apyrase and thienopyridine

Excessive platelet activation and accumulation can lead to vessel occlusion and thus present major therapeutic challenges in cardiovascular medicine. Apyrase, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ADP and ATP released from platelets and endothelial cells, thereby reducing platelet activation and recruitment. In the present study, we expressed a 68-kDa recombinant mosquito (Aedes aegypti) salivary apyrase using a baculovirus/insect cell expression system and purified it to homogeneity using anion-exchange chromatography on a large scale. A yield of 18 mg of purified recombinant apyrase was obtained from 1 litre of the medium. Kinetic analysis indicated that the recombinant apyrase had a K(m) of 12.5 microM for ADP and a K(m) of 15.0 microM for ATP. The recombinant apyrase inhibited ADP-, collagen- and thrombin-induced human platelet aggregation in a dose-dependent manner, indicating that the recombinant protein retained nucleotidase activity in a whole cell system, which suggests that it may serve as a therapeutic agent for inhibition of platelet-mediated thrombosis.

Topics: Aedes; Animals; Apyrase; Baculoviridae; Gene Expression; Genetic Vectors; Humans; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Pyridines; Recombinant Proteins; Salivary Proteins and Peptides