CONTINOUOUS FLOW EFFECT ON BLOOD COAGULATION PROTEIN

大多数凝血和纤溶的功能研究都是在静态实验条件下的试管中进行的，其中所有的蛋白质试剂要么处于均相溶液中，要么存在于磷脂囊泡表面，而磷脂囊泡本身处于均相悬浮液中。该项目的目标是开发和利用KinExa/TM流动荧光光谱仪的新检测方法，以在定义良好的连续流动系统中研究凝血和纤溶反应，该系统更真实地模拟了血管内存在的动态条件。KinExA的毛细管流动/观察细胞的内表面将作为管状酶反应器，磷脂和选定的凝血蛋白将被吸附在其中，以模拟凝血开始时存在的层流条件。或者，流动池将充满涂有相同试剂的珠子，以模拟凝血后期存在的湍流流动条件。在任何一种情况下，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将以不同的流速通过观察细胞。凝血蛋白以不同流速选择性激活或保留在毛细管流动/观察细胞内。在毛细管流动/观察细胞中选择性激活或保留的凝血蛋白将使用针对单个蛋白酶或针对感兴趣的凝血蛋白的荧光标记抗体的特异性荧光底物进行定量。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。此外，在观察细胞中形成纤维蛋白凝块，并灌注选定的纤维蛋白溶酶和抑制剂，以研究纤维蛋白凝块溶解过程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荧光标记的抗体针对感兴趣的纤溶蛋白进行量化。预计这些实验将在更接近生理条件下确定血流参数对凝血和纤溶级联中单个反应的重要性。

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.