Non-Canonical PAR3 Activation Induces Tie2-Dependent Endothelial Barrier Protective Effects
Authors: Stavenuiter Fabian , Mosnier Laurent O. 
Published: November 2019

Abstract
Introduction: Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease activated receptor 1 (PAR1), and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Non-canonical PAR1 and PAR3 activation by APC versus canonical activation by thrombin provide an explanation for the functional selectivity of these proteases. APC induces non-canonical PAR3 activation at Arg41 and synthetic peptides representing the tethered-ligand sequence of PAR3 after non-canonical cleavage (P3R) induce barrier protective effects in vitro and vascular integrity in vivo. However, signaling mechanisms employed by PAR3 remain undefined. To obtain better insights into the relation between coagulation proteases with endothelial barrier protective effects and canonical/non-canonical PAR1 and PAR3 activation, the PAR proteolysis analysis was extended to factor Xa (FXa). Similar to APC, FXa-mediates endothelial barrier protective effects that involve both PAR1 and EPCR. To date, however, no role for PAR3 in FXa-induced barrier integrity has been implicated.
Results: In the presence of EPCR, FXa cleaved PAR1 at Arg41 similar to thrombin and not at Arg46 alike APC, whereas FXa cleaved PAR3 at the non-canonical Arg41 similar to APC but not at the canonical Lys38 corresponding to cleavage by thrombin. Surprisingly, changes in electric cell-substrate impedance sensing (ECIS) using the iCelligence system showed FXa induced an immediate drop in endothelial cell index (~60%) comparable to that induced by thrombin, indicating that FXa induced a loss of cell barrier function. Notwithstanding, after incubation of endothelial cells with FXa for 3 hours, FXa protected (~40%) against TRAP-induced loss of barrier function, similar to that induced by APC, confirming barrier protective effects of FXa. PAR1 blocking antibodies prevented the early FXa-mediated loss of barrier function, indicating that PAR1 cleavage at Arg41 was responsible for this.In contrast,a combinationofPAR1 and PAR2 blocking antibodies was needed to inhibit late (3h) FXa-mediated barrier protection. Blocking antibodies against PAR3 confirmed that canonical PAR3 activation enhanced PAR1-mediated barrier disruptive effects of thrombin (~15%). PAR3 blocking antibodies also significantly reduced the barrier protective effect of FXa (~15%), indicating a functional role for non-canonical PAR3 activation by FXa. Neither canonical (P3K) nor non-canonical (P3R) PAR3 tethered-ligand peptides directly induced significant phosphorylation of ERK1/2 or Akt in endothelial cells. The P3K however, but not the P3R peptide, enhanced TRAP induced ERK1/2 phosphorylation. No Akt phosphorylation was observed in endothelial cells treated with TRAP in the presence of either P3K or P3R. Interestingly, both APC and FXa but not thrombin induced prolonged activation of the endothelial cell specific Tie2 receptor, determined by phosphorylation of Y992 and S1119. Tie2 activation by FXa required PAR3 and EPCR with a partial contribution of PAR1 and PAR2. P3R induced potent activation of Tie2 achieving maximal activation at ~0.8 µM P3R, whereas P3K failed to do so. Additionally, neither (non-)canonical PAR1 nor PAR2 tethered-ligand peptides induced activation of Tie2. Activation of Tie2 by P3R was relatively fast and reached half-maximal activation in about 5 minutes. Blocking antibodies against Tie2 reduced FXa-mediated barrier protective effects by approximately 34%, whereas inhibition of Tie2 did not affect thrombin mediated barrier disruption. Immunohistochemistry indicated that Tie2 activation by FXa and P3R resulted in clustering of activated Tie2 at the cell borders. Accordingly, Tie2 activation by FXa and P3R resulted in changes in the cellular distribution of the tight-junction-associated protein zona occludens (ZO-1) in time.
Conclusion: Here we identified a novel pathway for Tie2 activation by non-canonical PAR3 activation that promoted tight-junction formation and endothelial barrier protective effects. In contrast, canonical activation of PAR3 enhanced PAR1-mediated barrier disruptive effects by thrombin. These results exemplify the novel dimensions that non-canonical activation of PARs provides for the possible molecular mechanisms that are responsible for the functional selectivity of protease signaling.

Disclosures
No relevant conflicts of interest to declare.