FUNCTIONAL ANALYSIS OF VASCULAR NTPDASES

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CD39 is the prototype ATP/ nucleoside triphosphate diphosphohydrolase (ATPDase/NTPDase; EC 3.6.1.5) member of a family of E-type ectonucleotidases. CD39 modulates purinergic receptor-mediated signaling, following the hydrolysis of ATP and ADP, but also facilitates generation of adenosine for intracellular transport from extracellular adenine nucleotides. Although CD39 appears to be regulated and widely expressed within the vasculature, the functional significance of ectoenzyme expression by the endothelium is unclear. Ectonucleotidases could be involved in the regulation of vascular inflammatory reactions by influencing platelet microthrombi formation, endothelial cell activation or apoptosis and systemic purine homeostasis. To study the functional significance of the vascular NTPDase, the investigators have developed antisense reagents, recombinant adenoviruses and generated CD39-deficient and null mice by homologous recombination. These mutant mice have been shown to be fully viable but have a bleeding diathesis. Platelet hypofunction, secondary to selective purinergic P2Y1 receptor desensitization, can be demonstrated. In keeping with the predicted loss of this vascular thromboregulatory mechanism, fibrin deposition has been observed within the vasculature at multiple sites; the mutant mice also respond adversely to vascular insults and their cardiac xenotransplants rapidly fail. The role of CD39 in modulating differing endothelial cell purinoreceptor mediated effects in vitro and consequent thrombotic reactions in vivo will be further examined in this proposal. Vascular responsiveness to systemic platelet activation, ischemia-reperfusion injury and xenograft rejection will be evaluated in selected vascularized murine tissues over-expressing NTPDase biochemical activtity and in mutant mice deficient in CD39. Reconstitution experiments, using soluble NTPDases, will be performed in parallel. Purinergic mechanisms by which CD39 influences platelet and cellular activation appear to differ from those associated with endothelial constitutive nitric oxide synthase (eNOS), a second aspirin-insensitive thromboregulatory system. These pathways may be additive or synergistic and will be studied by in vitro systems and by modulating NO-activity in CD39 mice. The generation of mice deficient in both eNOS and CD39 will also be undertaken. These double knock-outs may be viable; however, a lethal phenotype will be also informative. These experiments should indicate the involvement of the vascular NTPDase/CD39, and also elucidate interactions with NOS systems, in certain vascular inflammatory disorders observed in human disease states.