Thrombin-mediated proteolysis in neuroinflammatory disease

描述(由申请人提供)：这项研究计划的长期目标是了解关键止血因素在中枢神经系统炎性脱髓鞘疾病(例如多发性硬化症；MS)进展中的作用。由于对炎症性中枢神经系统疾病等复杂过程的严格理解只能在易于操作的活体实验环境中实现，因此一个重要的实验资产将是具有选择性凝血酶原或凝血酶底物改变的基因靶向小鼠。这个项目的目标集中在一个普遍的假设上，即凝血酶除了支持血管完整性外，还是体内炎症过程的主要调节因子，并且多种凝血酶底物与炎性脱髓鞘疾病的进展有机械联系。这一研究方向是由强有力的初步数据推动的，这些数据表明凝血酶在实验性自身免疫性脑脊髓炎(EAE)中发挥了重要作用。凝血酶对局部小胶质细胞激活和导致运动功能丧失的髓鞘继发性破坏的总体重要性将通过对新建立的小鼠的神经炎性疾病的详细研究来确定，这些小鼠携带条件性凝血酶原基因敲除等位基因或表达突变形式的凝血酶原(FIIWE)，其蛋白酶特异性开关有利于抗凝底物蛋白C而不是促凝底物(目标1)。通过对PAR-1、PAR-4和纤维蛋白原存在结构性或功能性缺陷的小鼠的综合研究，包括缺乏形成纤维蛋白的能力或缺乏选定的纤维蛋白整合素受体结合模体的小鼠，将测试不同凝血酶靶标和信号通路的机制贡献(目标2)。最后，将使用具有不同信号和抗凝活性的重组小鼠APC衍生物(例如，5A-APC)以及具有蛋白C定向特异性的重组小鼠凝血酶(FIIaWE)来探索在APC或凝血酶水平上的药物干预在限制神经炎性疾病方面的潜在益处(目标3)。拟议的研究将提供更详细的了解体内止血和炎症系统之间的串扰，强调凝血酶在神经炎性疾病中的重要性，并阐明限制多发性硬化症临床表现的潜在治疗策略。公共卫生相关性：多发性硬化症(MS)是一种常见的神经炎症性疾病，会导致复发性瘫痪和视力丧失。血脑屏障的破坏和局部止血因子的激活似乎促进了中枢神经系统疾病的进展。这项研究的目的是利用现代分子遗传学对关键凝血因子在神经炎症性疾病中的作用进行更详细的机制理解，并确定新的治疗机会。

DESCRIPTION (provided by applicant): The long-term objective of this research program is to understand the role of key hemostatic factors in the progression of inflammatory demyelinating disease within the central nervous system (e.g., multiple sclerosis; MS). Since a rigorous understanding of a complex process such as inflammatory CNS disease can only be achieved in a easily-manipulated in vivo experimental setting, an important experimental asset will be gene-targeted mice with selected alterations in prothrombin or thrombin substrates. The aims of this project center on the general hypothesis that thrombin, in addition to supporting vascular integrity, is a master regulator of inflammatory processes in vivo and that multiple thrombin substrates are mechanistically tied to the progression of inflammatory demyelinating disease. The research direction is driven by strong preliminary data pointing to a seminal role of thrombin in experimental autoimmune encephalomyelitis (EAE). The overall importance of thrombin to local microglial activation and the secondary destruction of myelin sheaths that leads to loss of motor function will be established through detailed studies of neuroinflammatory disease in newly-established mice carrying either a conditional prothrombin knockout allele or expressing a mutant form of prothrombin (fIIWE) with a protease specificity switch favoring the anticoagulant substrate protein C over procoagulant substrates (Aim 1). The mechanistic contribution of distinct thrombin targets and signaling pathways will be tested through comprehensive studies of mice with constitutive or functional deficits in PAR-1, PAR-4 and fibrinogen, including mice lacking the capacity to form fibrin or lacking selected fibrin integrin receptor engagement motifs (Aim 2). Finally, the potential benefit of pharmacological intervention at the level of APC or thrombin in limiting neuroinflammatory disease will be explored using recombinant murine APC derivatives (e.g., 5A-APC) with distinct signaling and anticoagulant activities, as well as recombinant murine thrombin (fIIaWE) with a protein C-directed specificity (Aim 3). The proposed studies will provide a more detailed understanding of the crosstalk between the hemostatic and inflammatory systems in vivo, underscore the importance of thrombin in neuroinflammatory disease and illuminate potential therapeutic strategies for limiting the clinical manifestations of MS. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a common neuroinflammatory disease resulting in relapsing paralysis and vision loss. Blood-brain barrier breakdown and the local activation of hemostatic factors appear to contribute to the progression of CNS disease. The goal of this research is to use modern molecular genetics to develop a more detailed mechanistic understanding of the role of key coagulation factors in neuroinflammatory disease and identify novel therapeutic opportunities.