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• W2007667881 abstract "Over the past few years there has been considerable interest, and also controversy, surrounding the cell signaling function of activated protein C (APC). Whereas the anticoagulant function of this serine protease has been well characterized through the work of many different laboratories, the mechanisms underlying the ability of APC to specifically impart cytoprotective signaling in endothelial cells remain less well understood. The endothelial cell protein C receptor (EPCR) and protease activated recptor-1 (PAR-1) dependent cell signaling function of APC was first described by Ruf and co-workers in 2002 [1]. Since that time, several groups have endeavored to dissect the molecular mechanisms that enable this process. EPCR is expressed primarily by the vascular endothelium, and preferentially so in that of larger blood vessels [2-5], where it associates in caveolae/glycosphingolipid-rich rafts contained in the endothelial surface membrane [6]. EPCR has also been detected in other cell types, including monocytes and hematopoietic stem cells [7, 8]. Protein C binds EPCR via specific interactions with the Gla domain [9, 10]. In so doing, EPCR enhances the activation of protein C by the thrombin-thrombomodulin complex on the endothelial surface [11]. In addition to this, APC bound to EPCR is able to cleave, and so signal through, the integral membrane protein, PAR-1 [1]. PAR-1 is the prototypical thrombin receptor that belongs to a family of 7-transmembrane G-protein-coupled receptors [12, 13]. PAR-1 is activated by enzymatic cleavage that induces exposure of a new extracellular N-terminus that acts as a tethered activation ligand. Both thrombin and APC cleave and activate signaling through PAR-1 in the same way. However, whereas thrombin can do so unassisted, APC-mediated PAR-1 signaling requires EPCR as it enhances the affinity of protein C/APC for endothelial cell membranes, and also localizes APC to caveolae/glycosphingolipid-rich rafts, which are also enriched in PAR-1 [6]. Intriguingly, when APC signals through PAR-1 on endothelial cells it confers cytoprotective effects, including anti-apoptotic pathways and endothelial barrier protection [14]. Conversely, when thrombin signals through the very same receptor (which it does with about ∼3 orders of magnitude higher efficiency than APC) it imparts proinflammatory signals. How then can two different serine proteases activate PAR-1 in the same way, and yet transduce quite distinct signals/cellular effects? This has been explored by several groups. Bae et al. elegantly demonstrated that EPCR co-localizes with PAR-1 and caveolin-1 in cavaolae/lipid rafts [6]. They hypothesized that it was this co-localization that enabled PAR-1 cleavage/signaling by APC and suggested that the binding of the APC Gla domain to EPCR in some way promoted cytoprotective signaling through PAR-1 [15]. Schuepbach et al. demonstrated that APC could mediate PAR-1 dependent cytoprotective signaling in endothelial cells even in the presence of thrombin [16]. Together, these results implied the presence of distinct cellular pools of PAR-1 that are perhaps coupled to different intracellular signaling partners. A further demonstration of the difference between APC and thrombin mediated PAR-1 activation came from the finding that although thrombin readily cleaves PAR-1, the activated receptor is also rapidly internalized and degraded – a mechanism that serves to turn off the signal. Unlike thrombin, however, PAR-1 that has been activated by APC exhibits a longer half-life on the cell surface and can accumulate there, even when thrombin is present [16]. In this issue of the Journal of Thrombosis and Haemostasis, Bae et al. report their findings on the specificity of PAR-1 cleavage based on its caveolae/lipid raft localization [17]. To do this, the authors generated different classes of PAR-1 cleavage reporter constructs in which soluble alkaline phosphatase was fused to the PAR-1 exodomain that contains the APC/thrombin cleavage site. This in turn was fused to either the rest of PAR-1, or other different transmembrane domains that directed the fusion protein to different compartments of the transfected endothelial cell membrane (i.e. either lipid rafts/caveolae or a more general membrane distribution). Using this approach to quantify PAR-1 cleavage, the authors examined the relative ability of thrombin, APC or Gla domainless APC to proteolyse the fusion protein. Similar to previous studies, the specific proteolysis of the PAR-1 fusion protein by APC was dependent upon the interaction of the APC Gla domain with EPCR. This dependency was itself associated with the localization of the reporter construct