Molecular Dissection of Effector Cell Protease Receptor-1 Recognition of Factor Xa

Receptor-mediated assembly of blood proteases on vascular cells maintains the hemostatic balance and initiates intracellular signal transduction. Effector cell protease receptor-1 (EPR-1) is an ∼62-kDa vascular cell membrane receptor for the clotting protease factor Xa, participating in thrombin formation and lymphocyte activation. Here, recombinant EPR-1 fragments were engineered in the frame of intercellular adhesion molecule-1, transfected in mammalian cells, and analyzed for antibody recognition and ligand binding. Chimeric transfectants containing the EPR-1 sequence Met1-Arg60 bound the immunosuppressive anti-EPR-1 monoclonal antibody (mAb) 2E1. In contrast, transfected cells expressing the EPR-1 sequence Pro120-Ala154 were recognized by the functionally inhibitory anti-EPR-1 mAbs 9D4 and B6, bound 125I-factor Xa in a reaction quantitatively indistinguishable from that of wild-type EPR-1 transfectants, and promoted factor Xa concentration-dependent prothrombin activation in the absence of exogenous factor V/Va. Chimeric transfectants expressing the COOH terminus end of the EPR-1 extracellular domain (Ala157-Glu221) did not bind anti-EPR-1 mAbs and did not associate with factor Xa. Mutagenesis of Asn131 or Lys133 in the EPR-1 ligand recognition domain abolished factor Xa binding by 80 ± 5.5 and 96 ± 4%, respectively, while mutation of Lys126, Gly128, Asn129, and Asn134 was without effect. A synthetic peptide duplicating the EPR-1 sequence S123PGKPGNQNSKNEPP137 dose dependently inhibited factor V/Va-independent thrombin generation of resting endothelium (IC50∼ 1 μM), while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was ineffective. These findings demonstrate that EPR-1 contains two spatially distinct functional domains implicated in lymphocyte activation (Met1-Arg60) or factor Xa binding and prothrombin activation (Pro120-Ala154). These interacting sequences may provide a novel potential target for inhibition of factor Xa-dependent vascular cell responses. Receptor-mediated assembly of blood proteases on vascular cells maintains the hemostatic balance and initiates intracellular signal transduction. Effector cell protease receptor-1 (EPR-1) is an ∼62-kDa vascular cell membrane receptor for the clotting protease factor Xa, participating in thrombin formation and lymphocyte activation. Here, recombinant EPR-1 fragments were engineered in the frame of intercellular adhesion molecule-1, transfected in mammalian cells, and analyzed for antibody recognition and ligand binding. Chimeric transfectants containing the EPR-1 sequence Met1-Arg60 bound the immunosuppressive anti-EPR-1 monoclonal antibody (mAb) 2E1. In contrast, transfected cells expressing the EPR-1 sequence Pro120-Ala154 were recognized by the functionally inhibitory anti-EPR-1 mAbs 9D4 and B6, bound 125I-factor Xa in a reaction quantitatively indistinguishable from that of wild-type EPR-1 transfectants, and promoted factor Xa concentration-dependent prothrombin activation in the absence of exogenous factor V/Va. Chimeric transfectants expressing the COOH terminus end of the EPR-1 extracellular domain (Ala157-Glu221) did not bind anti-EPR-1 mAbs and did not associate with factor Xa. Mutagenesis of Asn131 or Lys133 in the EPR-1 ligand recognition domain abolished factor Xa binding by 80 ± 5.5 and 96 ± 4%, respectively, while mutation of Lys126, Gly128, Asn129, and Asn134 was without effect. A synthetic peptide duplicating the EPR-1 sequence S123PGKPGNQNSKNEPP137 dose dependently inhibited factor V/Va-independent thrombin generation of resting endothelium (IC50∼ 1 μM), while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was ineffective. These findings demonstrate that EPR-1 contains two spatially distinct functional domains implicated in lymphocyte activation (Met1-Arg60) or factor Xa binding and prothrombin activation (Pro120-Ala154). These interacting sequences may provide a novel potential target for inhibition of factor Xa-dependent vascular cell responses.