Expression of functional recombinant mosquito salivary apyrase: a potential therapeutic platelet aggregation inhibitor.
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.
Sun
Dongfeng
D
Department of Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, Manitoba, Canada.
McNicol
Archibald
A
James
Anthony A
AA
Peng
Zhikang
Z
D000330:Aedes / Q000201:enzymology*; D000818:Animals; D001081:Apyrase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000627:therapeutic use; D058919:Purinergic P2 Receptor Antagonists; D011725:Pyridines / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000627:therapeutic use; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000627:therapeutic use
D010975:Platelet Aggregation Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D011725:Pyridines; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C446540:thienopyridine; D001081:Apyrase