The diagnosis, treatment, and prevention of platelet-induced occlusive diseases of the coronary and cerebral circulation are a major challenge to the VA patient care mission. Excessive platelet activation and recruitment underlies the pathogenesis of platelet-induced occlusive vascular disease. Our proposal focuses on development of a novel approach toward prevention and treatment of these disorders, namely the use of the thromboregulatory ecto-apyrase ATPDase/CD39/NTPDase1. CD39 functions as the principal regulator of blood fluidity by metabolizing prothrombotic ATP and ADP released from activated platelets and other cells to prevent thrombus formation. We previously reported prothrombotic alterations in CD39 activity in patients with verified coronary artery disease. Subsequently, we examined leukocyte CD39 activity in stroke patients and found that lymphocytes from these subjects displayed changes in the ADPase/ATPase activity ratio, as compared to controls. This observation led to our hypothesis that alterations in CD39 activity can result in a predisposition for stroke. This application extends our previous research and focuses on three main areas. First, we will define the molecular biological mechanisms underlying regulation of expression and activity of CD39, and alterations therefrom that influence cellular function in vascular disease. Our preliminary data indicate that maximal CD39 enzyme activity requires formation of higher molecular weight complexes localized in cholesterol-rich membrane subdomains via a process requiring cleavage of the protein. We plan to fully confirm and extend these observations, including elucidation of the site of cleavage in CD39 and the subcellular location of the cleavage event. We have also determined that our recently discovered CD39 splice variants (V1-4) regulate apyrase activity at the cell surface. We will elucidate the role of these CD39 alternative splice variants in regulatio of CD39 intracellular trafficking and cleavage. We will also determine whether CD39 splice variant generation and translation is under tissue- and stimulus-specific regulation. CD39 splice variants modulate formation of enzymatically active CD39 species in the plasma membrane. This determines the pro- or antithrombotic phenotype of the cell. Second, we will further establish the relationship between alterations in CD39 activity, platelet reactivity, and stroke. W will extend our stroke research to 3 different patient groups: (1) Cryptogenic stroke patients (stroke in a young population without clinical etiology);(2) """"""""pre-stroke"""""""" patients who have verified obstructive sleep apnea;and (3) classical atherothrombotic stroke patients. We have identified cryptogenic stroke and stroke-prone patients who have increased responsiveness to platelet agonists and increased expression of platelet and leukocyte activation markers. De-identified blood samples from these patients will be processed for isolation of platelets, lymphocytes and neutrophils. These will then be analyzed by several methods, including flow cytometry and our newly developed Broad Range Platelet Aggregometry System. In depth classification of these patients with regard to their threshold for platelet reactivity will increae our comprehension of the pathogenesis of stroke. This will also allow us to test our hypothesis that cryptogenic stroke may, in large part, represent a disorder of enhanced platelet reactivity. Third, we will determine CD39 surface expression and splice variant profiles in leukocytes of stroke patients versus controls by flow cytometry, quantitative real-time PCR, and DNA Fragment analyses. These studies will establish the relationship between stroke etiology and CD39 variant profile. This will further verify our hypothesis that altered CD39 activities play a role in cryptogenic stroke and provide a mechanistic basis for this role. Our proposed studies represent a multidisciplinary approach to the pathogenesis, prevention and treatment of ischemic stroke as well as to the regulatory aspects of our newly discovered CD39 splice variants. This research will enhance our knowledge of the mechanisms of action of CD39 and ultimately lead to development of a safe and effective treatment of platelet-driven occlusive vascular disorders in patients.
Stroke, myocardial infarction, and peripheral vascular disease are circulatory disorders afflicting the majority of VA patients. Stroke is the most common life-threatening neurological disorder and the third leading cause of death in the US. Blood vessel obstruction due to the pathological accumulation of blood platelets is the major etiological factor in these disorders. Current antithrombotic and neuroprotective therapeutic measures are useful, but require further improvement to decrease morbidity and mortality. CD39 is a cell surface enzyme that controls thrombosis by modulating excessive platelet reactivity. Because of its ability to control platelet reactivity and thereby maintain blood fluidity, CD39 should serve as a therapeutic modality for platelet-driven thrombotic disorders. This has been demonstrated in animal models. Our multidisciplinary and multifaceted approach will provide new information on the treatment of stroke. Our studies will also establish predictive parameters for stroke predilection/recurrence and will suggest indications for CD39 therapeusis.