In preliminary experiments, I have identified a previously unrecognized protease inhibitor in human plasma. Initial studies indicate that this inhibitor is a protein with an apparent molecular weight of 65,6000, that it inactivates thrombin by forming a very stable 1:1 stoichiometric complex with the protease, and that it is immunologically distinct from all of the known plasma protease inhibitors. A striking property of the new inhibitor is that its action is markedly accelearted by heparin. In addition, at relatively high concentrations of heparin, this inhibitor competes quite effectively for thrombin with antithrombin III, although antithrombin III has previously been considered to be the only quantitatively significant inhibitor of thrombin in plasma. In this application, I propose to do the following: 1) Purify a sufficient quantity of the inhibitor for structural studies. 2) Raise antibodies in rabbits and develop a specific immunoassay for the new inhibitor in plasma. 3) Determine the partial NH2- and C00H-terminal amino acit sequences of the protein. 4) Determine the ability of the purified inhibitor to inactivate the following purified proteases: coagulation factors VIIa, IXa, XIIa (fragments), kallikrein, plasmin, and activated protein C. 5) Establish the heparin-dependence and stoichiometry of inhibition of the proteases in (4). 6) Perform detailed kinetic studies to determine the 2nd-order rate constant and/or the equilibrium dissociation constant of the inhibitor-protease complex in the presence and absence of heaprin. 7) Fractionate a crude heparin preparation by size and by affinity of binding to the inhibitor, and compare the ability of each fraction to activate both the new inhibitor and antithrombin III. 8) Label a highly active fraction(s) of heparin with 14C or 3H, and determine its dissociation constant and stoichiometry of binding to the new inhibitor. 9) Determine whether the new inhibitor undergoes limited proteolysis during complex formation with a protease. 10) Isolate and sequence the proteolytic gragments, if found, in (9) to investigate the structure of the reactive site of the inhibitor.
