""""""""Thrombosis and thrombolysis are maintained in a delicate balance by a series of enzymatic reaction. The component enzymes are produced as needed from zymogens, inactive forms which circulate in the blood at relatively high concentrations or in the case of t-PA secreted from cells as an active enzyme. Identifying and evaluating patients at risk for clinical thrombotic disorders will require accurate measurement of plasma levels of these active factors. In the Phase I project period, a bifunctional probe has been developed and characterized which irreversibly binds to serine proteases, and which contains a secondary recognition site for binding to a solid phase. This probe allows for the selective labelling and subsequent quantitation of active serine proteases at the precise time of sample collection. Assays using the probe and enzyme conjugated monoclonal antibodies have been developed for Factor Xa and t-PA. The Phase II proposal has the following specific aims: 1) To continue development of the CASSIA technology for t-PA toward the goal of a marketable product; 2) To expand and optimize CASSIA technology for the assay of additional serine proteases including thrombin Factor VIIa, Factor IXa, Factor Xa, and activated protein C; and 3) To evaluate the diagnostic potential of each assay developed under aim #2 through in vitro and clinical studies.""""""""
