MOLECULAR BASIS OF BLOOD COAGULATION REGULATION

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The overall objective of this project is to elucidate the molecular mechanisms of regulation of blood coagulation proteinases by the serpin inhibitor, antithrombin (AT); i.e. what governs the specificity of AT for its target proteinases, what is the mechanism by which AT traps these enzymes in stable complexes, how does heparin enhance the rate of this trapping and what other modulating factors act to protect proteinases at their site of action and to promote their inactivation when they escape from these sites? We will determine whether the specificity of AT for its target proteinases is dictated by the complementarity of an exposed reactive-site loop of AT for the active-site region of the enzyme and whether heparin activates AT by enhancing this complementary interaction with proteinases. The detailed mechanism of AT trapping of enzymes in stable complexes will further be elucidated. The role of the conformational change, in which the exposed reactive-site loop of AT is inserted as a central strand into the A beta-sheet of the protein core, in this trapping will be assessed. We will determine whether this conformational change is induced by a substrate-like interaction of AT with the enzyme, whether the conformational change traps the enzyme at an inter-mediate stage of this substrate reaction and whether some of the enzyme can escape the trapping by completing the cleavage of the reactive bond before trapping occurs. We will further elucidate the mechanism of heparin activation of AT. The specific heparin binding region of AT will be localized and the interactions responsible for inducing a conformational change in AT will be delineated. We will determine whether the heparin-induced conformational change is responsible for the enhanced reactivity of AT with certain proteinases and whether it acts only to promote tight binding of AT to heparin for other proteinase reactions, with the additional binding of the proteinase to heparin being responsible for the enhanced reactivity of AT with these latter enzymes. We will finally assess whether cofactor proteins and/or proenzyme domains released upon enzyme activation promote the inactivation of proteinases by AT and AT-heparin and whether the binding of these proteinases to phospholipid surfaces antagonizes this promotion. These studies will utilize classical thermodynamic and kinetic approaches for evaluating the interactions and reactions between AT, proteinases, heparin and other effectors and site-directed mutagenesis of recombinant AT for mapping the functional sites of AT which mediate these interactions and reactions. The studies are expected to provide new information on the structural basis of AT anticoagulant action as well as the general mechanism of action of serpin inhibitors. Elucidation of the factors which modulate AT-proteinase reactions should further provide an increased understanding of the mechanisms of localization of clotting and the expression of the activity of clotting proteinases. Finally, a rational basis for the design of recombinant ATs or of heparins with improved anticoagulant efficacy for anticoagulant therapy is anticipated.