Thrombin-mediated proteolysis in neuroinflammatory disease

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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.