Protease-activated receptor-1: key player in the sepsis coagulation-inflammation crosstalk

Protease-activated receptors (PARs) belong to the family of G protein-coupled receptors. Among the four members, PAR1 plays a major role in orchestrating the interactions between coagulation and inflammation. PAR1 has opposing functions during sepsis, and PAR1 blockade or activation may be alternatively beneficial at early or late stages of different sepsis models. Studying molecular mechanisms of the crosstalk between inflammation and coagulation may lead to the identification of new targets for therapies in sepsis. However, the time-dependent switch of PAR1 from an exacerbating proinflammatory receptor to a protective anti-inflammatory receptor needs to be investigated before clinical trials can be recommended. Finally, as PAR1 seems to play a singular role in Streptococcus pneumoniae-induced sepsis through a crosstalk between PAR1 and platelet-activating factor receptor, the exact role of PAR1 needs to be investigated in other models of sepsis.


Introduction
In the previous issue of Critical Care, Schouten and colleagues show the critical involvement of proteaseactivated receptor (PAR)-1 in a lethal Streptococcus pneumoniae pneumonia model [1]. PARs belong to the family of G protein-coupled receptors [2]. Four PARs are currently known (PAR1, PAR2, PAR3, PAR4). In contrast with other G protein-coupled receptors, PARs are not activated in vivo by binding of a soluble ligand but, instead, are activated by proteolysis triggered by extracellular proteases. PARs use a fascinating mechanism to convert an extracellular proteolytic cleavage event into a transmembrane/intracellular signal: the receptors carry their own tethered ligands, which remain cryptic until unmasked by receptor N-terminal cleavage, and then an intramolecular rearrangement allows the ligand and the receptor moieties to interact. A considerable body of evidence supports a prominent role for PARs in a variety of human physiological and pathophysiological processes, and thus substantial attention has been paid to develop new drug-like molecules either activating or blocking PARs [2].

PAR1 is involved in the interactions between infl ammation and coagulation
Th e crosstalk of infl ammation and coagulation has emerged as a major mechanism controlling the host response to invading microorganisms, and poor regu lation of this mechanism is held responsible for the occurrence of multiple organ failure and eventually death in patients with severe sepsis/septic shock. PAR1 plays a major role in orchestrating the interplay between coagulation and infl ammation [2][3][4][5].
PAR1 is the primary cell-surface receptor responsible for thrombin-mediated platelet aggregation in humans, but is also activated by many other proteases -including activated protein C (APC) and its receptor, the endothelial protein C receptor. All of these diff erent proteases, which are released during activation of the clotting cascade, can regulate PAR signalling by either activation or inactivation. PAR1 then has an intriguing dual function: PAR1 activation by thrombin results in a pro infl ammatory/endothelial-permeability enhancing res ponse, while the same receptor activated by APC/endothelial protein C receptor results in an anti-infl ammatory, endothelial-integrity preserving response [3]. Nevertheless, understanding the role of PAR1 signalling in sepsis remains complex due to the multiple and, in part, opposite eff ects ascribed to this receptor.
A further complexity is present when many types of cells, all present in the same environment, express this receptor -platelets, leukocytes, macrophages, endo thelial cells, epithelial cells and fi broblasts, for example. Moreover, thrombin has a much higher affi nity for PAR1 Abstract Protease-activated receptors (PARs) belong to the family of G protein-coupled receptors. Among the four members, PAR1 plays a major role in orchestrating the interactions between coagulation and infl ammation. PAR1 has opposing functions during sepsis, and PAR1 blockade or activation may be alternatively benefi cial at early or late stages of diff erent sepsis models. Studying molecular mechanisms of the crosstalk between infl ammation and coagulation may lead to the identifi cation of new targets for therapies in sepsis. However, the time-dependent switch of PAR1 from an exacerbating proinfl ammatory receptor to a protective anti-infl ammatory receptor needs to be investigated before clinical trials can be recommended. Finally, as PAR1 seems to play a singular role in Streptococcus pneumoniae-induced sepsis through a crosstalk between PAR1 and platelet-activating factor receptor, the exact role of PAR1 needs to be investigated in other models of sepsis.
with a higher catalytic effi ciency relative to APC. How APC activates PAR1 is unclear when thrombin is also present in the same environment.
Finally, PAR1 can take up multiple conformational states, each state triggering diff erent downstream signalling pathways and cellular responses. For example, compart mentalisation of PAR1 in lipid rafts and localisation to caveolae are critical for the APC-biased agonist of PAR1 [2].

