Our studies imply that the cell signaling activities of APC, mediated by the receptors EPCR and PAR1, are much more important for APC's ability to reduce sepsis mortality than its anticoagulant activities. These findings may help resolve the debate about the controversial role of APC-initiated cell signaling mechanisms in sepsis (20
). Based on mechanistic insights gained from the analysis of mouse endotoxemia, we provide proof of principle that a selective reduction of APC's anticoagulant activity by site-directed mutagenesis retains APC's efficacy in mortality reduction in endotoxemia, as well as in three different models of bacterial sepsis. The latter observation is consistent with the notion that the EPCR and PAR1 dependence of APC efficacy demonstrated for endotoxemia also plays a dominant role in mortality reduction in models of bacterial infection.
A first important implication of our findings is that variants such as 5A-APC may provide a safer alternative for the treatment of patients with severe sepsis by reducing the risk of severe bleeding associated with currently practiced APC therapy. Although our data strongly imply that the EPCR- and PAR1-mediated effects of APC are both necessary and sufficient for mortality reduction in mouse models of endotoxemia and bacterial sepsis, human patients with severe sepsis and multiorgan failure may nevertheless benefit from anticoagulation. In particular, in patients with sepsis complicated by disseminated intravascular coagulation, anticoagulation may reduce mortality risk and reduce morbidity secondary to thrombotic organ damage. The availability of variants such as 5A-APC, in which APC-mediated cell signaling activity required for mortality reduction and anticoagulant potency are dissociated, should enable the managing of APC infusion and anticoagulation as separate therapeutic entities, tailoring each to the specific needs of a given patient. Second, compared with normal APC, the efficacy of APC variants with selectively reduced anticoagulant activity likely can be explored in different modes of administration (i.e., continuous infusion vs. bolus) over a much greater dose range without a concomitant increase in associated bleeding risk. Indeed, although it was found that APC failed to protect septic children and adult patients with less than severe sepsis, there was no attempt of dose optimization in these groups, providing a potential explanation for the lack of APC efficacy. The peak plasma concentrations of APC achieved by bolus injections in our experiments exceed by one to two orders of magnitude the level of APC maintained in septic patients by 96 h of continuous infusion (~45 ng/ml) (24
), approaching the plasma concentration of endogenous protein C. Because protein C zymogen and APC bind EPCR with comparable affinity (25
), exogenously administered APC must outcompete endogenous protein C to engage EPCR. This may explain, on the one hand, why patients with low levels of endogenous protein C are most likely to benefit from APC therapy (25
); on the other hand, this suggests that engagement of EPCR is at best suboptimal under conditions currently established for APC therapy. Collectively, these considerations provide a strong rationale to reevaluate the optimal dose and mode of administration of APC and variants thereof in septic patients.
The primary, successfully achieved objective of this study was to gain initial insight into the role of receptor-mediated APC effects on sepsis survival. Accordingly, we chose 7-d mortality as the endpoint of our experimental analyses. A pressing, as yet unanswered question remains about the nature of the survival-enhancing downstream effects triggered by APC through EPCR and PAR1. Although we document APC-dependent alterations of coagulation, inflammation, apoptosis, and microvascular permeability in endotoxemia through premortem and survival analyses, it is unclear whether the observed changes can indeed account for the improved survival of APC-treated mice. For example, it appears doubtful whether the rather minor reduction of apoptotic cell abundance in tissue parenchyma suffices to improve survival. In contrast to observations of reduced lymphocyte apoptosis in human patients undergoing APC therapy (26
), which could in theory explain the survival-promoting effect of APC (27
), we were unable to correlate apoptosis or abundance of lymphocyte subpopulations with APC treatment or survival in mouse models. It remains to be determined whether this discrepancy is caused by species-specific APC effects, differences between mouse endotoxemia and human sepsis, or dosing differences. Likewise, with the exception of APC-treated EPCR-deficient mice, we only observed minor changes in the plasma cytokine profile and only moderate reductions of lung inflammation in APC-treated mice. The drastic APC-induced increase of IL-6 and MCP-1 in EPCR-deficient mice might instead constitute a nonphysiological, experimental artifact reflecting direct engagement of PAR1 by high bolus doses of APC. We note that endothelial-associated EPCR has, at least in mice, the ability to sequester a stable pool of APC that is excluded from the circulation and accounts for approximately one third of the total APC pool (28
). Near absence of EPCR from endothelial cells may therefore lead (but only in EPCR-deficient mice) to a further increase of APC available for PAR1 interaction. APC modulation ofd
-dimer follows a similar mouse strain–specific pattern, with unknown underlying mechanism and relevance for survival.
The identification of EPCR and PAR1 as the necessary and sufficient mediators of APC's therapeutic benefit, as established by this study, constitutes an essential first step toward the identification of the as yet elusive downstream effects controlled by this signaling pathway that are critical for survival. For example, the importance of the documented endothelial permeability barrier protection by APC for survival of endotoxemia can be examined by testing 5A-APC efficacy in mice with endothelial cell–restricted loss of EPCR or PAR1 function. Identification of the critical cell populations targeted by APC, characterization of APC effects on this cell population, validation of findings in models of bacterial sepsis, and hypothesis-based analysis of human patients will provide further insight into the survival-enhancing function of APC.