INHIBITION OF PLATELET-FUNCTION BY RECOMBINANT SOLUBLE ECTO-ADPASE CD39/

Excessive platelet accumulation and recruitment, leading to vessel occ lusion at sites of vascular injury, present major therapeutic challeng es in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme w ith ADPase and ATPase activities, rapidly metabolizes ATP and ADP rele ased from activated platelets, thereby abolishing recruitment. Therefo re, a soluble form of CD39, retaining nucleotidase activities, would c onstitute a novel antithrombotic agent. We designed a recombinant, sol uble form of human CD39, and isolated it from conditioned media from t ransiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown un der serum-free conditions was subjected to anti-CD39 immunoaffinity co lumn chromatography, yielding a single similar to 66-kD protein with A TPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platele t reactivity. Kinetic analyses indicated that, while soluble CD39 had a K-m for ADP of 5.9 mu M and for ATP of 2.1 mu M, the specificity con stant k(cat)/K-m was the same for both substrates. Intravenously admin istered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data ind icate that soluble CD39 is a potential therapeutic agent for inhibitio n of platelet-mediated thrombotic diatheses.