von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC₅₀ values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or α-thrombin. ¹²⁵1-jararaca GPIb-BP binding to GPIb was not altered by the presence of human α-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear- induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamidomethylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH²-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca²⁺-dependent lectins, especially to those of two functionally opposite venom proteins, botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb- modulator).

^* This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (to Y.F. and K.T.), from the Fujita Health University (to K.T.), and from the Saichi Nakajima Memorial Foundation (to Y.F.).

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