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A Comparative Study of the Protein C Pathway in Septic and Nonseptic Patients with Organ Failure

¹ INSERM Unité 765, Paris, France; ² AP-HP, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique, Paris, France; ³ Université Paris-Descartes, Paris, France; ⁴ AP-HP, Hôpital Européen Georges Pompidou, Service de Réanimation Médicale, Paris, France; ⁵ Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands; and ⁶ Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, The Howard Hughes Medical Institute, Oklahoma City, Oklahoma

Correspondence and requests for reprints should be addressed to Pr. Jean-Luc Diehl, M.D., INSERM U765, Service de Réanimation Médicale, Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75908 Paris, Cedex 15, France. E-mail: jldiehl{at}invivo.edu

	ABSTRACT

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Rationale: Severe sepsis is associated with an exacerbated procoagulant state with protein C (PC) system impairment. In contrast, the inflammatory and coagulation status of nonseptic patients with organ failure (OF) is less documented.

Objectives: To compare coagulation activation, focusing on the PC system, and inflammatory status in septic and nonseptic patients with OF.

Methods: Thirty patients with severe sepsis and 30 nonseptic patients were recruited at the onset of OF and compared with 30 matched healthy subjects. We performed an extensive analysis of the PC pathway, including plasma protein measurements and quantification of leukocyte expression of PC system receptors. In addition, we analyzed the inflammatory status, based on inflammation-related gene leukocyte expression.

Measurements and Main Results: We observed coagulation activation, reflected by a similar increase in tissue factor mRNA expression, in the two patient groups when compared with the healthy subjects. Soluble thrombomodulin levels were higher in septic patients than in healthy control subjects, whereas PC, protein S, and soluble endothelial cell PC receptor levels were lower. Similar results were obtained in nonseptic patients with OF. Monocyte thrombomodulin overexpression, together with increased circulating levels of activated PC, suggests that the capacity for PC activation is at least partly preserved in both settings. No difference in the inflammatory profile was found between septic and nonseptic patients.

Conclusions: The pathogenesis of OF in critical care patients is characterized by an overwhelming systemic inflammatory response and by exacerbated coagulation activation, independently of whether or not infection is the triggering event.

Clinical trial registered with www.clinicaltrials.gov (NCT 00361725).

Key Words: protein C • organ failure • coagulation • inflammatory profile

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject Patients with severe sepsis and organ failure have abnormalities of coagulation and protein C pathways, but little is known about these pathways in patients with organ failure who are not septic. What This Study Adds to the Field Severely ill patients with organ failure have similar coagulation and protein C pathway abnormalities, whether or not sepsis is present, suggesting that common mechanisms are involved in the pathogenesis of organ failure.

 
Sepsis, defined as a systemic inflammatory response to infection, carries a very high mortality rate, especially when associated with organ failure (OF) and/or septic shock. In the United States, the yearly incidence of severe sepsis (SS; sepsis with acute organ dysfunction) is approximately 750,000 cases, with approximately 215,000 deaths (1). The incidence and mortality rate both increase with age (1, 2). The global incidence of SS in industrialized countries is projected to increase by 1.5% per year, mainly because of aging of the population (1). The activation of coagulation and the exacerbated inflammation observed in this setting have led to therapeutic use of activated protein C (APC), a natural anticoagulant protein with antiinflammatory properties.

The protein C (PC) anticoagulant pathway is one of the main systems that inhibit blood coagulation. It involves two circulating proteins, PC and protein S (PS), and two endothelial receptors, thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR). Conversion of PC to APC is initiated by thrombin bound to TM on endothelial surfaces. When bound to EPCR, PC activation is accelerated by a factor of about 20 (3). Once activated, APC has anticoagulant effects (potentiated by its cofactor, PS), antiinflammatory effects (3), and antiapoptotic activity (4).

