The synthesis and/or activation of matrix degrading proteinases play an essential role in the migration of monocytes and macrophages through tissue. Foremost among these proteinases is the urokinase type plasminogen activator (uPA)-plasminogen system. Plasmin binds to and degrades fibrin and several components of the extracellular matrix (ECM). In addition, plasmin activates several members of the family of matrix metalloproteinases (MMP), which are responsible for the degradation of collagen and other components of the ECM. Despite earlier observations that binding to the cell surface imparts a kinetic advantage to plasminogen activation and protection of plasmin from inhibition, the biology of membrane-bound plasmin remains largely unexplored, and its role as a therapeutic target in chronic inflammatory diseases remains untested. The overall purpose of experiments described in this proposal is to test the hypothesis that plasmin binding sites on the surface of macrophages are the primary regulator of their pericellular proteinase cascade, and blocking plasmin(ogen) binding to their surface is an effective strategy to reduce macrophage accumulation, and its sequelae in chronic inflammatory diseases. Two complimentary strategies are proposed: (1) Utilizing mice deficient in a defined plasminogen binding site (annexin II), we will directly determine the role of plasmin binding sites in macrophage activation of MMP-9, degradation of ECM and MCP-l-dependent migration through ECM in vitro (Specific Aim 1). Also, we will determine the effect of annexin II deficiency on macrophage recruitment in vivo utilizing thioglycollate-induced peritonitis and foreign body-induced granuloma models and recruitment to atherosclerotic lesions in Apo E-/- IAnx II-/- mice (Specific Aim 2). (2) We will determine the therapeutic effectiveness of an inactive cell-binding fragment of plasminogen to block macrophage activation of MMP-9, ECM degradation and MCP-1- dependent migration through ECM in vitro, as well as macrophage recruitment in vivo in the thioglycollate-induced peritonitis and foreign body-induced granuloma models (Specific Aim 3). We believe that the results of proposed in vitro and in vivo experiments will provide a basis for novel therapeutic strategies targeting plasmin-binding sites to modulate chronic inflammation, tissue remodeling and atherosclerosis.
