IMMUNOSELECTIVE TARGETING OF AN ANTI-THROMBIN AGENT TO THE SURFACE OFCYTOKINE-ACTIVATED VASCULAR ENDOTHELIAL-CELLS

An immunoconjugate was designed to target hirudin, a potent and specif ic inhibitor of thrombin, to the surface of activated endothelial cell s. Hirudin was covalently crosslinked to the monoclonal antibody H18/7 that recognizes the extracellular domain of E-selectin (CD62E), an en dothelium-leukocyte adhesion molecule that is expressed only on cytoki ne-activated endothelium. The hirudin-H18/7 immunoconjugate selectivel y bound to interleukin-1-activated but not to unactivated cultured hum an umbilical vein endothelial cells with a temporal profile similar to that of inducible cell-surface procoagulant activity. When bound to a ctivated endothelial cells, the hirudin-H18/7 immunoconjugate signific antly inhibited endogenous thrombin activity generated from coincubate d human plasma and fibrin clot formation on the monolayer surface. Cel lular responses that are mediated via the thrombin receptor, such as i ncreases in cytoskeletal F-actin content, also were significantly down regulated, and monolayers were protected from thrombin-induced disrupt ion by this treatment. The ability to selectively antagonize thrombin- dependent processes at the endothelium-blood interface may provide new insights into complex pathophysiological processes, such as thrombosi s, inflammation, and atherogenesis. These studies also demonstrate the general feasibility of selective targeting of therapeutic agents to e ndothelial cells based on recognition of an activation-dependent surfa ce phenotype.