SS is associated with systemic inflammation and a procoagulant state mediated by the tissue factor (TF) pathway (5). Anticoagulant pathways such as PC become activated and consumed during SS. APC was preferred to PC for therapeutic use in this setting, because the capacity for PC activation was believed to be diminished by endothelial dysfunction. However, recent studies show that effective PC activation can also occur in patients with SS (6, 7). The therapeutic potential of APC was shown in the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study (8), in which APC was especially beneficial in the most severely ill patients, defined as those with an Acute Physiology and Chronic Health Evaluation (APACHE) II score of 25 or greater or multiple organ failure; in contrast, APC was not beneficial in patients with less severe disease (9).

The pathogenesis of sepsis is characterized by an overwhelming systemic inflammatory response and by exacerbated coagulation activation. Because OF in nonseptic critical care patients may be due to a variety of underlying diseases, few pathophysiologic studies have focused on these patients as a distinct group. Previous reports have involved patients with heat stroke, exacerbated chronic obstructive pulmonary disease (COPD), pulmonary embolism or cardiac arrest, and trauma, and neurosurgery patients, and showed a systemic inflammatory response and hypercoagulability (10–13). A better understanding of the role of the PC pathway in nonseptic critical care patients could pave the way for therapeutic use of PC or APC in this setting.

In the present study, we evaluated coagulation activation and extensively explored the PC system in septic and nonseptic critical care patients with OF. In addition, given the interactions between inflammation and coagulation, we compared the inflammatory status in the two patient groups, based on inflammation-related gene expression analysis. Part of this study was presented at the American Thoracic Society 2006 International Conference (14).

	METHODS

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Subjects
The design of this case-control study has been described in detail elsewhere (15), Briefly, the cases were 30 consecutive patients studied at the onset of SS, defined using standard criteria (8), and 30 consecutive patients with OF (defined by the same criteria) but no evidence of infection. As an inclusion criterion, the delay between the onset of organ failure(s) and the study blood sampling was less than 24 hours for all patients. Previous treatment with vitamin K antagonists was retained as a noninclusion criterion. All patients were recruited in a 20-bed medical intensive care unit of a university hospital (Hôpital Européen Georges Pompidou, Paris, France). Controls were healthy subjects matched for age and sex with patients with SS. The APACHE II score was used as an indicator of disease severity (16).

The study was approved by the ethics committee of the Société de Réanimation de Langue Française. Written, informed consent was obtained from the participants or their next of kin before blood collection.

Sample Preparation
Venous blood was collected between 8:00 and 12:00 A.M.. Plasma was obtained by immediate centrifugation (20 min at 2,300 x g at 15°C) of blood collected in tubes containing either 0.105 M sodium citrate (1:9 vol/vol; BD Vacutainer; Becton Dickinson, Le Pont-de-Claix, France) or 0.105 M sodium citrate and 20 mM benzamidine (final concentration). Samples were kept at –80°C until use.

For flow cytometry, monocytes were tested immediately after isolation from blood collected in cell preparation tubes (BD Vacutainer; Becton Dickinson) by centrifugation for 30 minutes at 1,500 x g at 20°C.

Total RNA was extracted from whole blood collected with the PAXgene Blood RNA System (Becton Dickinson/Qiagen, Courtabœuf, France).

Laboratory Methods
PC and PS activity were measured with clotting assays, using STA or STA compact analyzers (Diagnostica Stago, Inc., Asnières, France). Diagnostica Stago test kits for PC activity (Staclot Protein C) and PS activity (Staclot Protein S) were used.

Soluble TM (sTM) and soluble EPCR (sEPCR) were measured in plasma by using specific ELISA assays (Asserachrom TM and Asserachrom EPCR, respectively; Diagnostica Stago, Inc.).

