Most functional studies of blood coagulation and fibrinolysis have been conducted in test tubes under static experimental conditions where all of the proteinaceous reagents are either in homogeneous solution or present on the surface of phospholipid vesicles that are themselves in homogeneous suspension. The goal of this project is to develop and exploit new assays on the KinExa/TM flow spectrofluorimeter to investigate coagulation and fibrinolysis reactions in a well-defined continuous flow system that simulates more realistically the dynamic conditions that are present intravascularly. The inner surface of the capillary flow/observation cell of the KinExA will serve as a tubular enzyme reactor to which phospholipids and selected coagulation proteins will be adsorbed to simulate the laminar flow conditions present in the initiation of coagulation. Alternatively, the flow cell will be filled with beads coated with the same reagents to simulate the turbulent flow conditions present in the later stages of coagulation. In either case, individual mixtures of soluble coagulation factors and/or physiological serine protease inhibitors will then be drawn through the observation cell at different flow rates. Coagulation proteins selectively activated or retained within the capillary flow/observation cell at different flow rates. Coagulation proteins selectively activated or retained within the capillary flow/observation cell will be quantified using fluorigenic substrates that are specific for individual proteases or fluorescently-labeled antibodies directed against the coagulation protein(s) of interest. Phospholipid- dependent coagulation proteases to be studied include intrinsic Xase, extrinsic Xase, and pro-thrombinase. In addition, fibrin clots will be formed in the observation cell and perfused with selected fibrinolytic enzymes and inhibitors to investigate the binding and degradation reactions involved in the dissolution of fibrin clots. Binding reactions that occur to and within the fibrin network will be quantified with fluorescently-labeled antibodies directed against the fibrinolytic proteins of interest. It is anticipated that these experiments will determine the importance of flow parameters on individual reactions within the blood coagulation and fibrinolysis cascades under conditions that more closely approximate those of physiological.
Showing the most recent 10 out of 34 publications