PAR1 and Streptococcus pneumoniae pneumonia
In the lungs, the role of PARs in infl ammatory processes remains controversial [4]. PAR1 activation contributes to the pathogenesis of infl uenza A virus infection [6]. PAR1 signalling inhibition decreases infl ammation, early virus replication and mortality after infection with multiple infl uenza A virus strains, and is eff ective even when dosing was initiated at day 3 after inoculation. Additionally, PAR1 activation exacerbates ventilation injuryinduced pulmonary oedema [7]. Also, Par1 -/mice are protected from ventilation lung injury in a setting of high-tidal volume ventilation and bleomycin-induced lung injury [7][8][9]. Th ese observations suggest that PAR1 signalling contributes to proinfl ammatory responses to injury in the lungs.
In the study by Schouten and colleagues, PAR1 impairs the host defence response, as refl ected by a reduced lethality, lower bacterial loads, less pulmonary neutrophil infl ux and less lung damage in PAR1 knockout mice [1]. Even if the mechanisms underlying these diff erences remain to be elucidated, crosstalk between PAR1 and platelet-activating factor receptor (PAFR) may partly explain these diff erences. PAR1 and PAFR have been shown to cooperate together, as PAR1 induced the expres sion of platelet-activating factor and PAFR [10]. Indeed, PAFR plays a crucial role in the pathogenesis of pneumococcal disease. Th e biological activity of plateletactivating factor is mainly determined by phosphorylcholine, which binds specifi cally to PAFR. Phosphorylcholine is also a prominent part of the cell wall of S. pneumoniae and specifi cally binds to PAFR expressed on human respiratory epithelial cells, which facilitates pneumococcal entry into these cells and transcytosis to the basal surface of endothelial cells [11]. Using PAFR knockout mice, the same group demonstrated that PAFR was used by S. pneumoniae to induce severe lethal pneumonia, as refl ected by a reduced mortality, attenuated bacterial outgrowth in the lungs and diminished dissemination of the infection in PAFR knockout mice [11]. PAR1 may then amplify PAFR-dependent pneumococcal dissemination and induce severe pneumonia. Further studies are defi nitely needed to dissect the exact PAR1 signalling mechanisms in S. pneumoniae pneumonia. Nevertheless, as underlined by the authors, the therapeutic eff ect and impact of PAR1 signalling inhibition with concurrent antibiotic treatment for S. pneumoniae pneumonia needs to be evaluated.
Other studies examining the role of PAR1 in sepsis have revealed confl icting results. Whereas studies using blocking agents confi rmed the present results, others showed detrimental eff ects. Th ese discrepancies may be explained by the diff erent sepsis models (pneumonia, polymicrobial sepsis induced by caecal ligation and puncture and endotoxinemia), diff erent severity and inoculum (lethal dose 90 vs. lethal dose 100) and diff erent stages of the disease. PAR1 has opposing, temporally controlled functions during the progression of the sepsis, and PAR1 blockade or activation may be alternatively benefi cial at early or late stages of diff erent sepsis models. Th e complexity of PAR1 signalling mechanisms supports the assessment of the immunological status of the patients. Immunological monitoring could help to guide therapies with immunomodulatory drugs with an antiinfl ammatory eff ect or an immunostimulatory eff ect [12].

Conclusion
Increased insight into the molecular mechanisms of the tight relationship between infl ammation and coagulation may lead to the identifi cation of new targets for therapies that can modify the excessive activation that leads to dysregulation of these systems. PAR1 has a pivotal role in mediating protective APC eff ects. Th e time-dependent switch of PAR1 from an exacerbating proinfl ammatory receptor to a protective anti-infl ammatory receptor needs to be investigated in other experimental models before clinical trials can be recommended.