PAR Regulation of Platelet Function in Diabetic Patients

Overview

Thrombin is the most potent activator of platelets, and platelet activation is a hallmark of thrombosis. Coronary artery disease (CAD) is the major cause of mortality and morbidity in the United States and other industrialized countries, and thrombotic sequelae are the key cause of death in diabetes. The accumulation of thrombin at sites of vascular injury provides one of the major mechanisms of recruiting platelets into a hemostatic plug. Thrombin works by activation of the G protein-coupled protease activated receptors PAR1 and PAR4 on human platelets to initiate signaling cascades leading to increases in \[Ca\]i, secretion of autocrine activators, trafficking of adhesion molecules to the plasma membrane, and shape change, which all promote platelet aggregation. The thrombin receptors work in a progressive manner, with PAR1 activated at low thrombin concentrations, and PAR4 recruited at higher thrombin concentrations. As direct thrombin inhibitors become widely used in clinical practice, it is important to assess their effects on vascular function. Our hypothesis is that PAR1 and PAR4 do not signal through the same G protein pathways, and that PAR4 is not a strong platelet agonist. To investigate this hypothesis, the investigators will study the G protein pathways downstream of PAR4, and assess ex-vivo platelet responsiveness to thrombin, PAR1, and PAR4 agonist peptides, both in normal human subjects, and along the stages of pathology, from patients with stable angina as well as unstable angina who are undergoing angioplasty. Similarly, the investigators will examine platelet function in patients with metabolic syndrome as well as diabetes, along the continuum from insulin resistance to full-blown disease. These studies will provide deeper insight into the G protein pathways used by PARs. They will elucidate the contribution of PAR receptors to normal platelet function as well as the abnormal platelet activation in thrombotic states. The long term goal is to understand the implications for PAR receptors as therapeutic targets for anti-platelet therapies that may carry less bleeding risk.

Given the known roles of proteases and PARs in coagulation, inflammation, pain, healing and protection, the need for development of a PAR antagonist as a therapeutic agent for treatment of thrombosis, atherosclerosis and inflammation is well-recognized. Thus, blocking PAR action by inhibiting the PAR-G protein interface is an alternative target for blocking downstream consequences of thrombin-mediated cellular activation. Since there are two PARs on human platelets, PAR1 and PAR4, it is critical to define the roles of both receptors in several likely clinical settings where PAR antagonists would be used. In this proposal both the G protein pathways underlying PAR signaling mechanisms as well as their roles in pathologies characterized by activated platelets will be studied in detail. We propose in our grant to investigate the specific roles of individual PARs in mediating the events leading to the multi-stage process of platelet activation and clot formation. The long term goals of these studies are to determine novel PAR-specific anti-platelet therapies. The 2002 estimates of the DHHS suggest that 6.3% of the population of the United States (18.2 Million people) have diabetes, with estimated annual costs of $132 Billion (Centers for Disease Control and Prevention). The complications of coronary artery and of cerebrovascular disease account for up to 65% of deaths in patients with diabetes. The prevalence, complexity and complications of cardiovascular disease are increased in patients with diabetes. This appears related to the frequent association of diabetes and of insulin resistance with traditional risk factors for atherosclerosis. Recent studies by Haffner and other investigators have established the concept of diabetes as a coronary risk equivalent; this is reflected in the guidelines for therapeutic targets set by the ADA, AHA and ACC. The clinical importance of platelet activation is reflected by the benefits of aspirin and clopidogrel and GPIIbIIIa inhibitors in diabetics. Epidemiologic data demonstrates that the metabolic syndrome is associated with an increased incidence of atherosclerotic events. Type 2 diabetes shares many features in common with the metabolic syndrome, and may represent a precursor condition, Stern described this in the "common soil hypothesis". Hsueh and Law have more recently proposed that the progression of insulin resistance to type 2 diabetes parallels the progression of endothelial dysfunction to atherosclerosis. These studies were designed to study PAR signaling and G protein activation states in patients with platelet activation in the setting of the continuum of the metabolic syndrome and diabetes mellitus. The degree of platelet activation as determined by assays of platelet reactivity and by the expression of markers of platelet activation will be correlated with the changes in PAR signaling. These studies will provide a comprehensive assessment of platelet activation in the setting of the metabolic syndrome, and will compare the extent of activation in this setting with that in a group of contemporaneously studied patients with diabetes mellitus. Finally, the influence of the direct thrombin inhibitor, bivalirudin on platelet activation and signaling will be assessed.