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.
Highlights
¶ This work was done during the tenure of an American Heart Association Established Investigator Award
A recombinant factor IX/X chimera, and a sequence-specific antibody, we found that the interconnecting EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88(Gly) in factor Xa mediates ligand binding to Effector cell protease receptor-1 (EPR-1) and becomes surface exposed only after zymogen activation
Aliquots (10.8 nM) of 125I-factor Xa were incubated with 100 nM concentrations of the indicated factor Xa inhibitors for 20 min at 22 °C, before addition to HUVEC monolayers or suspensions of Chinese hamster ovary (CHO) cells transiently transfected with the EPR-1 cDNA in a total volume of 300 l for an additional 20-min incubation at 22 °C
Summary
Proteins and Protein Labeling—The experimental procedures for the isolation and characterization of human plasma factors IX and X and the generation of the corresponding active proteases IXa and Xa have been reported [8]. Thrombin generation under the various conditions tested was quantitated by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 (Chromogenix, Molndal, Sweden) at A405 [29] and converted to thrombin concentrations (nanomolar) using a standard curve constructed with serial increasing concentrations of bovine thrombin In another series of experiments, stable EPR-1 transfectants or HUVEC were mixed with increasing concentrations (0.1–200 M) of the factor X-derived peptides 83– 88, its control scrambled variant, or the -COOH terminus EGF peptide (Gly)-His101-Glu102-Glu103-Gln104-Asn105-Ser106Val107-Val108-(Gly), for 20 min at 22 °C, before addition of factor Xa (43 nM) and prothrombin (138 nM), and determination of S-2238 hydrolysis. The suboptimal concentration of prothrombin of 138 nM used in these ex-
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