Abstract The long-term objective of this research program is to better understand how the mechanisms by which plasmin- mediated proteolysis modifies the neuroinflammatory disease, multiple sclerosis (MS). Plasmin is the premier fibrinolytic enzyme. Fibrin(ogen) contributes to demyelinating disease progression via interaction with the macrophage/microglial αMβ2 receptor. Thus, while the contribution of plasmin seems predictable, we have found that plasminogen deficiency is strongly protective from the initiation and progression of a murine model of MS. The mechanistic contribution of these processes to the initiation and progression of neuroinflammation is not well defined. Our overarching hypothesis is that plasmin-mediated proteolysis is a fundamental determinant of neuroinflammation through distinct mechanisms that differentially support disease initiation and progression. This hypothesis is supported by strong preliminary data that establishes that plasminogen deficiency significantly protects from the initiation and progression of a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Protection from initiation of disease development is dependent on the presence of fibrin(ogen). Plasmin localization to the cell surface is vital for normal macrophage migration. We will test the differential requirement for either intracellular (externalized on macrophage activation) or transmembrane plasminogen receptors. The impact of the preservation of fibrin matrices will be examined on early events in EAE disease initiation, including peripheral macrophage recruitment to the central nervous system (CNS) and blood brain barrier breakdown (Aim 1). In this aim, we will also assess the impact of inhibition of inhibition of plasmin activation on established neuroinflammatory disease. Plasmin has many substrates other than fibrin. One particular substrate that may contribute to neuroinflammatory disease is matrix metalloprotease 9 (MMP9). We will assess plasmin-mediated MMP9 activation in the development of neuroinflammatory disease (Aim 2). The requirement for localization of plasminogen to the cell surface (via plasminogen receptors) for successful macrophage recruitment to the CNS will also be tested (Aim 3). We will further assess blockage of plasminogen receptors on reversal of established neuroinflammatory disease. Our preliminary data supports non-fibrinogen proteolytic targets as driving EAE disease progression. We anticipate that this research program will reveal insights into the development and progression of neuroinflammation that could provide the basis for novel therapeutic modalities for MS.