Determinants of the specificity of protease‐activated receptors 1 and 2 signaling by factor Xa and thrombin

Abstract

Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase. In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.

The emerging role of coagulation proteases in kidney disease

“…In addition to their essential role in blood clotting, both factor Xa and thrombin can elicit multiple cellular effects via PARs and their co-receptors. Thrombin activates PAR1, whereas factor Xa can also signal via PAR2, directly or together with the TF–factor VIIa complex 122,123,126 . Consistent with these observations, inhibition of factor Xa but not of thrombin suppressed expression of the proinflammatory cytokine IL-6, emphasizing the beneficial effects of protease-specific inhibition in a mouse model of sickle cell disease 123 .…”

Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from lipopolysaccharide-stimulated myeloid cells

“…In this study, a peptide mimicking this FXa amino acid sequence (FX 83-88 ), previously shown to be crucial for PAR2-dependent barrier protective and anti-inflammatory activity of FXa on endothelial cells, 22,23 produced a dose-dependent decrease in FXa regulation of LPS-stimulated cytokine production from THP-1 cells, such that FXa anti-inflammatory activity could be completely blocked by the presence of this peptide. Interestingly, bovine FXa, whose inter-EGF region amino acid sequence is not similar to its human counterpart, was unable to mount an anti-inflammatory response similar to that of human FXa, providing a possible explanation for the observed species-specific loss of function.…”

“…In contrast, the FXa Gla domain may not be required for PAR2-dependent FXa signaling on endothelial cells. 22 It is not clear at this stage whether FXa Gla domain truncation confers long-range structural changes that disrupt FXa myeloid receptor binding sites on the Gla domainless protease, or itself represents a crucial binding site for FXa myeloid cell surface receptors.…”

“…Although structurally homologous to APC, FXa has been described both as a driver 20,21 and an inhibitor 22,23 of TLR-and cytokine-induced inflammation depending on the cell type and signaling receptors activated. FXa can activate both PAR1, PAR2 and to a lesser extent, PAR4.…”

In silico thrombin generation: Plasma composition imbalance and mortality in human immunodeficiency virus

“…In this study, it is the suppression of the anticoagulants PC and AT that allows for the continued formation of the procoagulant complexes by not inactivating FVa and FVIIIa, which converts FX to FXa and allows for the continuation of thrombin generation. The increase in these critical enzymes (FXa and thrombin) also further promotes the observed hypercoagulation in part through the activation of protease‐activated receptors 1 and 2,55, 56, 57 which is seen in this population by the increased proinflammatory cytokines such as IL‐6. This combined effect may be driving the excess thrombin generation that is seen among the mortality cases.…”