PURIFICATION AND CHARACTERIZATION OF PLATELET-AGGREGATION INHIBITORS FROM SNARE VENOMS

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet agg regation have been purified from the venom of two snake species. A sma ll platelet aggregation inhibitor (pl.AI), multisquamatin (Mr=5,700), was purified from Echis multisquamatus venom by hydrophobic interactio n HPLC and two steps on C18 reverse phase HPLC. A larger pl.AI, contor trostatin (Mr=15,000), was purified by a similar HPLC procedure from t he venom of Agkistrodon contortrix contortrix. Both pl.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 ha s an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine rabbit PRP, respectively. In a competitive binding assay using I-125-7E3 (a monocl onal antibody to GPIIb/IIIa that inhibits platelet aggregation) both c ontortrostatin and multisquamatin demonstrated GPIIb/IIIa specific bin ding to human and canine platelets. The IC50 for contortrostatin displ acement 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, respecti vely. Our results indicate that both pl.AIs inhibit platelet aggregati on by binding with high affinity to GPIIb/IIIa.