Activation-dependent exposure of the inter-EGF sequence Leu(83)-Leu(88) in factor Xa mediates ligand binding to effector cell protease receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of I-125-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu(83)-Phe(84)-Thr(85)-Arg(86)-Lys(87)-Leus(88)-(Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of I-125-factor Xa to these cells in a dose-dependent manner (IC50 similar to 20-40 mu M). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu(83)-Leu(88) within a factor IX backbone inhibited binding of I-125-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma Ma had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited I-125-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or Ma was observed, Furthermore, binding of I-125-factor Xa to immobilized JC15 was inhibited by mo lar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or Ma. These findings identify the inter-EGF sequence Leu(83)-Leu(88) in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.