Purification and characterization of platelet aggregation inhibitors from snake venoms

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (p1.AI), multisquamatin (Mr = 5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger p1.AI, contortrostatin (Mr = 15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both p1.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb-/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.