Fibrinogen and Factor XIII in Venous Thrombosis

 描述(由申请人提供)：静脉血栓形成(VT)每年影响100多万美国人。即使接受治疗，30%-50%的室性心动过速患者仍会复发或长期患病，包括慢性疼痛、浮肿和溃疡(所谓的“血栓后综合征”)。目前的抗凝治疗以凝血因子Xa或凝血酶为靶点，并与灾难性出血的重大风险相关。在不增加出血风险的情况下减少血栓形成的下一代药物的开发，将对这一重大的临床问题产生深远的影响。静脉“红色”血栓的显著特征是红细胞(RBC)和纤维蛋白含量高。现有的范例表明，红细胞在纤维蛋白聚合物形成过程中被动地被困在血栓中。然而，我们最近发现，凝血因子XIII(FXIII)调节红细胞在血栓中的滞留，并决定血栓的大小。我们还发现了促进与FXIII相互作用的纤维蛋白原残基，这是正常的FXIII激活和纤维蛋白交联所必需的。重要的是，我们已经证明，抑制FXIIIa活性或阻断FXIII-纤维蛋白原相互作用可以减少凝血红细胞含量，从而显著减少体外和体内血栓大小。这些令人兴奋的发现表明，抑制FXIII或阻断FXIII-纤维蛋白原相互作用代表了降低VT的全新和新颖的策略。这项应用的目的是确定FXIII和纤维蛋白原介导室速的分子机制。总体假设是FXIII(A)活性调节红细胞在凝块中的滞留。因此，抑制FXIII的活性或干扰FXIII与纤维蛋白原之间的相互作用将降低VT。这一假说将在三个具体目标上得到验证：1)确定FXIII结合所需的纤维蛋白原残基，并确定破坏FXIII与纤维蛋白原相互作用的多肽是否会减少血栓中的红细胞滞留；2)确定FXIIIa是否通过纤维蛋白交联促进红细胞在血栓中的滞留；以及3)确定FXIII的作用以及FXIII(A)抑制室速和止血的效果。这项研究将使用生化、遗传学和药理学工具来确定FXIII-纤维蛋白原轴，并确定FXIII活性和红细胞滞留在全血凝块形成过程中的作用。FXIII-纤维蛋白原相互作用和FXIIIa功能将使用重组蛋白、溶液相结合分析和创新的纤维蛋白分析方法进行研究。血栓的形成将使用室速和止血的体外和体内模型进行研究，包括活体动物成像(例如，超声波成像和活体显微镜)。这些研究将极大地扩展我们对FXIII(A)和纤维蛋白原在血栓形成和稳定性中新认识的作用的理解。这项研究具有很高的创新性，因为它挑战了目前红细胞被血栓被动缠绕的范式，并提出可以减少血栓红细胞的含量来预防室性心动过速。这项拟议的研究具有临床意义，因为它可能揭示一种全新的减少室性心动过速的策略。

 DESCRIPTION (provided by applicant): Venous thrombosis (VT) affects over 1 million Americans annually. Even with treatment, 30-50% of VT patients develops recurrent VT or suffer debilitating long-term morbidity including chronic pain, edema, and ulcers (so- called "post-thrombotic syndrome"). Current anticoagulation therapies target factor Xa or thrombin and are associated with a significant risk of catastrophic bleeding. The development of next generation agents which reduce thrombosis without increasing the risk of hemorrhage, would have a profound impact on this significant clinical problem. The defining characteristics of venous "red" thrombi are their high red blood cell (RBC) and fibrin content. The existing paradigm suggests RBCs are passively trapped in the clot during fibrin polymer formation. However, we have recently discovered that factor XIII (FXIII) mediates RBC retention in clots and determines clot size. We have also identified fibrinogen residues that promote interactions with FXIII and are required for normal FXIII activation and fibrin crosslinking. Importantly, we have shown that inhibiting FXIIIa activity or blocking FXIII-fibrinogen interactions reduces clot RBC content, and consequently, significantly reduces thrombus size in vitro and in vivo. These exciting discoveries suggest that inhibiting FXIII or blocking FXIII-fibrinogen interactions represent entirely new and novel strategies for reducing VT. The goal of this application is to determine the molecular mechanisms by which FXIII and fibrinogen mediate VT. The overall hypothesis is that FXIII(a) activity mediates RBC retention in clots. Thus, inhibiting FXIII activiy or disrupting the interaction between FXIII and fibrinogen will reduce VT. This hypothesis will be tested in three Specific Aims: 1) Define the fibrinogen residues required for FXIII binding and determine whether peptides that disrupt FXIII interactions with fibrinogen decrease RBC retention in clots, 2) Determine whether FXIIIa promotes RBC retention in clots via fibrin crosslinking, and 3) Determine the role of FXIII and effect of FXIII(a) inhibition in VT and hemostasis. This study will employ biochemical, genetic and pharmacologic tools to define the FXIII- fibrinogen axis and determine the role of FXIII activity and RBC retention during whole blood clot formation. FXIII-fibrinogen interactions and FXIIIa function will be examined using recombinant proteins, solution phase binding assays, and innovative fibrin analytical methods. Clot formation will be studied using in vitro and in vivo models of VT and hemostasis, including live-animal imaging (e.g., ultrasound imaging and intravital microscopy). These studies will significantly expand our understanding of newly-recognized roles for FXIII(a) and fibrinogen in thrombus formation and stability. This study is highly innovative because it challenges the current paradigm that RBCs are passively entangled in thrombi and proposes that thrombus RBC content can be reduced to prevent VT. The proposed research is clinically significant because it may reveal an entirely new strategy to reduce VT.