Production of a human apyrase protein as an antithrombotic agent is proposed. Thrombotic vascular occlusion due to abnormal platelet aggregation can lead to many diseases, including ischemic stroke, myocardial infarction, and restenosis after angioplasty or bypass surgery. Many of the current anti-platelet or fibrinolytic agents are undesirable due to the lack of specificity, narrow time window of administration, or severe adverse effects. Human ecto-apyrase has recently emerged as a critical endothelial thromboregulator with outstanding premise as an anti-platelet drug. The apyrase metabolizes extracellular ADP released by activated platelets, thereby inhibiting platelet aggregation and recruitment. We have cloned the human apyrase and expressed its extracellular domain in mammalian cells. Our recombinant apyrase exhibits an outstanding ADPase activity in vitro. We plan to integrate cutting edge technologies to create a robust CHO-cell based expression system suitable for large-scale production and efficient purification of biologically active recombinant proteins. We expect this research to provide an advanced system for producing the apyrase, and other proteins of therapeutic importance, at a substantially reduced cost, thus creating a reliable source of protein necessary for pre-clinical/clinical studies.
