Structural Studies of Blood Clotting Proteins

该项目的总体目标是确定纤维蛋白溶解物理过程的分子机制。尽管人们对纤维蛋白溶解的生物化学了解很多，但对纤维溶解时纤维蛋白凝块结构的变化以及消化去除的碎片的外观知之甚少。 我们的初步结果表明，纤维蛋白凝块的纤维蛋白溶解是通过横向切割纤维来进行的，而不是从外到内消化纤维，并去除由非共价力结合在一起的大块。 共焦显微镜揭示了裂解前沿发生的复杂变化。 第一个具体目标测试了以下假设：纤维蛋白溶解是通过裂解前沿的一系列作用进行的，包括纤维的横向切割，但这些过程受到裂解条件的影响，包括酶/激活剂的性质及其引入方式，以及其他蛋白质的存在，例如 Lp(a)、PAI-1 和 TAFI。 主要方法之一是使用共焦显微镜在反射和荧光模式下实时跟踪消化。 我们还将通过扫描电子显微镜检查消化的凝块表面。 从凝块中取出的切割碎片将通过负对比或旋转阴影样本的透射电子显微镜进行表征。 内源性纤维蛋白溶解过程，即在纤溶酶原和 tPA 存在的情况下形成凝块，以便它们在内部被消化，将通过共聚焦显微镜和跟踪凝块的浊度和硬度随时间的变化来研究。 第二个具体目标测试了以下假设：纤维蛋白溶解的速率和性质取决于凝块结构，而不仅仅是纤维直径的函数。 总体方法是制备具有不同结构的凝块，对其进行定量表征，然后使用几种不同的方法测量纤维蛋白溶解速率，并测量纤溶酶原和 tPA 的结合以及纤溶酶原在裂解前沿的激活。 第三个具体目标测试了以下假设：血小板聚集对纤维蛋白结构和性质的影响对裂解前沿的事件具有重大影响。纤维网在血小板聚集体附近更加致密，并且纤维在血小板周围更加定向。此外，纤溶酶原激活剂抑制剂-1 从血小板中释放出来，并与血小板聚集体附近的纤维蛋白结合，从而延缓这些区域的溶解。 尽管这些现象众所周知，但所涉及的物理过程和局部影响尚未得到研究。 还将在阿昔单抗存在的情况下研究富含血小板的凝块溶解的物理过程，阿昔单抗是纤维蛋白（原）与血小板整合素 αIIbbeta3（在血小板聚集中结合纤维蛋白原的整合素）之间相互作用的抑制剂。 这些研究的结果将帮助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。

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.