The long-term objective of the proposed research is to elucidate the mechanism and regulatory factors involved in the interactions of proteases of the blood coagulation system with circulating protein inhibitors, and to define the effects of heparin and related compounds on these processes. this should lead to a better understanding of various physiological and pathological aspects of coagulation and will delineate the effects that administration of heparin can produce when utilized in the treatment of thrombotic disorders. The specific reactions to be investigated will be those between thrombin and antithrombin III; Factor Xa and antithrombin III; and Factor Xa and alpha1-Proteinase Inhibitor. A study will be made on the specific effects of heparin on each of these reactions. The actions of heparan sulfate and dermatan sulfate as physiological effectors in promoting the neutralization of procoagulants by antithrombin will also be investigated. Additionally, the possible contribution of glucosyl ceramide to the activity of antithrombin III will be determined. Detailed studies will also be conducted on the location and distribution in high molecular weight heparin, of the oligosaccharide sequences that function as the recognition or binding sites for target proteins. These investigations will include novel procedures involving reactions on matrix-bound heparin and covalently linked fluorescent labels. The binding of heparin with coagulant proteins and inhibitor-proteins, as well as the reactions between coagulant and anticoagulant proteins will be studied by means of fluorescent-labeled components. Fluorescence intensity and polarization techniques will be employed. These investigations are expected to define the relationship between the chain-length of heparin and the binding stoichiometries, equilibrium binding constants, and rates of the abovementioned processes.
