Thrombosis is a major cause of morbidity and mortality in Western man. It is important to understand how the body regulates both thrombin activity and protection. The goal of this research is to delineate the pathobiological role of protein C inhibitor (PCI), a serine protease inhibitor (serpin) whose significance is not clearly defined. We will accomplish this goal using molecular biological/protein biochemical approaches, coagulation-based assays in the absence and presence of endothelial cells, immunohistochemical/in situ hybridization studies on vessel wall components, and by preparing transgenic mice that over-express PCI. The protein C pathway is an important component in regulating thrombin activation. Protein C is proteolytically activated by thrombin complexed to thrombomodulin (TM). The primary inhibitor of this activated protein C (APC) is PCI. Recently, it has been shown that PCI is a potent inhibitor of thrombin bound to TM and that PCI plasma levels are elevated in male survivors of myocardial infarction. This means that PCI could regulate both the activity and production of APC (suggesting that PCI is """"""""procoagulent""""""""). We hypothesize that PCI plays a role in the pathogenesis of thrombosis by preventing both the activation and activity of the protein system of proteases. To test this hypothesis, we proposed to one, study the mechanism of PCI inhibits the hemostatic proteases thrombin (+/- TM) and APC; two, determine the mechanism of how PCI sustains thrombin generation in plasma and using a purified coagulation assay in the presence of endothelial cells; three, study the in vivo localization of PCI antigen and mRNA in the vasculature and examine how PCI activity is modulated in vitro by vessel wall components; and four, characterize the contribution of PCI over- expressing transgenic mice to regulate APC and thrombin activity in response to vascular injury. While it is evident that inhibition of plasma-derived proteases is achieved by specific interactions, there are many essential molecular and cellular details that remain to be described. The information provided by the experiments outlined in this grant proposal will further define the mechanism of how PCI functions in hemostasis and thrombosis. Understanding the properties of PCI may lead to new insight into the pathophysiology and therapies of cardiovascular diseases.
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