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Published on: July 3, 2019
Reconstitution of CD39 in liposomes amplifies nucleoside triphosphate diphosphohydrolase activity and restores
Carolyn A Haller1, Wanxing Cui, Jing Wen
1Department of Surgery, Emory University, Atlanta, GA 30322, USA.
Background:
CD39 (nucleoside triphosphate diphosphohydrolase [NTPDase-1]) expressed on the luminal surface of endothelial cells rapidly metabolizes extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), and abrogates platelet reactivity. Optimization of CD39 enzymatic activity appears dependent upon the expression of both transmembrane domains within plasma membranes. Thus, motivation exists to examine therapeutic antiplatelet formulations that consist of liposomal CD39.
Methods:
Full-length human CD39 was produced by using a yeast expression system, purified, and reconstituted within lipid vesicles. The catalytic efficiency (kcat/Km) of CD39-mediated phosphohydrolysis of ADP and ATP was determined both for detergent-solubilized and protein-reconstituted CD39 within lipid membranes. The capacity of CD39-containing lipid vesicles to inhibit platelet activation induced by ADP, collagen, or thrombin was determined in vitro by platelet aggregometry. A murine model of thromboplastin-induced thromboembolism was used to determine the effectiveness of intravenous liposomal CD39 in limiting platelet consumption and mortality.
Results:
Reconstitution of human CD39 in lipid vesicles was associated with a decrease in Km of nearly an order of magnitude over the detergent-solubilized form. There was a concomitant increase in both ADPase and ATPase catalytic efficiencies (kcat/Km ADPase: sol CD39: 2.7 x 10(6) vs liposomal CD39: 1.4 x 10(7) min/ M; kcat/Km ATPase: sol CD39: 7.2 x 10(6) vs liposomal CD39: 2.0 x10(7) min/M). Furthermore, CD39 lipid vesicles effectively inhibited platelet aggregation when activated by ADP, collagen, or thrombin, and also promoted platelet disaggregation (60.4% +/- 6.1%). Treatment with CD39 lipid vesicles preserved platelet counts after thromboplastin injection (pretreatment, 906.8 +/- 42.9 platelets/microm3; empty vesicles, 278.6 +/- 34.8 platelets/microm3; CD39 vesicles, 563.6 +/- 42.2 platelets/microm3; n = 10 mice/test group; P < .0001). In parallel survival studies, liposomal CD39 reduced mortality from 73% to 33% (P < or = .05; n = 12 mice/experimental test group, n = 15 mice/control test group).
Conclusions:
Incorporation of solubilized CD39 into a lipid bilayer restores enzyme activity and optimizes thromboregulatory potential. Treatment with CD39 in liposomal formulations decreased mortality in a murine model of thromboplastin-induced thromboembolism by limiting intravascular platelet aggregation and thrombosis.
