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Volume 272, Number 13, Issue of March 28, 1997 pp. 8340-8345
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Activation-dependent Exposure of the Inter-EGF Sequence Leu⁸³-Leu⁸⁸ in Factor Xa Mediates Ligand Binding to Effector Cell Protease Receptor-1

(Received for publication, October 23, 1996, and in revised form, January 15, 1997)

Grazia Ambrosini , Janet Plescia , Kirk C. Chu ^§ , Katherine A. High ^§ and Dario C. Altieri

From the  Molecular Cardiobiology Program and Department of Pathology, The Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536 and ^§ Departments of Pediatrics and Pathology and Laboratory Medicine, University of Pennsylvania and the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

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 ~ 20-40 µM). 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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