Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function.

Abstract

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.