title: Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1.
abstract: 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.
authors.affiliation: Molecular Cardiobiology Program and Department of Pathology, The Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: G
authors.initials: G
authors.lastname: Ambrosini
authors.forename: J
authors.initials: J
authors.lastname: Plescia
authors.forename: K C
authors.initials: KC
authors.lastname: Chu
authors.forename: K A
authors.initials: KA
authors.lastname: High
authors.forename: D C
authors.initials: DC
authors.lastname: Altieri
mesh_terms: D001665:Binding Sites; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D007930:Leucine; D011516:Prothrombin / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000077022:Survivin
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000077022:Survivin; D011516:Prothrombin; D012697:Serine Endopeptidases; D015951:Factor Xa; D007930:Leucine