The ability of the vasculature

The ability of the vasculature to determine the nature and magnitude of a stimulus is essential for the appropriate and proportionate response. The vascular response to injury include rapid changes to blood flow to reduce the loss of blood, followed by increased contact with the sub-endothelial layer, in order for the formation of a stable and effective clot [[29], [30], [31], [32]]. In agreement with this JSH-23 the incubation of the endothelial monolayers with fXa resulted in an initial reduction in permeability followed by significantly higher levels of leakage across to the lower chamber. Interestingly, incubation of endothelial monolayers with sub-optimal levels of fXa (5 nM) did not produce any response while at 10 nM, the enzyme produced full response in the cells. The magnitude of increased permeability at 60 min, following activation with fXa (10 nM) was higher than that obtained with PAR2-AP. Furthermore, the response to PAR2-AP did not produce the initial tightening of the endothelial layer at 30 min. Activation of fX within the blood is initiated by the TF-fVIIa complex and therefore the relatively lower amounts of fXa generated by the extrinsic pathway may be incapable of causing vascular permeability. Conversely, full activation of the coagulation involving the intrinsic pathway can convert a large quantity of the available fX at the locality of the injury. Therefore, it is plausible that under these conditions the adaptations in the permeability of the endothelial layer to allow contact between plasma and the sub-endothelial layer, becomes an essential step in the completion and strengthening of the clot. This mechanism appears to be dependent on the proteolytic activity of fXa and was significantly diminished in the presence of Rivaroxaban. As stated above, while some studies report that the activation of PAR2 signalling promotes endothelial permeability [8,21,22], others did not find a response [23,24]. Furthermore, Feistritzer and Riewald [5] suggested that PAR1 and PAR2 act in conjunction to desensitize endothelial response to further activation. Our data indicate that the activation of PAR2 appears to be a major inducer of permeability and may participate in the original reduction endothelial tightness. A recent report has shown that the activation of PAR2 can disrupt cellular tight junctions through the activation of p38 MAPK alone [33]. However, concurrent activation of ERK1/2 and Akt pathways in response to PAR2 activation may occur independently [33,34]. Furthermore, some PAR2 signalling appears to be dose dependent even when using the activating peptide [35]. These data are in accord with our finding that the induction of PAR2 using the activating peptide results in the activation of p38 MAPK the magnitude of which can further be modulated by other factors including the presence of TF [36]. Moreover, the activation of PAR2 alone using the agonist peptide did not produce the initial tightening of the endothelial layer at 30 min. Consequently, PAR1 appears to be an essential contributor to the initial reduction in permeability. Therefore, the responses of PAR2, in conjunction with PAR1 appear to constitute a means of converting the signal arising from fXa into the converse but canonical and time-dependent modifications in endothelial permeability. It has been recognised that while PAR2 constitutes the main receptor for fXa induced signalling, PAR1 also acts as a minor target for fXa [37] and may be involved in fXa signalling [38]. The protective influence of PAR1 [5,39,40] in thrombin-mediated signalling, on endothelial function has been documented [4,5]. Moreover, the ability of PAR1 to induce endothelial permeability is known [41]. However, thrombin appears to influence vascular permeability through multiple mechanisms including direct signalling, by influencing other cells through the nitric oxide-mediated pathways. Moreover, the outcomes of response to thrombin appear to be both cell-specific and target-selective, affecting various different proteins within the cellular tight junction [42]. In addition, some of the differences in the outcomes appear to arise from the requirement for cell-surface effectors such as APC or PAR3 [4,43] although some reports dispute a direct role for PAR3 in the response to PAR1 or the expression of PAR4 on some endothelial cells [44]. Finally, it has been suggested that the magnitude of activation of PAR1 is amplified by the proteolytic action of thrombin, in comparison to for example by PAR1-activating peptide [45,46]. Therefore, we are not able to present a conclusive explanation for the lack of alterations in permeability in response to PAR1-activating peptide. Additionally, the focus of our study has been the role of fXa as a first messenger in regulating the induction of endothelial permeably in response to the activation of coagulation. Consequently, the discussions on the multiple roles of thrombin and the magnitude of PAR1 activation, together with the multiple second messengers and pathways involved in PAR signalling [3,4] is beyond the scope of our study. Consequently, the outcome of PAR1 activation was not further pursued in this study. However, in addition to the contribution from PAR1, co-operative signalling from other receptors such as EPCR [53,54] may also contribute to PAR2 signalling and require clarification.