APC
We used the APC assay described by Liaw and colleagues (6) with HAPC 1555, a monoclonal antibody specific for APC. Briefly, 96-well plates (Nunc Immuno Maxisorp; Polylabo, Strasbourg, France) were coated with 5 µg/ml HAPC 1555 in coating buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid], 150 mM NaCl, 5 mM CaCl₂) for 2 hours at 37°C. The plates were then blocked for 1 hour at 37°C with 200 µl of blocking buffer (coating buffer containing 1% bovine serum albumin). Before APC assay, the citrate/benzamidine-containing plasma samples were anticoagulated and recalcified by adding heparin, HEPES pH 7.5, and CaCl₂ at final concentrations of 2 U/ml, 20 mM, and 5 mM, respectively. Plasma samples were then transferred to coated microtiter plates and incubated at room temperature for 30 minutes. The wells were washed twice for 10 minutes and the chromogenic activity of bound APC was determined by adding 0.5 mM Spectrozyme APC (Chromogenix, Milan, Italy) in coating buffer. Substrate hydrolysis was monitored at 405 nm and 37°C in endpoint mode over time. A standard curve was constructed with increasing amounts of APC (0–100 ng/ml), purified and activated as previously described (17), in blocking buffer with 20 mM HEPES, pH 7.5, 2 U/ml heparin, and 20 mM benzamidine.

Quantitative Flow Cytometry
TM and EPCR expression on monocytes was quantified by means of quantitative flow cytometry with a calibrator kit (Platelet Calibrator; Biocytex, Marseille, France). The kit includes a mixture of four calibration beads coated with increasing concentrations of mouse IgG (360, 8,600, 29,000, and 90,000 molecules for the batch used throughout the study). Cells were stained with an indirect immunofluorescence technique with mouse IgG1 monoclonal antibodies (mAbs) against TM (kindly provided by Diagnostica Stago, Asnières, France), and against EPCR. All mAbs were used at saturating conditions (10 µg/ml final concentration), as determined in preliminary experiments with concentrations ranging from 1 to 20 µg/ml. An IgG1 negative isotype control was included in each series. The staining reagent was a polyclonal anti-mouse IgG–phycoerythrin antibody. A calibration curve was constructed for each sample series. A gate was applied to monocytes, defined as CD14-positive cells. Monocytes were stained with anti-CD14–fluorescein isothiocyanate (Immunotech, Marseille, France) in a direct immunofluorescence technique. Five thousand events were acquired on this gate on a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). Receptor numbers were derived from the calibration curve, after subtracting the negative isotype control value.

Real-Time Quantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from 2.5 ml of whole blood collected with the PAXgene Blood RNA System, and eluted in 30 µl of RNase-free water. Eight microliters of RNA solution was immediately used for cDNA synthesis as previously described (18), and the cDNA was stored at 20°C until use. The theoretical and practical aspects of quantitative reverse transcription–polymerase chain reaction are described elsewhere (18). The endogenous RNA control used here was the TATA box-binding protein (TBP gene) (Genbank accession no. NM_003194), and each test result was normalized to the sample's TBP content. Primers for TF, TM, EPCR, and TBP amplification (Table 1) were chosen with the assistance of Oligo 5.0 software (National Biosciences, Plymouth, MN). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons or in a different exon.

Statistical Analysis
Continuous data are expressed as medians and interquartile ranges and were compared by using the Mann-Whitney test. To detect correlations between continuous data, we used the Spearman correlation coefficient. Categorical data are shown as proportions and were compared with the ² test. We used StatView software (Abacus Concepts, Berkeley, CA) for all analyses, and differences with P values below 0.05 were considered significant.

	RESULTS

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Study Population
Between March 2003 and October 2003 we studied 30 adult critical care patients at the onset of SS (SS group), as well as 30 adult critical care patients with at least one acute OF not related to infection (OF group), and 30 healthy control subjects. The clinical charts of the patients with SS and OF were carefully reviewed by two intensivists not involved in the study for confirmation of the diagnosis and of inclusion criteria. None of the patients with OF had positive blood cultures at the time of the study. None of the patients with SS were receiving APC and only three patients with SS were receiving corticosteroids at the time of the study. The clinical characteristics of the study patients and the microbiological characteristics of the patients with SS are listed in Tables 2 to 4. Healthy control subjects were comparable to both patient groups in terms of age and sex (Table 2).

We also found that, among the 60 critical care patients, PC and PS activities were significantly lower in patients with acute respiratory distress syndrome (ARDS) versus non-ARDS patients (PC activity: 27% [10–57%] and 58% [49–87%], respectively; P = 0.0005; PS activity: 34% [20–43%] and 49% [30–57%], respectively; P = 0.045).

