Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

Abstract

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF⅐FVIIa complex and by FXa in the ternary TF⅐FVIIa⅐FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF⅐FVIIa-Xa complex normally but were severely impaired in binary TF⅐FVIIa⅐PAR2 signaling. The residues identified were located in the model-predicted S2 pocket of FVIIa, and complementary PAR2 P2 Leu-38 replacements demonstrated that the P2 side chain was indeed crucial for PAR2 cleavage by TF⅐FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF⅐FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactorassociated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.Coagulation factor VIIa (FVIIa) 2 in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through proteaseactivated receptors (PARs). TF⅐FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2). Activation of PAR2 by the TF⅐FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF⅐FVIIa⅐FXa (3). In the latter complex, FXa is the primary activator for PAR2 (4). PAR2 triggers typical G-protein-coupled as well as ␤-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional respo...

The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors

“…Expression of EPCR markedly enhanced PAR2 cleavage when FX was added simultaneously. The nematode inhibitor NAPc2 stabilizes the ternary TFFVIIa-FXa product complex, leaving the active site of FXa accessible to substrates, including PARs (21,38). PAR2 cleavage by the NAPc2-stabilized ternary complex was similar on EPCR-expressing versus control cells.…”

“…Signaling Assays and Quantification of PAR Cleavage-Signaling assays in serum-free DMEM used ERK1/2 phosphorylation after 10 min or induction of immediate early gene mRNA levels after 90 min as readouts (38). Briefly, HaCaT cells or murine SMCs were switched for 3 h to serum-free DMEM followed by the addition of proteases in the presence of 2 M hirudin to prevent thrombin signaling due to coagulation factor carryover from the culture medium (39).…”

“…Cells were lysed in sample buffer for SDS-PAGE and Western blotting for phospho-ERK1/2, or mRNA was isolated from cells with TRIzol reagent (Invitrogen) for quantitative mRNA determinations (4). PAR cleavage was quantified in cells transiently transfected with FLAG-tagged PAR1 or PAR2 as described (38).…”

“…We considered the potential caveat that the chosen readouts for cell signaling were insensitive to detect effects of EPCR on the TF-FVIIa binary signaling complex. Therefore, we measured PAR cleavage by TF-associated proteases using surface detection of FLAG-tagged PAR constructs (38). In addition, antibody blockade of EPCR might have produced nonspecific steric hindrance of TF signaling complexes.…”

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice

“…To clarify whether 5A-aPC inhibited Peli1 induction by proteolytic "disarming" of PAR2 via cleavage at R38 in the PAR2 extracellular domain, we tested BMDCs derived from mice homozygous for the R38E mutation that renders PAR2 resistant to cleavage by both fXa and aPC. [34][35][36] As shown earlier, 27 LPSinduced Peli1 expression in R38E-PAR2 cells was blunted but was restored by the PAR2 agonist SLIGRL. aPC inhibited Peli1 induction by LPS/SLIGRL in R38E-PAR2 cells to a similar extent as in wild-type cells, ruling out a role for R38 cleavage ( Figure 4C).…”