to caveolae, as when the targeting of the fusion protein to these membrane microdomains was disrupted the specificity of PAR-1 proteolysis associated with the APC Gla domain-EPCR interaction was lost. The authors also found that the caveolae targeted PAR-1 fusion construct could be made susceptible to proteolysis by Gla domainless APC (i.e. the EPCR dependency of proteolysis was lost) if cells were co-incubated with inactive thrombin. This effect was dependent upon the interaction of exosite I of thrombin with a hirudin-like sequence in the exodomain of PAR-1. These results implied that the binding/interaction of thrombin with PAR-1 induced either a change in its membrane localization, or a conformational change in PAR-1 that enabled its proteolysis by APC in an EPCR independent manner. Similar results were obtained when inactive protein C and Gla domainless APC were co-incubated with transfected endothelial cells. The authors therefore contend that the binding of protein C to EPCR also induced a redistribution of PAR-1 that enhanced its recognition/susceptibility to proteolysis. Evidence for the redistribution of PAR-1 out of the caveolae in response to incubation of endothelial cells with thrombin or APC was previously provided by the same group [6], suggesting that these reporter constructs may be behaving in an analogous manner to endogenous PAR-1. What then are the implications of these findings? Could they give mechanistic insight into the different cellular effects observed when either thrombin or APC signal through PAR-1? The authors suggested a model in which the redistribution of PAR-1 induced by either the direct binding of thrombin, or the binding of the APC Gla domain to EPCR, caused PAR-1 to associate with either proinflammatory or cytoprotective signaling partners, respectively. Although the current work does not provide information on exactly how this might be mediated, it does provide a framework for the understanding of this process. Naturally, the use of reporter constructs that are very likely to be expressed at elevated levels to the endogenous PAR-1 means that caution must be taken in extrapolating these findings to the physiological situation. To address this, the authors endeavored to corroborate their hypothesis by examining the effects of APC signaling through endogenous PAR-1 in endothelial cells [17]. Endothelial cells incubated with Gla domainless APC in the presence of inactive thrombin exhibited increased cell permeability and elevated apoptosis after exposure to TNF-α, suggesting that the binding of thrombin to PAR-1 had not only made it susceptible to Gla domainless APC cleavage but also directed its signaling actions toward proinflammatory pathways. In stark contrast, endothelial cells incubated with Gla domainless APC in the presence of inactive protein C exhibited reduced cell permeability and were protected against apoptosis. This suggested that the binding of the inactive protein C Gla domain to EPCR had also made PAR-1 susceptible to activation by Gla domainless APC cleavage but that this had directed its signaling actions toward cytoprotective signaling pathways. Whereas the results presented by Bae et al. elegantly support the presented hypothesis, there still remain unanswered questions. The evidence for PAR-1 membrane redistribution is rather inferential and direct visualization of the dynamics of any EPCR and PAR-1 relocalization in response to different agonists will invariably be necessary to confirm this. Furthermore, why cannot factor VII, which was recently demonstrated to bind EPCR, induce PAR-1 redistribution [10, 18]? It is clear that further studies are now required to ascertain how any such redistribution, exposure of the PAR-1 scissile bond or association with different intracellular signaling molecules actually takes place. That aside, the data from Bae et al. do make an important suggestion. In vivo, it might be assumed that much of the endothelial EPCR is constitutively occupied by protein C. Based on the hypothesis presented in their manuscript, this infers that PAR-1 on endothelial cells may thus be constitutively directed toward cytoprotective signaling. The cytoprotective properties of APC are increasingly being suggested to have therapeutic benefit; if so, the findings by Bae et al. may take on increasing significance. The authors state that they have no conflict of interest." @default.
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• W2007667881 title "Cytoprotective effect of activated protein C: specificity of PAR-1 signaling" @default.
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• W2007667881 doi "https://doi.org/10.1111/j.1538-7836.2008.02951.x" @default.
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