TM Modulation in SS and OF
sTM levels were significantly higher in patients with SS and those with OF (78 [43–174] and 86 [46–227] ng/ml, respectively) than in the healthy control subjects (43 [31–54] ng/ml; P = 0.0037 and 0.0010, respectively) (Table 5). No significant difference was found between the patients with SS and those with OF (P = 0.7394).

To determine if an increase in TM expression could compensate for membrane shedding of sTM, we measured TM mRNA levels in whole blood. mRNA levels were not increased in patients with SS when compared with healthy control subjects (0.54 [0.27–0.75] and 0.48 [0.35–0.62] AU, P = 0.6491). In contrast, TM mRNA levels were higher in patients with OF than in control subjects (0.87 [0.51–1.95] vs. 0.48 [0.35–0.62] AU, P = 0.0005) and in patients with SS (vs. 0.54 [0.27–0.75] AU, P = 0.0068). TM mRNA levels correlated positively with the neutrophil count (but not with the monocyte count) in both the SS group ( = 0.508, P = 0.0071) and the OF group ( = 0.535, P = 0.0055). The difference in TM mRNA expression between the two patient groups might have been due in part to the lower neutrophil counts in patients with SS (median: 8.95 [4.4–14.5] vs. 12.1 [7.2–18.2] x 10⁹ cells per liter).

The importance of circulating cells in the pathology of SS has been shown in animal models (19). To further explore the role of PC system receptors, and given the critical role of monocytes in the initiation of severe sepsis, we used flow cytometry to measure monocyte outer membrane expression of TM.

TM was similarly overexpressed on monocytes of both SS patients (816 [539–961] molecules per cell) and OF patients (779 [436–1212] molecules per cell) compared with control subjects (373 [189–765] molecules per cell; P = 0.0005 and 0.0008, respectively).

EPCR Levels
sEPCR levels were lower in patients with SS and those with OF (79 [56–123] and 81 [61–127] ng/ml, respectively) compared with healthy control subjects (119 [91–211] ng/ml; P = 0.0014 and 0.0039, respectively) (Table 5), but no significant difference was found between patients with SS and those with OF (P = 0.6788).

We found lower levels of EPCR mRNA in patients with SS (0.47 [0.34–0.86] AU) and those with OF (0.80 [0.62–1.05] AU) compared with healthy control subjects (1.26 [1.02–1.58] AU, P < 0.0001 for both comparisons). EPCR mRNA levels were significantly lower in the SS group than in the OF group (P = 0.0021).

EPCR expression on the monocyte surface was below the detection limit in all the subjects.

Circulating APC Levels Increase during SS and OF
The existence of a functional PC system is confirmed by APC production, and we thus measured circulating APC levels. APC levels in the healthy subjects ranged from 0.58 to 0.98 ng/ml (0.72 [IQR, 0.66–0.75] ng/ml) (Table 5). This normal range is lower than previously described (6), possibly due to the use of different APC standards.

Even though nine patients (five SS and four OF) received heparin (unfractionated heparin in five patients, low-molecular-weight heparin in four patients), which is known to lower PC activation and to accelerate APC inhibition through the PC inhibitor (PCI) (20), APC levels were significantly higher in patients with SS (0.79 [0.73–0.91] ng/ml) and patients with OF (0.85 [0.70–0.97] ng/ml) compared with healthy subjects (P = 0.0107 and 0.0078, respectively). The increase was similar in the two patient groups (P = 0.5497), reflecting activation of the PC system in both SS and OF.

