Tissue injury requires rapid and focal response of blood coagulation factors and cellular proliferation for repair. The cysteinyl-rich and growth factor-like domains in many blood coagulation factors may play a central role in such a process. However, the functional roles of these domains are presently unknown and uncharacterized. The goals of this proposal are the attainment of knowledge of the role of these cysteinyl-rich domains in the mechanism of blood coagulation pathway, stimulation of plasminogen activator for vasculature, and the proliferative responses in injury. The proposal includes chemical synthesis, conformational study by NMR and biological evaluation of the cysteinyl-rich domains in Factor IX and Factor X of the coagulation pathway. Synthetic cysteinyl-rich domains of these blood coagulation factors and their variants will be prepared by the solid-phase method. The solution conformation of these synthetic molecules will be studied by 2D-NMR to correlate the structure-activity studies. To determine the functional role of these cysteinyl-rich domains in the blood coagulation pathway, we propose to study the Ca2+- binding activity, the activation of tissue plasminogen activator and urokinase, and the inhibition of binding of Factor IX to its endothelial cell receptor on the endothelial cell-dependent coagulation pathway. Synthetic analogs are tested for their selective activity on mitogenicity, stimulation of tissue plasminogen activator, or inhibitory activity against the binding of Factor IX to its receptor on the endothelial cell. Our long term goal is that the chemical, physical and biological studies of these cysteinyl-rich, growth factor domains and their analogs will aid the understanding, on a molecular level, of the interaction and activation of various blood coagulation factors. This may ultimately lead to the design and synthesis of suitable analogs that function as inhibitors for the in vivo regulation of a specific coagulation factor and tissue plasminogen activator. Such inhibitors may provide a very different approach to the interruption of vasculative of tumor progression or thrombosis.
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