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 stru ctural requirements of this recognition were investigated. Factor Xa o r catalytically inactive 5-dimethylaminonaphthalene-1-sulfonyl (dansyl ) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguisha bly to HUVEC and EPR-1 transfectants, and inhibited equally well the b inding 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 83)-Phe(84)-Thr(85 )-Arg(86)-Lys(87)-Leus(88)-(Gly) inhibited factor V/Va-independent pro thrombin 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 p eptide 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-facto r Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, whi le recombinant factor IX or plasma Ma had no effect. An antibody gener ated against the factor X peptide 83-88, and designated JC15, inhibite d 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 inhi bited by mo lar excess of unlabeled factor Xa, but not by comparable c oncentrations of factors X or Ma. These findings identify the inter-EG F sequence Leu(83)-Leu(88) in factor Xa as a novel recognition site fo r EPR-1, and suggest its potential role as a protease activation depen dent neo-epitope. This interacting motif may help elucidate the contri bution of factor Xa to cellular assembly of coagulation and vascular i njury.