The H3 Haplotype of the EPCR Gene Determines High sEPCR Levels in Critically Ill Septic Patients

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infections [1]. The protein C (PC) anticoagulant pathway controls thrombosis and inflammation [2], and activation of PC to activated PC (APC) is enhanced by the presence of the endothelial protein C receptor (EPCR) [3]. Apart from an anti-thrombotic role, APC exerts anti-inflammatory, anti-apoptotic and cytoprotective functions that are EPCR-dependent [2, 4‐7]. The presence of a soluble form of EPCR (sEPCR) in human physiologic plasma, which is elevated in inflammatory conditions [8], strengthens the idea of EPCR shedding. sEPCR can be generated rapidly via metalloprotease tumor necrosis factor-α-converting enzyme/metallopeptidase domain 17 (TACE/ADAM17) shedding as a response to an inflammatory stimulus [9, 10] and slowly via alternative mRNA splicing [11]. sEPCR binds both PC and APC with similar affinity to membrane-bound EPCR [12, 13], blocking, however, the anti-thrombotic activity of the latter [12‐14]. While the role of the membrane form of EPCR is clearly anti-thrombotic and anti-inflammatory, the physiologic significance of sEPCR in vivo remains to be clarified. Apart from sEPCR, functionally relevant mutations in the EPCR gene have been reported, resulting in either reduced expression or dysfunction of the receptor, which can induce thrombosis development. These include a rare 23-bp insertion in exon 3 (position 6367) [15] and single-nucleotide polymorphisms (SNPs) at coding and non-coding regions of the EPCR gene; based on the SNPs that are present, human EPCR genotypes can be divided into four haplotypes, H1, H2, H3 and the very rare H4. The H2 consists of all the common alleles [16, 17]. The functional importance of these mutations has been mainly studied in patients with venous and coronary thrombosis [18‐24], and results from these studies have suggested that the H3 haplotype may be a candidate risk factor for deep vein thrombosis, possibly by increasing the levels of sEPCR and promoting its procoagulant effects because of APC sequestration [13, 16, 24]. On the other hand, the role of the EPCR gene haplotypes in sepsis has only been studied to some degree [25, 26], and even fewer have studied correlations between plasma sEPCR levels and EPCR gene polymorphisms in sepsis [26]. Therefore, the aim of the present study was to measure sEPCR levels in critically ill septic patients at admission to the intensive care unit (ICU) and to evaluate potential associations of sEPCR with patient genotype. We genotyped one haplotype-tagging single-nucleotide polymorphism (htSNP) belonging to H1 (C6333T) and two htSNPs belonging to H3 (A6936G and C1651G) (numbering according to GenBank accession no. AF106202) [15‐17]. To investigate the role of the above EPCR mutations as predisposing factors to higher sEPCR levels, we performed multivariate logistic regression analysis controlling for potential confounding factors, such as disease severity, age, sex and diagnostic category.

Since the question still remains whether sEPCR responds to changes in disease, and plasma sEPCR levels have been shown to be lower in patients with increased disease severity when controlling for rs867186-G variant (H3 haplotype) [29], we reasoned that EPCR mutations are solely responsible for increased sEPCR levels in critically ill septic patients rather than sepsis severity or the risk of poor outcomes. The H3 haplotype has been shown to be a candidate risk factor for deep vein thrombosis, possibly by increasing the levels of sEPCR and promoting the procoagulant effects due to APC sequestration [13, 16, 24]. In a previous investigation the frequency of H3 in critically ill septic patients was not associated with severe sepsis and/or septic shock development [28]. However, it has been shown that deep vein thrombosis patients and critically ill septic patients have a higher H3 haplotype frequency compared with healthy controls [16, 24, 28].

sEPCR increases in response to injury and selective regulatory mechanisms exist upon inflammation. The mechanisms controlling the expression of cell-bound and circulating soluble EPCR are still unclear and include chronic and pathological processes. There is evidence that the H3 haplotype-associated Gly219 involves increased EPCR shedding from the endothelium [30], and Guitton et al. [31] demonstrated the cumulative effect of gender and the H3 haplotype on sEPCR in primary cultures of vascular endothelial cells. In previous studies, sEPCR levels in septic patients were found to be significantly higher [8, 32], unchanged [33] or lower [34] compared with healthy volunteers. This inconsistency among studies could be partially explained by different frequencies of the EPCR H3 haplotype in the various study populations. Our laboratory has shown that sEPCR may act as an early indicator of sepsis prior to its clinical appearance [35], supporting the hypothesis that elevated sEPCR levels are implicated in the pathogenesis of the septic syndrome. The results of the present study indicate that severe sepsis, septic shock, higher qSOFA prior to ICU admission or high APACHE II scores are not associated with increased plasma sEPCR levels. Our results rather indicate that in our critically ill septic patient cohort H3 carriers have higher levels of sEPCR at ICU admission compared with non-H3 carriers. Our results additionally demonstrate that sEPCR levels at admission are not predictive of hospital mortality.

Our study has the following limitations: first, it was a single-center study with a modest sample size. Also, we did not obtain sEPCR measurements prior to sepsis appearance. We also only studied in-hospital mortality and not 30-day mortality. Finally, it was a pilot study, exploratory in nature and observational, not allowing conclusions about causative mechanisms. However, our patients were well characterized and all sEPCR measurements were made in septic patients. Our study was also conducted in a general ICU. We additionally genotyped a small non-septic patient cohort with systemic inflammatory response syndrome (SIRS) [27] and observed that their sEPCR levels are not dependent on haplotype (data not shown).