Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: The unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon pi scivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectiv ely. Piscivostatin had two binding motifs recognized by platelet glycoprote in IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegri n piscivostatin toward platelet aggregation differed ft-om those of other m onomeric disintegrins such as trimestatin and echistatin. We measured plate let aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins i nhibited the formation of small (9 to 25 mum in diameter), medium-sized and large aggregates (25 to 70 mum in diameter) in a dose-dependent manner. Th e platelet aggregates disaggregated after reaching a maximal number on eith er treatment with ADP alone or monomeric disintegrin/ADP. However, the smal l aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-G PIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion protei ns without platelet activation, piscivostatin induced a platelet shape chan ge alone with no aggregate formation. The present study indicated that pisc ivostatin has two unique contradictory activities; acting as a double inhib itor of platelet aggregation and platelet aggregate dissociation.