Mechanisms linking the hemostatic protease thrombin to arthritic disease

描述（由申请人提供）：类风湿性关节炎（RA）是一种常见且极度衰弱的炎症性疾病，导致关节组织的侵蚀和降解，最终导致功能丧失。止血系统的激活和凝血酶原随后转化为活性丝氨酸蛋白酶凝血酶是RA的一个突出特征。受影响关节内的纤维蛋白沉积是凝血酶活性旺盛的一个病理结果，纤维蛋白（原）最近被证明可以驱动与关节炎发病相关的炎症事件。重要的是，纤维蛋白原似乎只是凝血酶调节关节炎发病中心事件的众多下游效应物之一。本建议的目的将集中于以下具体假设：(1)凝血酶促进与关节炎疾病相关的局部炎症、滑膜增生、肠膜形成和软骨/骨破坏；(2)凝血酶诱导的纤维蛋白聚合和与纤维蛋白交联的fXIII转谷氨酰胺酶的激活是凝血酶加重炎性关节疾病的两种机制。(3)“重新设计”凝血酶底物特异性，以促进抗凝/抗炎活性超过促凝活性，将酶从促关节炎因子转化为抗关节炎因子。这些假设将使用最近建立的基因靶向小鼠系和已建立的胶原诱导关节炎的实验设置进行测试。基因强加的循环凝血酶原减少与纤维蛋白原缺陷小鼠的特异性作用将确定凝血酶-纤维蛋白原轴在关节炎发病中的作用（目的1）。通过使用表达对凝血酶蛋白酶活性不敏感的纤维蛋白原形式或携带转谷氨酰胺酶fXIIIA催化亚基基因基因消融的小鼠，将特别评估凝血酶介导的纤维蛋白聚合和交联在关节炎进展中的机制作用（目的2）。最后，携带凝血酶原基因位点定向突变的小鼠将导致底物特异性从促凝剂转移到抗凝/抗炎靶点，以研究凝血酶可以被重定向作为抗关节炎酶的概念（目的3）。拟议的研究将填补我们目前对关节炎发病机制的理解的重大空白，通过定义驱动关节炎发病机制的止血和炎症系统之间的相互作用机制，并可能为关节炎的治疗提供新的治疗机会。公共卫生相关性：止血系统的激活，包括中央止血丝氨酸蛋白酶凝血酶的产生，是人类类风湿关节炎和实验性炎症性关节炎的一个突出特征。这些研究的长期目标是确定凝血酶在炎症性关节疾病发病机制中的作用。提出的工作将填补我们对凝血酶-纤维蛋白原轴与关节炎疾病之间相互作用的理解中的重大空白，并可能为使用具有选定底物特异性的新型“定制”凝血酶变体治疗关节炎提供原理证明。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is a common and extremely debilitating inflammatory disease that results in erosion and degradation of joint tissue and ultimately loss of function. Activation of the hemostatic system and the subsequent conversion of prothrombin to the active serine protease thrombin is a prominent feature of RA. Robust fibrin deposition within affected joints is one pathological consequence of exuberant thrombin activity, and fibrin(ogen) has recently been shown to drive inflammatory events associated with arthritis pathogenesis. Importantly, fibrinogen appears to be just one of many downstream effectors by which thrombin regulates events central to arthritis pathogenesis. The aims of this proposal will focus on the following specific hypotheses: (1) thrombin promotes the local inflammation, synovial hyperplasia, pannus formation, and cartilage/bone destruction associated with arthritic disease, (2) thrombin-induced fibrin polymerization and activation of the fXIII transglutaminase which cross-links fibrin are two mechanisms by which thrombin exacerbates inflammatory joint disease, and (3) "re-engineering" thrombin substrate specificity to promote anticoagulant/anti-inflammatory activity over procoagulant activity will convert the enzyme from a pro-arthritic to an anti-arthritic factor. These hypotheses will be tested using recently established gene-targeted mouse lines and the well-established experimental setting of collagen-induced arthritis. The specific effect(s) of a genetically-imposed reduction in circulating prothrombin in conjunction with fibrinogen-deficient mice will define the role of the thrombin-fibrinogen axis on arthritis pathogenesis (Aim 1). By employing mice that either express a form of fibrinogen insensitive to thrombin protease activity or that carry a genetic ablation of the transglutaminase fXIIIA catalytic subunit gene, the mechanistic role of thrombin-mediated fibrin polymerization and cross-linking on arthritis progression will be specifically evaluated (Aim 2). Finally, mice carrying a site- directed mutation in the prothrombin gene that results in a shift in substrate specificity away from procoagulant to anticoagulant/anti-inflammatory targets will be used to investigate the concept that thrombin can be redirected to serve as an anti-arthritic enzyme (Aim 3). The proposed studies will fill significant gaps in our current understanding of arthritis pathogenesis by defining mechanisms of cross-talk between the hemostatic and inflammatory systems that drive arthritis pathogenesis and may highlight novel therapeutic opportunities for the treatment of arthritis. PUBLIC HEALTH RELEVANCE: Activation of the hemostatic system, including generation of the central hemostatic serine protease thrombin, is a prominent feature of both human rheumatoid arthritis and experimental inflammatory arthritis. The long-term goal of these studies is to determine the role of thrombin in the pathogenesis of inflammatory joint disease. The proposed work will fill significant gaps in our understanding of the interplay between the thrombin- fibrinogen axis and arthritic disease, and may provide the proof-of-principle for the use of novel "customized" thrombin variants with selected substrate specificity to treat arthritis.