Continued support is requested for biochemical studies designed to provide new basic knowledge about proteins responsible for the regulation of the intrinsic coagulation pathway, the kinin-forming pathway and the intrinsic fibrinolytic pathway of plasma. This research proposal addresses basic questions about the initiation and regulation of the contact activation pathways of plasma. The overall strategy of approach is to isolate human plasma proteins that are involved in these pathways, to define relationships between molecular structure and function, and then to study these proteins in plasma experiments in order to define the behavior of these molecules in a physiologic milieu. The long-term objectives of this research project and of the laboratory of the principal investigator involve systematic efforts to elucidate the molecular mechanisms responsible for the regulation of thrombosis and hemostasis. The studies proposed on Factor XII, Factor XI, Factor IX, prekallikrein, high MW kininogen and plasma prourokinase will give new information about the structure and function of these plasma proteins.
The specific aims i n this proposal include studies designed to expand our basic knowledge of the chemical, physical, immunochemical and functional properties of Factors XII, XI, and IX, prekallikrein, high MW kininogen and prourokinase. Experiments are designed to provide further insight to the molecular mechanisms responsible for surface-dependent activation of Factor XII, Factor XI, and prekallikrein. The molecular basis for protein-protein interactions among these plasma components on negatively charged surfaces will be studied. Proteolytic fragments of each of these molecules will be prepared and the expression of functional activity will be related to molecular structure. Abnormal variants of prekallikrein, prekallikrein-Long Beach, and of Factor XII, Factor XII-Bar1, will be isolated and further studied to relate functional abnormalities to structural abnormalities. The recent controversial hypothesis that high MW kininogen, the contact activation cofactor, must be activated by limited proteolysis and subsequently inactivated by proteolysis will be experimentally tested. The contributions of Ca++ ions to the molecular mechanisms responsible for the activation of Factor IX by Factor XIa will be investigated. Quantitative immunoblotting studies of fragments of Factors XII, XI, IX, prekallikrein and high MW kininogen in activated whole plasma will be developed using Western blot techniques. These studies will allow a critical assessment of current concepts and hypotheses for the activation and proteolysis of these proteins in the plasma milieu. The activation of plasma prourokinase by Factor XIIa, Factor XIa, and kallikrein will be studied to help define mechanisms of Factor XII-dependent intrinsic fibrinolysis. It is anticipated that further insights into the molecular mechanisms responsible for contact activation of coagulation, kinin-formation, and fibrinolysis will come from these basic studies.
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