In order to function physiologically, most of the enzymes involved in stimulating or limiting blood clot formation (procoagulant or anticoagulant, respectively) must bind simultaneously to a phospholipid surface, calcium ions, and a non-enzymatic protein cofactor. The experiments proposed in this application address two primary issues: (i) what are the mechanisms by which the required protein cofactors stimulate the zymogen activation activity of procoagulant and anticoagulant enzymes; and (ii) what are the topographies of the procoagulant and anticoagulant complexes. As our most recent results have demonstrated, these issues are closely coupled: factor Va, the cofactor in the prothrombinase complex, alters both the conformation of the active site of the enzyme factor Xa and the location of the active site above the membrane surface. Fluorescence spectroscopy will be used to examine the interactions between the components of two procoagulant (factor Xa- factor Va and factor IXa-factor VIIIa) and two anticoagulant (thrombin-thrombomodulin and activated protein C-protein S) complexes. The emission of fluorescent probes covalently attached to residues in the active sites of the enzymes will be monitored to determine whether the conformation of the active site is altered by the association of the enzyme with calcium ions, the membrane surface, and/or its cofactor. The influence of other proteins (e.g., inactivated cofactors, individual cofactor subunits, activation peptides) will also be investigated. When possible, spectral changes will be used to monitor complex formation and determine the affinity between the enzyme and its ligands. Singlet-singlet energy transfer will be used to measure directly the distance between the membrane surface and the active sites of the membrane-bound enzymes. This will indicate the molecular arrangement in the thrombin-thrombomodulin complex, about whose topography very little is known. In the other complexes, the dependence of the measured distance on the presence of the cofactor will indicate the magnitudes of the conformational changes that accompany the assembly of the complexes on the membrane surface. These results will also indicate whether the structurally dissimilar cofactors function homologously by altering the location of the active site, and thereby contribute to the substrate specificity of the enzyme. Other energy transfer experiments will include the location of the Gla-independent calcium ion binding sites in protein C and factor IX above the membrane surface.
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