Vascular injury in coronary, cerebral, and peripheral arteries evokes local platelet activation, recruitment and thrombotic occlusion, reversal of which presents a major therapeutic challenge. Platelets are consistently unresponsive to agonists in the presence of endothelial cells (EC), even in the absence of eicosanoids and nitric oxide. This observation culminated in our characterization of endothelial cell CD39/ecto-ADPase as the prime thromboregulator. CD39 rapidly metabolizes ADP released from activated platelets, thereby abolishing aggregation and recruitment. A recombinant, soluble form of human CD39, solCD39, was developed which potently blocked agonist-induced human platelet aggregation in vitro, and prolongs bleeding time in mice in potently blocked agonist- induced human platelet aggregation in vitro, and prolongs bleeding time in mice in vivo. CD39-/- mice exhibited a latent pro-thrombotic phenotype with increased susceptibility to ischemic cerebral thrombosis and injury, demonstrating a critical role for CD39 in cerebral thromboregulation. This collaboration will decipher the pivotal biological role of endogenous CD39 in inhibiting ischemia-driven thrombosis, and will develop CD39 as a novel anti-thrombotic agent. Structure-function studies will: Develop specific information about the CD39 active site, to identify which amino acids are required for enzyme catalysis; Map critical regions in CD39 which contribute to its tertiary structure; Establish the contribution of glycosylation to CD39 enzymatic activity; Determine biophysical and structural properties of solCD39 to comprehend mechanisms of nucleotide dephosphorylation; Genetic multi- valent derivatives of the extracellular domain of CD39 to define the structural basis for oligomerization-mediated promotion of enzymatic activity. The role of endogenous CD39 in microvascular thrombosis will be studied in CD39-/- mice, subjected to ischemic stroke with our without reconstitution with solCD39. SolCD39 will also be investigated as a potential therapeutic agent in an established baboon model of ischemic stroke. Using endothelial cells from control and CD39-/- mice, as well as ischemic murine and baboon brain tissue, ischemia- or hypoxia-driven modulation of CD39 expression will be studied. The research represents a multi-disciplinary approach to understanding the critical role of CD39 as the prime regulator of platelet-mediated occlusive arterial thrombosis. This collaboration, based on compelling feasibility data and historical collaborative success, will advance the understanding of CD39 thromboregulation, and lead to a novel therapeutic agent for thrombotic diatheses.
| Dai, Jiayin; Liu, Jie; Deng, Yiqun et al. (2004) Structure and protein design of a human platelet function inhibitor. Cell 116:649-59 |
