Molecular dissection of effector cell protease receptor-1 recognition of factor Xa - Assignment of critical residues involved in antibody reactivity and ligand binding

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 similar to 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 Met(1)-Arg(60) bound the immunosuppressive anti-EPR-l monoclonal antibody (mAb) 2E1. In contrast, transfected cells expressing the EPR-1 sequence pro(120)-Ala(154) mere recognized by the functionally inhibitory anti EPR-1 mAbs 9D4 and B6, bound I-125-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 ex ogenous factor V/Va. Chimeric transfectants expressing the COOH terminus end of the EPR-1 extracellular domain (Ala(157)-Glu(221)) did not bind anti-EPR-1 mAbs and did not associate with factor Xa. Mutagenesis of Asn(131) or Lys(133) in the EPR-1 ligand recognition domain abolished factor Xa binding by 80 +/- 5.5 and 96 +/- 4%, respectively, while mutation of Lys(126), Gly(128), Asn(129), and Asn(134) was without effect. A synthetic peptide duplicating the EPR-1 sequence S(123)PGKPGNQNSKNEPP(137) dose dependently inhibited factor V/Va-independent thrombin generation of resting endothelium (IC50 similar to 1 mu M), while the adjacent EPR-1 sequence p(136)PKK- RERERSSHCYP(150) was ineffective. These findings demonstrate that EPR-1 contains two spatially distinct functional domains implicated in lymphocyte activation (Met(1)-Arg(60)) or factor Xa binding and prothrombin activation (pro(120)-Ala(154)). These interacting sequences may provide a novel potential target for inhibition of factor Xa-dependent vascular cell responses.