Structural Studies of Blood Clotting Proteins

凝血蛋白的结构研究

• 批准号: 6656243
• 负责人: JOHN W WEISEL
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6656243
• 关键词: blood coagulation; confocal scanning microscopy; fibrin; fibrinolysis; plasminogen; plasminogen activator; plasminogen activator inhibitors; platelet aggregation; scanning electron microscopy; thrombin; transmission electron microscopy

The overall goal of this project is the determination of molecular mechanisms of the physical process of fibrinolysis. Although much is known about the biochemistry of fibrinolysis, much less is known about the changes in fibrin clot structure as fibers are lysed and the appearance of the pieces removed by digestion. Our preliminary results have shown that fibrinolysis of fibrin clots proceeds by transverse cutting of fibers, rather than digestion of fibers from the outside-in, and removal of large pieces held together by non-covalent forces. Confocal microscopy has revealed complex changes that take place at the lysis front. The first specific aim tests the hypothesis that fibrinolysis proceeds by a series of actions at the lysis front including transverse cutting of fibers, but these processes are affected by the conditions of lysis, including the nature of the enzyme/activator and the mode of its introduction, and the presence of other proteins, such as Lp(a), PAI-1, and TAFI. One of the principal approaches will be to follow digestion in real time using confocal microscopy in both reflectance and fluorescence modes. We also will examine the digested clot surface by scanning electron microscopy. The cleaved pieces removed from the clots will be characterized by transmission electron microscopy of negatively contrasted or rotary shadowed specimens. The process of intrinsic fibrinolysis, in which clots are formed in the presence of plasminogen and tPA so that they are digested internally, will be studied by confocal microscopy and by following turbidity and rigidity of the clots as a function of time. The second specific aim tests the hypothesis that the rate and nature of fibrinolysis is dependent on clot structure, but is not simply a function of fiber diameter. The overall approach will be to make clots with different structures, characterize them quantitatively and then to measure the rates of fibrinolysis using several different methods and to measure the binding of plasminogen and tPA and the activation of plasminogen at the lysis front. The third specific aim tests the hypothesis that the influence of platelet aggregation on fibrin structure and properties has a major impact on events at the lysis front. The fiber meshwork is much denser in the vicinity of platelet aggregates and the fibers are more oriented around the platelets. In addition, plasminogen activator inhibitor-1 is released from platelets and binds to fibrin in the vicinity of platelet aggregates, retarding lysis in these areas. Although these phenomena are well known, the physical processes involved and the local effects have not been studied. The physical process of lysis of platelet-rich clots will also be studied in the presence of abciximab, an inhibitor of interactions between fibrin(ogen) and the platelet integrin, alphaIIbbeta3, the integrin that binds fibrinogen in platelet aggregation. The results of these studies will help us to understand molecular mechanisms of fibrinolysis, which may have clinical implications for the treatment and prevention of thrombotic disorders.

该项目的总体目标是确定纤溶物理过程的分子机制。虽然人们对纤溶的生物化学了解很多，但人们对纤维溶解时纤维蛋白凝块结构的变化以及消化去除纤维碎片的外观知之甚少。我们的初步结果表明，纤维蛋白凝块的纤溶是通过横向切割纤维进行的，而不是从外到内消化纤维，并去除由非共价力粘合在一起的大块。共聚焦显微镜显示了裂解前沿发生的复杂变化。第一个特定目的验证了纤溶通过裂解前沿的一系列作用进行的假说，包括横向切割纤维，但这些过程受到裂解条件的影响，包括酶/激活剂的性质和引入的方式，以及其他蛋白质的存在，如Lp(A)、PAI-1和TAFI。主要方法之一将是在反射和荧光模式下使用共聚焦显微镜实时跟踪消化。我们还将用扫描电子显微镜检查消化后的血块表面。从凝块中取出的解理碎片将通过阴性对比或旋转阴影标本的透射电子显微镜来表征。内源性纤溶过程，即在纤溶酶原和tPA存在的情况下形成凝块，使它们在内部被消化，将通过共聚焦显微镜和跟踪凝块的混浊和硬度随时间的变化来研究。第二个具体目的是检验纤溶的速度和性质取决于血栓结构的假设，但不是简单的纤维直径的函数。总的方法是制作具有不同结构的血栓，对其进行定量表征，然后使用几种不同的方法测量纤溶速率，并测量纤溶酶原与tPA的结合以及纤溶酶原在溶解前沿的激活。第三个特定目的是检验这样一种假设，即血小板聚集对纤维蛋白结构和性质的影响对溶解前沿的事件有重大影响。在血小板聚集体附近，纤维网络更致密，纤维更多地定向在血小板周围。此外，纤溶酶原激活物抑制物-1从血小板释放，并与血小板聚集物附近的纤维蛋白结合，延缓这些区域的溶解。虽然这些现象是众所周知的，但所涉及的物理过程和局部效应还没有被研究过。在阿昔单抗的存在下，还将研究富含血小板的凝块溶解的物理过程。阿昔单抗是一种抑制纤维蛋白(原)和血小板整合素之间相互作用的物质，αIIbbeta3是一种在血小板聚集中结合纤维蛋白原的整合素。这些研究结果将有助于我们了解纤溶的分子机制，这可能对血栓性疾病的治疗和预防具有临床意义。