The regulation of coagulation is central to many diseases, including heart disease and stroke. The objective of this research is to better understand the different mechanisms by which the serpins antithrombin (AT) and protein C inhibitor (PCI) inhibit free thrombin, and thrombin bound to thrombomodulin (TM). Specifically, the role of the reactive site loop of PCI in the inhibition of thrombin bound to TM will be examined. A second objective will be to compare the function of the H-helix of PCI with the H-helix of AT. The first aim of this proposal is to substitute the P3-P3' reactive site loop residues of PCI for those of AT to determine whether the high rates of inactivation are due merely to the reactive site loop differences, or some other portion of PCI. The second aim is to determine the role of the H-helix in AT. To do this, glutamate residues in the H-helix of AT will be replaced with arginine and lysine, mimicking the structure of the H-helix in PCI. In the third aim, position 314 of AT will be mutated to an arginine. These proteins will then be assayed with thrombin in the presence and absence of TM to determine whether these mutations alter the activity of AT. The outcome of these experiments may provide a clearer understanding of the different mechanisms by which PCI and AT inhibit thrombin complexed with TM.