Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Abstract

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.

Figure 1

PAR activation and cofactor dependence. Xenopus oocytes were injected with cRNAs encoding TF (TF) and PAR1, PAR2, PAR3, or PAR4, and agonist-triggered ⁴⁵Ca²⁺ release was assessed. Surface expression of TF and PARs was confirmed by anti-FLAG antibody binding (not shown). (A) Activation of PARs by FVIIa (50 nM), FXa (174 nM), SLIGRL (100 μM), or thrombin (αT, 10 nM). TF expression was necessary for FVIIa responses (C), but did not affect responses to FXa (not shown). Data shown are fold increase ⁴⁵Ca release (mean ± SE of 2–10 independent experiments each done in triplicate). (B) Effect of the thrombin inhibitor hirudin (10 units/ml) on PAR1 activation by other proteases. Protease concentrations were as in A. *, TF cRNA was co-injected with PAR1 cRNA when FVIIa was used as an agonist. Representative experiment (mean ± SD, n = 3). (C) Concentration responses to FVIIa and FXa in oocytes expressing PAR2 with or without TF as indicated. Average of 2–3 independent experiments done in triplicate (mean ± SE).

Figure 2

Mechanism of PAR2 activation. Xenopus oocytes were injected with cRNAs encoding wild-type or mutant TF and/or PAR2, and agonist-triggered ⁴⁵Ca²⁺ mobilization was measured. Representative experiments (mean ± SD, n = 3) are shown. (A) FVIIa-induced responses in oocytes expressing PAR2 with or without wild-type or mutant TF. (B) Responses in oocytes expressing PAR2 or the PAR2 cleavage site mutant (PAR2_S37P). Receptors were co-expressed with TF where indicated. ND, not determined.

Figure 3

Activation of PAR2 in stably transfected fibroblasts by FVIIa and FXa. KOLFs expressing TF, PAR2, or TF + PAR2 were used. Results shown are representative of several lines tested. Figures show representative experiments (mean ± SD; n = 3). (A) PI hydrolysis induced by FVIIa (Top), FXa (Middle), or a combination of picomolar FVIIa and zymogen FX (Bottom), all relative to saturating PAR2 agonist (SLIGRL, 100 μM). The maximal response to SLIGRL varied with PAR2 expression and the lower maximal response in KOLF_PAR2 relative to KOLF_TF+PAR2 is likely caused by different PAR2 expression levels in the particular clones shown. (B) Dose responses for FVIIa-induced PI hydrolysis in KOLF_TF+PAR2 in the presence or absence of plasma concentrations of FX. (C) Calcium transients induced by FXa (1 unit/ml) or FVIIa (100 pM) + FX (1 unit/ml) in Fura-2 loaded KOLF_TF+PAR2 cells. 1 unit/ml FXa or FX = 174 nM. Increases in cytosolic calcium were measured fluorometrically. Agonists were added at time indicated by arrows. (D) Dose-response for FXa- or Gla-domainless FXa-induced PI hydrolysis. (E) Inhibition of FXa-induced PAR2 activation by preincubation of cells with the indicated concentrations of active site-inhibited FXa .

Figure 4

Protease responses in keratinocytes and vascular endothelial cells. Data shown are fold increase in ³H-inositol phosphate accumulation (mean ± SD, n = 3) and are representative of at least three separate experiments. (A) PI hydrolysis in response to proteases and peptide agonists in keratinocyte line HaCaT, which expresses TF and PAR2 constitutively. Where indicated, anti-TF mAb was added 1 h before agonists. (B) PI hydrolysis in response to proteases and peptide agonists in naïve HUVEC or HUVEC pretreated for 6 h with tumor necrosis factor α (10 ng/ml) and vascular endothelial growth factor (1 nM). Cytokines induce TF expression and increase PAR2 expression in these cells. Cell surface TF expression as determined by surface ELISA with a TF mAb is shown in the inset. Where indicated, active site-inhibited FVIIa was added 1 h before agonists.