Our present findings support an important role for PAR1 in mouse models of IAV infection. Studies with PAR1-AP indicated that PAR1 activation increased inflammation, early virus production, weight loss, and mortality after infection (Figures and ), and studies using Par1–/– mice indicated that PAR1 contributed to the pathogenesis of IAV infection (Figure ). The observation that SCH79797, a drug that inhibits PAR1 signaling, decreased inflammation, early virus production, weight loss, and mortality after infection was in accord with the PAR1-AP and Par1–/– results. Moreover, the observation that SCH79797 decreased mortality after infection with multiple IAV strains (H1N1, H3N2, and H5N1), and was effective even when dosing was initiated at day 3 after inoculation, suggests that PAR1 inhibition should be explored in additional preclinical studies and, if appropriate, in humans as a possible treatment for influenza.
To our knowledge, a role for PAR1 in the response to, and the pathogenesis of, virus infections has not been previously described. PAR1 activation in endothelial cells, fibroblasts, and other cell types triggers various responses, many of which are proinflammatory (e.g., chemokine and cytokine production, adhesion molecule display, prostaglandin production, and permeability increases; refs. 14
). In accord with our observations, intratracheal delivery of PAR1 agonist was not sufficient to trigger inflammation in the lungs of otherwise normal mice (35
), but did exacerbate ventilation injury–induced pulmonary edema (36
). Additionally, Par1–/–
mice are protected from ventilation injury–induced and bleomycin-induced lung injury (36
). Like our results, these observations suggest that PAR1 signaling contributes to inflammatory responses to injury in the lung, the major target in our IAV infection model.
PAR1 activation did not exacerbate the effects of IAV infection in Plg–/–
mice (Figure ). It is possible that PLG is simply playing a permissive role for the effect of PAR1 activation in IAV infection; that is, PLG supports infection and injury, and PAR1 activation exacerbates their effects. Interestingly, however, PAR1-AP did promote PLG-dependent HA cleavage in lung epithelial cultures, suggestive of a possible interaction of PAR1 signaling with the ability of IAV to become infectious and hence replicate. These findings are consistent with the prior observation that PLG contributes to the pathogenesis of IAV infection (27
). Additionally, PAR1 signaling may promote PLG activation to plasmin (29
), thereby providing a possible link to increased HA cleavage and IAV production. It is also possible that PAR1 activation contributes to proinflammatory functions of PLG (25
), by promoting its conversion to plasmin or by other mechanisms.
Additional considerations suggest that PAR1 activation’s abilities to promote early virus replication and to enhance a harmful inflammatory response in the respiratory tract are, at least in part, independent of each other. When PAR1-AP was delivered 3 days after infection, despite similar virus replication in the lungs, treatment still had a deleterious effect (data not shown). Additionally, based on critical residues in HA involved for cleavage by plasmin, it is unlikely that the replication of highly pathogenic H5N1 and 2009 pandemic H1N1 are modulated by plasmin (42
), yet SCH79797 treatment still decreased mortality.
As noted above, we found that in IAV-infected A549 cells, activation of PAR1 increased PLG-dependent HA cleavage, an essential step for virus infectivity. Indeed, only the cleaved form of HA permits pH-dependent fusion of the viral envelope within the endosomal membranes and subsequent release of the genome into the cytosol and virus replication. In vivo, PAR1 also promoted virus replication shortly after infection. However, at 48 hours after infection, no difference in lung virus titers was observed between PAR1-AP–stimulated and unstimulated mice, which suggests that HA cleavage could be compensated by other proteases that are either recruited or activated by infection in the lungs.
Therefore, we propose a model (Figure ) in which PAR1 promotes activation of PLG into plasmin. Subsequently, plasmin acts on virus replication through HA cleavage, enhancement of which likely enhances inflammation via pathogen-associated molecular patterns. Simultaneously, plasmin also acts as a proinflammatory mediator that accounts for the deleterious lung inflammation. Additionally, PAR1 triggers a variety of proinflammatory responses, independent of PLG and virus, that may exacerbate inflammation and injury. Because PAR1 couples coagulation to inflammation (14
) and coagulation to fibrinolysis (30
), further studies are needed to investigate the overall impact of hemostasis dysregulation in PAR1-mediated inflammation during IAV infection.
Proposed model for PAR1-mediated influenza virus pathogenesis.
Our observation that a PAR1 agonist (43
) exacerbated the effects of IAV infection suggests that PAR1 activation is capable of promoting inflammation and tissue damage in this setting. Moreover, our observation that Par1–/–
mice and SCH79797-treated mice were protected from IAV infection suggests that PAR1 activation contributes to the pathogenesis of IAV infection and that PAR1 is endogenously activated during IAV infection. Accordingly, the natural PAR1 activator thrombin was generated in IAV-infected lungs (45
), and elevated levels of PAR1 were observed in the airways of IAV-infected mice (17
). It is worth noting, however, that SCH79797 is known to have off-target effects on cell proliferation and survival (46
); thus, we cannot exclude PAR1-independent effect of SCH79797. However, SCH79797 was capable of inhibiting PAR1 signaling (Figure A and ref. 18
), and the concordance of our KO and inhibitor studies — and the fact that their effects were opposite from those of PAR1-AP — suggest that the effects of SCH79797 in our model could be related to its ability to block PAR1 signaling.
Besides PAR1, other PARs may be involved in the pathogenesis of IAV infection (48
). Identification of the exact nature and amount of proteases present at the site of infection, and how virus strain differences alter the immune response and its interactions with PARs, may advance our understanding of the pathogenesis of IAV infection.
Current treatments for IAV infection target the viral proteins M2 and NA. These drugs suffer from a number of disadvantages, including the rapid development of resistant virus variants as a result of selective pressure, which highlights the need for new pharmacological strategies against IAV infection. Because targeting host proteins would not be subject to resistance, and because severe infections with IAV are associated with a deleterious host inflammatory response, drugs regulating inflammation are appealing as potential treatments for IAV infection (51
). In our present study, blocking PAR1 signaling almost fully protected mice from a highly pathogenic, oseltamivir-resistant 2009 pandemic H1N1v virus isolated from a severely diseased oseltamivir-treated patient (34
). Additionally, inhibition of PAR1 signaling up to 3 days after inoculation protected mice from a detrimental outcome of infection with various IAVs, including H1N1 and H3N2 strains. Because IAVs of the H1N1 and H3N2 subtypes are currently circulating in the human population, it is reasonable to assume that PAR1 antagonists are most likely also effective against seasonal influenza viruses. Interestingly, the PAR1 antagonist vorapaxar has been studied as a potential antithrombotic drug in approximately 40,000 patients over 3 years (53
). The most serious side effect, increased incidence of intracranial bleeding, occurred mainly in patients with a history of prior stroke. In the absence of such a history, the increase in the incidence of intracranial bleeding was less than 1 per 1,000 treatment-years. Thus, short periods of PAR1 antagonism would appear to be relatively safe. This observation, in consideration with our results, suggests that PAR1 antagonism should be further explored for the treatment of IAV in additional preclinical models and, if appropriate, human studies.