AGGRETIN, A NOVEL PLATELET-AGGREGATION INDUCER FROM SNAKE (CALLOSELASMA-RHODOSTOMA) VENOM, ACTIVATES PHOSPHOLIPASE-C BY ACTING AS A GLYCOPROTEIN IA/IIA AGONIST

A potent platelet aggregation inducer, aggretin, was purified from Malayan-pit-viper (Calloselasma rhodostoma) venom by ionic-exchange chromatography, gel-filtration chromatography and HPLC. It is a heterodimeric protein (29 kDa) devoid of esterase, phospholipase A and thrombin-like activity. Aggretin (> 5 nM) elicited platelet aggregation with a lag period in both human platelet-rich plasma and washed platelet suspension. EDTA (5 mM), prostaglandin E(1) (1 mu M) and 3,4,5-trimethoxy-benzoic acid 8-(diethylamino)octyl ester ('TMB-8'; 100 mu M) abolished its aggregating activity, indicating that exogenous bivalent cations and intracellular Ca2+ mobilization are essential for aggretin-induced platelet aggregation. Neomycin (4 mM) and mepacrine (50 mu M) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 mu M) did not significantly affect its aggregating activity. Aggretin caused a significant increase of [H-3]InsP formation in [H-3]Ins loaded platelets, intracellular Ca2+ mobilization and thromboxane B-2 formation. Neomycin, a phospholipase C inhibitor, completely inhibited both the increase of [H-3]InsP and intracellular Ca2+ mobilization of platelets stimulated by aggretin. A monoclonal antibody (6F1) directed against glycoprotein Ia/IIa inhibited platelet shape change and aggregation induced by aggretin.I-125-aggretin bound to platelets with a high affinity (K-d = 4.0 +/- 1.1 nM), and the number of binding sites was estimated to be 2119 +/- 203 per platelet. It is concluded that aggretin may act as a glycoprotein Ia/IIa agonist to elicit platelet aggregation through the activation of endogenous phospholipase C, leading to hydrolysis of phosphoinositides and subsequent intracellular Ca2+ mobilization.