Mechanisms linking the hemostatic protease thrombin to arthritic disease

描述(申请人提供)：类风湿性关节炎(RA)是一种常见的、极其虚弱的炎症性疾病，会导致关节组织的侵蚀和退化，最终导致功能丧失。激活止血系统和随后将凝血酶原转化为活性丝氨酸蛋白酶凝血酶是类风湿关节炎的一个显著特征。病变关节内纤维蛋白沉积旺盛是凝血酶活性旺盛的病理结果之一，而纤维蛋白(原)最近被证明是与关节炎发病机制相关的炎症事件的驱动因素。重要的是，纤维蛋白原似乎只是凝血酶调节关节炎发病关键事件的许多下游效应物之一。这项建议的目的将集中在以下特定假设：(1)凝血酶促进局部炎症、滑膜增生、血管疙瘩形成以及与关节炎相关的软骨/骨破坏，(2)凝血酶诱导的纤维蛋白聚合和激活FXIII转谷氨酰胺酶(它与纤维蛋白交织在一起是凝血酶加剧炎性关节疾病的两种机制)，以及(3)“重新设计”凝血酶底物的特异性，以促进抗凝/抗炎活性，而不是促凝剂活性，将该酶从促关节炎转化为抗炎因子。这些假说将使用最近建立的基因靶向小鼠品系和成熟的胶原蛋白诱导的关节炎实验环境进行验证。循环中凝血酶原的遗传性减少与纤维蛋白原缺陷小鼠共同作用的特异性效应(S)将确定凝血酶-纤维蛋白原轴在关节炎发病机制中的作用(目标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的小鼠，或携带转谷氨酰胺酶fXIIIA催化亚单位基因的遗传消融的小鼠，将专门评估凝血酶介导的纤维蛋白聚合和交联剂在关节炎进展中的机制作用(目标2)。最后，携带凝血酶原基因定点突变导致底物专一性从促凝剂转变为抗凝血剂/抗炎靶标的小鼠将被用于研究凝血酶可被重新定向以用作抗关节炎酶的概念(目标3)。拟议的研究将通过确定推动关节炎发病的止血系统和炎症系统之间的串扰机制来填补我们目前对关节炎发病机制的理解的重大空白，并可能突出治疗关节炎的新的治疗机会。公共卫生相关性：激活止血系统，包括产生中枢止血丝氨酸蛋白酶凝血酶，是人类类风湿性关节炎和实验性炎症性关节炎的显著特征。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。这项拟议的工作将填补我们对凝血酶-纤维蛋白原轴与关节炎之间相互作用的理解上的重大空白，并可能为使用具有选定底物特异性的新型“定制”凝血酶变异体治疗关节炎提供原则证明。

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.