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性疾病，可导致关节组织侵蚀和退化，最终丧失功能。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的突出特征。受影响关节内的稳健纤维蛋白沉积是凝血酶活性旺盛的一种病理结果，并且纤维蛋白（原）最近已显示驱动与关节炎发病机制相关的炎症事件。重要的是，纤维蛋白原似乎只是凝血酶调节关节炎发病机制中心事件的许多下游效应物之一。本提案的目标将侧重于以下具体假设：（1）凝血酶促进与关节炎疾病相关的局部炎症、滑膜增生、血管翳形成和软骨/骨破坏，（2）凝血酶诱导的纤维蛋白聚合和交联纤维蛋白的fXIII转氨酶的活化是凝血酶加剧炎性关节疾病的两种机制，和（3）“重新设计”凝血酶底物特异性以促进抗凝/抗炎活性超过促凝血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胶原诱导的关节炎的完善的实验设置进行测试。与纤维蛋白原缺陷小鼠联合的循环凝血酶原的遗传性降低的特异性作用将定义凝血酶-纤维蛋白原轴在关节炎发病机制中的作用（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氨酶fXIIIA催化亚基基因的基因消融的小鼠，将具体评价凝血酶介导的纤维蛋白聚合和交联对关节炎进展的机制作用（目的2）。最后，将使用在凝血酶原基因中携带定点突变的小鼠来研究凝血酶可以被重定向以用作抗关节炎酶的概念，所述定点突变导致底物特异性从促凝血剂向抗凝剂/抗炎靶标转变（Aim 3）。拟议的研究将通过定义驱动关节炎发病机制的止血和炎症系统之间的串扰机制，填补我们目前对关节炎发病机制的理解中的重大空白，并可能突出治疗关节炎的新治疗机会。公共卫生相关性：止血系统的活化，包括中心止血丝氨酸蛋白酶凝血酶的产生，是人类类风湿性关节炎和实验性炎性关节炎的突出特征。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。所提出的工作将填补我们对凝血酶-纤维蛋白原轴和关节炎疾病之间相互作用的理解中的重大空白，并且可以为使用具有选定底物特异性的新型“定制”凝血酶变体来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.