THE EFFICIENT CATABOLISM OF THROMBIN-PROTEASE NEXIN-1 COMPLEXES IS A SYNERGISTIC MECHANISM THAT REQUIRES BOTH THE LDL RECEPTOR-RELATED PROTEIN AND CELL-SURFACE HEPARINS

Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that ac ts as a suicide substrate for thrombin (Th) and urokinase-type plasmin ogen activator (uPA), PN1 forms 1:1 stoichiometric complexes with thes e proteases, which are then rapidly bound, internalized, and degraded, The low density lipoprotein receptor-related protein (LRP) is the rec eptor responsible for the internalization of protease-PN1 complexes, H owever, we found that the LRP is not significantly involved in the ini tial cell surface binding of thrombin-PN1, leading us to investigate w hat cellular component was responsible for this initial interaction, S ince Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibi lity that cell surface heparins were involved in initial complex bindi ng, Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin, To a scertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which th e heparin binding site was rendered non-functional, When added at equa l initial concentrations of complexes, Th-PN1(K7E) was catabolized B-t o 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes, These data d emonstrate the sizable contribution of cell surface heparins to Thromb in-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their s ubsequent LRP-dependent endocytosis.