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Abstract
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 Km of 12.5 µM for ADP and a Km of 15.0 µM 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.
| Abbreviations | ||
| ADP | = | adenosine diphosphate; |
| ATP | = | adenosine triphosphate; |
| Km | = | Michaelis constant; |
| GP | = | glycoprotein; |
| cDNA | = | complimentary DNA; |
| MOI | = | multiplicity of infection; |
| SDS–PAGE | = | sodium dodecyl sulfate– polyacrylamide gel electrophoresis |
| Abbreviations | ||
| ADP | = | adenosine diphosphate; |
| ATP | = | adenosine triphosphate; |
| Km | = | Michaelis constant; |
| GP | = | glycoprotein; |
| cDNA | = | complimentary DNA; |
| MOI | = | multiplicity of infection; |
| SDS–PAGE | = | sodium dodecyl sulfate– polyacrylamide gel electrophoresis |