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

Abstract

Assembly of coagulation and anticoagulation pathways occurs on vascular cells through the regulated ligand recognition of membrane protease receptors (1, 2). Belonging to structurally different and evolutionarily unrelated gene superfamilies, cell-surface receptors for thrombin (3), urokinase (4), protein S (5), or factor Xa (6) provide a controlled microenvironment for limited proteolytic activation of the clotting and fibrinolytic cascades (1, 2). Recent studies have also underscored the participation of protease receptors in pleiotropic mechanisms of vascular cell signal transduction, including transcription of activation-dependent genes (7), generation of intracellular second messengers (3), modulation of immune inflammatory responses (8), and cell proliferation (5, 9). Aberrations of protease-dependent signaling pathways may play a primary pathogenetic role in the establishment and progression of the atherothrombotic disease (9, 10) as well as in neoplastic transformation and tumor cell dissemination (5, 11).Binding of factor Xa to leukocytes (12) or endothelial cells 1 is contributed by effector cell protease receptor-1 (EPR-1), 2 a 337-amino acid single membrane-spanning glycoprotein lacking significant homologies to other protease receptors and expressed in a cell-type specific fashion by alternative mRNA splicing (13). Occupancy of EPR-1 with factor Xa participates in factor V/Va-independent mechanisms of prothrombin activation (6), generates intermediate product(s) of factor IX activation (14), or contributes an accessory pathway of lymphocyte costimulation (8).To gain insights into the potential pathophysiological relevance of EPR-1-factor Xa interaction in hemostasis and vascular cell signal transduction, we sought to identify the structural requirements implicated in receptor-ligand recognition. Using a eukaryotic expression strategy of EPR-1 chimeric constructs combined with site-directed mutagenesis and synthetic peptidyl mimicry, we have identified two spatially distinct regions in the EPR-1 extracellular domain separately involved in lymphocyte activation mechanisms or factor Xa binding and prothrombin activation. 2 The abbreviations used are: EPR-1, effector cell protease receptor-1; ICAM-1, intercellular adhesion molecule-1; PCR, polymerase chain reaction; bp, base pairs; CHO, Chinese hamster ovary; mAb monoclonal antibody; HUVEC, human umbilical vein endothelial cell(s). EXPERIMENTAL PROCEDURES Construction of EPR-1-ICAM-1 Chimeras-Three

Effector Cell Protease Receptor-1 Is a Vascular Receptor for Coagulation Factor Xa

“…The catalytic efficiency of prothrombin activation by the endothelium (in cell culture) is similar to platelets (44). Synthetic EPR-1 peptides inhibit prothrombinase activity on endothelial cells (45) and monoclonal anti-EPR-1 antibodies inhibit prothrombinase activity on platelets (46) in a dose-dependent manner (each in the absence of exogenous V/Va). However, the relationship of EPR-1 and factor Va and the relative importance of each to prothrombin formation on the endothelial cell surface in vitro remains to be determined.…”

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

“…In another series of experiments, stable EPR-1 transfectants or HUVEC were mixed with increasing concentrations (0. Background hydrolysis of S-2238 in the absence of prothrombin (8 -14%) was subtracted to calculate specific thrombin cleavage (29). The suboptimal concentration of prothrombin of 138 nM used in these ex-periments is nonsaturating for the system, and has been used for comparison with previous data obtained with other EPR-1 ϩ cells (9).…”

“…For antibody neutralization experiments, 10.8 nM aliquots of 125 I-factor Xa were preincubated with increasing concentrations of control non immune rabbit IgG (Zymed Laboratories, San Francisco, CA) or sequence-specific JC15 antibody (see below), for 15 min at 22°C before addition to HUVEC monolayers and determination of specific binding. For all experiments nonspecific binding was assessed in the presence of a 100 -150-fold molar excess of unlabeled factor Xa added at the start of the incubation and was subtracted from the total to calculate net specific binding (29). For peptide inhibition experiments, increasing concentrations (0.1-500 M) of the various factor X-derived peptides or their variants (24) were preincubated with HUVEC monolayers in serum-free RPMI 1640 medium for 20 min at 22°C before addition of 10.8 nM 125 I-factor Xa and determination of specific binding, as described above.…”

“…Cells were mixed with 138 M prothrombin, 1.2 mM CaCl 2 , and 43 nM factor Xa for 5 min at 22°C. Thrombin generation under the various conditions tested was quantitated by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 (Chromogenix, Molndal, Sweden) at A 405 (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.…”

“…Transfection Experiments and Purification of Recombinant Coagulation Proteins-Subconfluent cultures of Chinese hamster ovary (CHO) cells were transfected with 15 g of an EPR-1 cDNA clone in the mammalian cell expression vector pcDNA3 (Invitrogen, San Diego, CA) by electroporation, as described elsewhere (29). After a 48-h culture at 37°C, cells were washed, suspended in serum-free Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Walkersville, MD) to a final concentration of 5 ϫ 10 6 /ml, and analyzed for 125 I-factor Xa binding or factor V/Va-independent prothrombin activation (29), as described below.…”