CONTINOUOUS FLOW EFFECT ON BLOOD COAGULATION PROTEIN

大多数血液凝固和纤维蛋白溶解的功能研究都是在静态实验条件下在试管中进行的，其中所有的蛋白质试剂要么在均匀的溶液中，要么存在于磷脂囊泡的表面上，磷脂囊泡本身在均匀的悬浮液中。本项目的目标是开发和利用KinExa/TM流动荧光分光光度计上的新测定，以研究在定义明确的连续流动系统中的凝血和纤溶反应，该系统更真实地模拟血管内存在的动态条件。KinExA的毛细管流动/观察池的内表面将用作管状酶反应器，磷脂和选定的凝血蛋白将被吸附到该反应器中，以模拟凝血开始时存在的层流条件。或者，流动池将填充有涂覆有相同试剂的珠粒，以模拟凝结后期阶段中存在的湍流条件。在任一种情况下，可溶性凝血因子和/或生理丝氨酸蛋白酶抑制剂的单独混合物随后将以不同的流速被抽吸通过观察池。凝血蛋白在不同流速下选择性激活或保留在毛细管流动/观察池内。将使用对单个蛋白酶具有特异性的荧光底物或针对目标凝血蛋白的荧光标记抗体对选择性激活或保留在毛细管流动/观察池内的凝血蛋白进行定量。待研究的磷脂依赖性凝血蛋白酶包括内源性Xase、外源性Xase和凝血酶原酶。此外，将在观察室中形成纤维蛋白凝块，并灌注选定的纤维蛋白溶解酶和抑制剂，以研究纤维蛋白凝块溶解中涉及的结合和降解反应。发生在纤维蛋白网络上和纤维蛋白网络内的结合反应将用针对感兴趣的纤维蛋白溶解蛋白的荧光标记抗体进行定量。预计这些实验将确定在更接近生理条件下，血液凝固和纤维蛋白溶解级联中流动参数对个体反应的重要性。

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.