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 E1 (1 microM) and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester ('TMB-8'; 100 microM) 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 microM) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 microM) did not significantly affect its aggregating activity. Aggretin caused a significant increase of [3H]InsP formation in [3H]Ins-loaded platelets, intracellular Ca2+ mobilization and thromboxane B2 formation. Neomycin, a phospholipase C inhibitor, completely inhibited both the increase of [3H]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. 125I-aggretin bound to platelets with a high affinity (Kd = 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.
Huang
T F
TF
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Liu
C Z
CZ
Yang
S H
SH
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry*; D004789:Enzyme Activation; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D013929:Thromboxane B2 / Q000096:biosynthesis; D010738:Type C Phospholipases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D009842:Oligopeptides; D010716:Phosphatidylinositols; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D013929:Thromboxane B2; C047981:arginyl-glycyl-aspartic acid; D010738:Type C Phospholipases; D002118:Calcium; D007213:Indomethacin