Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 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-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-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 Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). 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 Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-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 IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 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.
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CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-N GENE-N GENE-Y
125I 125I 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone Gly 125I factor X Leu83-Leu88 factor IX factor Xa factor Xa EPR-1 factor IX IXa EPR-1 factor X factor Xa factor Xa factors X or IXa factor Xa factor Xa factors X or IXa inter-EGF sequence Leu83-Leu88 factor Xa EPR-1 factor Xa Factor Xa factor Xa EPR-1 factor Xa factor Xa EPR-1 factor X inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) factor V/Va prothrombin factor Xa factor X effector cell protease receptor-1 inter-EGF sequence effector cell protease receptor-1 inter-EGF sequence Leu83-Leu88 factor Xa