Patients with SS and Those with OF Have a Similar Inflammatory Profile
Inflammation and coagulation interact in multiple ways. Inflammatory processes promote coagulation, whereas coagulation activation amplifies inflammation (21). We thus compared the inflammatory status of the two patient groups by first quantifying plasma IL-6. IL-6 levels were significantly higher in both the OF group (90.5 [47.7–216] ng/ml) and the SS group (336.5 [129–906] ng/ml) than in healthy subjects (2.6 [1.8–3.9] ng/ml, P < 0.0001 for both comparisons). However, this increase was more pronounced in septic patients (P = 0.0013). In both patient groups, we observed a correlation between elevated IL-6 and TF mRNA levels ( = 0.517, P = 0.0072 for the OF group, and = 0.458, P = 0.0154 for the SS group). Finally, we also found that, among the 60 critical care patients, patients with ARDS had significantly higher IL-6 levels than non-ARDS patients (633.5 [274–10,427] and 108.5 [53–293.5] ng/ml, respectively).

Biological Differences between Day 28 Survivors and Nonsurvivors
Among the main biological parameters, we found a significant difference between survivors and nonsurvivors in IL-6 (78.8 [47.7–170.0] and 375.0 [205.8–567.5] ng/ml, respectively; P < 0.0001), TF mRNA (2.10 [1.26–3.26] and 4.19 [1.96–13.27] AU, respectively; P = 0.0199), and APC (0.73 [0.62–0.86] and 0.85 [0.75–1.07] ng/ml, respectively; P = 0.0309) levels.

	DISCUSSION

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We compared the inflammatory status and the coagulation activation status, focusing on the PC system, in septic and nonseptic patients with OF. Despite a relatively limited number of patients and a monocentric design, we believe that our two patient groups can be viewed as roughly representative of these critical care patient populations. Specifically, we found that the characteristics of our patients with SS were similar to those reported in two recent large therapeutic studies (8, 22).

As expected, we found that patients with SS had increased TF mRNA levels in blood cells and markedly decreased circulating PC and PS levels (<50% of normal). The reductions in PS and PC levels in patients with SS were of the same order of magnitude in the PROWESS study (23). Importantly, we observed the same trends in nonseptic patients with acute OF. Expression of TF mRNA by blood cells was similarly increased in the two groups of patients, leading to a state of hypercoagulability. Similar PC pathway variations were also found in the two groups, with significantly reduced levels of PC and PS and increased levels of sTM. However, it should be noted that the diminution in PS activity was significantly more marked in patients with SS than in patients with OF, and accordingly that the difference in PC activity between the two groups was near-significant. Considering the number of patients in each group (30), one cannot formally exclude the possibility of a more pronounced activation of coagulation in patients with SS than in patients with OF. Interestingly, we found that septic and nonseptic patients with ARDS had much lower PC levels (and to a lesser extent, PS levels) than non-ARDS patients, illustrating the fact that the type of OF could be as important as the occurrence or not of sepsis. Patients with ARDS also had higher levels of IL-6, confirming that ARDS was associated with an overwhelming systemic inflammation.

Coagulation parameters have been extensively explored in SS, and PC system impairment is known to influence vital outcome (24). In contrast, the role of the PC system in nonseptic patients with acute OF has rarely been studied. Our nonseptic OF group mainly included patients with heart failure, heat stroke, COPD exacerbation, pulmonary embolism, and cardiac arrest, reflecting the overall recruitment of our medical intensive care unit. Interestingly, even if this group were relatively heterogeneous in terms of the underlying disorders, our results point to a homogenous response in terms of coagulation activation, underlining the role of the TF pathway in the pathophysiology of acute OF unrelated to infection; similar variations in the PC pathway were also observed. Proinflammatory cytokine release, endothelial activation, and diffuse microvascular thrombosis have been observed in heat stroke (10), together with PC system perturbations (25), as well as after successful cardiopulmonary resuscitation (12) and during acute exacerbations of COPD (11). Likewise, coagulation activation is increased in all these situations. The induction of TF expression by monocytes may be triggered by inflammatory cytokines produced during the systemic inflammatory response that accompanies OF (26). Indeed, we found increased IL-6 plasma levels during OF, although this increase was stronger in the septic patients. The similar inflammatory response during septic and nonseptic OF was also reflected by the similar leukocyte inflammatory gene expression profile in the two groups (see the online supplement). Among the 25 inflammation-related genes available for analysis, including several involved in cytokine and chemokine synthesis, no difference was found according to septic status. It thus seems that OF is associated with a similar inflammatory response, whatever the triggering event. Further interpretation of gene expression modulations would have been questionable because of the small size of the present study and in the absence of a kinetic study.

