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在试管中进行的，在静态实验条件下，所有的蛋白质试剂要么在均匀的溶液中，要么存在于磷脂微囊的表面，而磷脂微囊本身就是均匀的悬浮液。该项目的目标是在KinExa/TM流动荧光分析仪上开发和开发新的分析方法，以在定义良好的连续流动系统中研究凝血和纤溶反应，该系统可以更真实地模拟血管内存在的动态条件。KinExA的毛细管流/观察池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到该反应器中，以模拟凝血启动过程中存在的层流条件。或者，在流动池中填充涂有相同试剂的珠子，以模拟凝聚后期阶段存在的湍流条件。在任何一种情况下，可溶性凝血因子和/或生理性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池。凝血蛋白以不同的流速选择性地激活或保留在毛细血管流动/观察细胞内。选择性激活或保留在毛细血管流动/观察细胞内的凝血蛋白将使用针对单个蛋白酶的荧光底物或针对感兴趣的凝血蛋白(S)的荧光标记抗体进行定量。需要研究的磷脂依赖凝血酶包括内源性Xase、外源性Xase和凝血酶原酶。此外，纤维蛋白凝块将在观察细胞中形成，并与选定的纤溶酶和抑制剂一起灌流，以研究与纤维蛋白凝块溶解有关的结合和降解反应。发生在纤维蛋白网络和纤维蛋白网络内的结合反应将用针对感兴趣的纤溶蛋白的荧光标记抗体进行量化。预计这些实验将在更接近生理条件下确定血液凝固和纤溶级联中流动参数对个别反应的重要性。

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.