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 production. Our long-term goal is to understand the interactions between the """"""""glycosaminoglycan (GAG)-dependent"""""""" serine protease inhibitor (serpin) heparin cofactor II (HCII), GAGs and thrombin and attempt to define how these interactions regulate the """"""""cytokine-like"""""""" effects of thrombin on thrombin responsive cells. The serine protease thrombin has a key role in blood coagulation and other host defense mechanisms. Thrombin has two distinct types of activities: (i) those related to hemostasis, which are primarily manifest at sites of intravascular injury; and (ii) cytokine-like activities, which are primarily manifest in the extravascular stroma. There is much clinical evidence to suggest that antithrombin (AT) controls the first type of thrombin activity. We have a theory that HCII in the presence of extravascular proteoglycans regulates the second type of thrombin activity. We also hypothesize that thrombin dysregulation in atherosclerotic vessels is partly due to the loss of HCII-GAG interactions because of changes to proteoglycans with the progression of disease. Finally, we have a hypothesis that HCII activity is dependent upon unique protein-GAG interactions to displace its acidic domain for thrombin inhibition. To test these hypotheses, we propose to one, examine the mechanism of HCII-thrombin inhibition in the presence of GAGs, and to """"""""transfer"""""""" the HCII structural elements for GAG-dependent activity to a non-GAG-dependent serpin (alpha1-protease inhibitor) by site-directed mutagenesis; two. crystallize HCII and crystallize thrombin-HCII acidic domain peptides, and to study structure-function relationships of the acidic domain; and three, characterize the ability of HCII to regulate thrombin's cytokine-like activity for mononuclear leukocytes and endothelial cells exposed to pro- and antiatherogenic substances, and to study the in vivo localization of HCII, AT, and thrombin antigen in normal and atherosclerotic vessel wall. 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 HCII functions in thrombosis, especially related to atherosclerosis. Understanding the molecular and cellular properties of HCII may lead to new insight into the pathophysiology and therapies of cardiovascular diseases.
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