The similar coagulation activation and inflammatory responses seen in septic and nonseptic patients could suggest that similar treatments might be beneficial in both settings. Based on our observations, we wondered if the PC pathway might also be involved in the pathophysiology of nonseptic OF. The existence of a functional PC system was confirmed by experiments showing APC production. As previously reported (6), most of our patients with SS, despite endothelial alterations, were able to produce APC, as shown by a significant increase in APC levels. Again, we observed a similar increase in APC levels in nonseptic patients with acute OF, further underlining the similar PC anticoagulant pathway modifications in critical care patients with and without SS.

We also examined the role of TM and EPCR, two receptors required for PC activation. The decrease in sEPCR levels that we observed in both patient groups relative to healthy control subjects contrasts with previous reports showing an increase (27) or no modification (6, 28) in sEPCR levels during SS. Possible explanations for these discrepancies include the use of different ELISA systems, and the bimodal distribution of sEPCR levels in normal subjects. The latter is related to genetic factors, because healthy subjects and patients carrying the A3 haplotype have significantly higher levels than noncarriers (29). In our study, this bimodal distribution was found in the healthy control group but not in the patients with SS. A difference in the A3 haplotype distribution in the different studies and patient groups might also explain certain discrepancies. Genetic variability might also be involved in the different profiles of sEPCR responses in the context of SS. Nevertheless, we found that EPCR mRNA expression was down-regulated in blood cells of patients with SS. This is consistent with in vitro data showing that endothelial cell exposure to TNF- results in decreased EPCR mRNA levels, and that TNF- might participate in the low EPCR expression observed on endothelial surfaces during meningococcal sepsis (28). However, EPCR mRNA expression was up-regulated in mice challenged with LPS, an effect related to thrombin production (30). It is also conceivable that the regulation of EPCR expression is tissue specific.

As previously reported (23), we observed an increase in sTM levels in patients with SS, and a similar increase in patients with OF. These increased levels of sTM are difficult to interpret. They may reflect increased TM cleavage from the cell surface, leaving the endothelium devoid of intact TM and thus unable to promote PC activation. However, this shedding could be compensated for by an increase in cell surface expression of TM and subsequent release of large amounts of the soluble form without affecting the cell's potential for PC activation. Although EPCR was barely detectable and nonquantifiable in our patients, TM could be quantified on the monocyte surface. In normal subjects, TM expression was estimated to represent 373 molecules per monocyte. We observed at least a doubling of monocyte surface TM expression in both patient groups. A similar increase in TM expression on monocytes stimulated in vitro with LPS has been reported (31). This suggests that the regulation of TM expression is tissue specific, because down-regulation has been described on cultured endothelial cells exposed to endotoxin (31, 32), and in human skin biopsies stimulated with TNF- (33) or during meningococcal sepsis infection (28). TM overexpression on the monocyte surface may contribute to preserving the potential to generate circulating APC in patients with SS, but may also reflect the potential for extravascular monocytes to promote APC production locally. Despite the clear increase in TM expression on monocytes in patients with SS compared with healthy subjects, no corresponding increase in TM mRNA was detected. In a previous report, TM was only minimally expressed on resting neutrophils, with approximately 80% of the protein being intracytoplasmic (34). The existence of an intracytoplasmic pool in monocytes might explain the increase in TM expression on the cell surface without a corresponding increase in mRNA levels. Another possibility is the existence of a different regulatory mechanism in neutrophils and monocytes, with up-regulation of TM mRNA in neutrophils only.

Interestingly, we found significant differences in initial IL-6, TF mRNA, and APC levels between critical care survivors and nonsurvivors. The more pronounced increase in IL-6 and TF mRNA could be seen as the reflection of a more severe insult, leading to a worse prognosis. The difference in APC levels between survivors and nonsurvivors is more difficult to understand. In a previous series of critical care neutropenic patients, Mesters and colleagues reported a trend toward higher concentrations of APC in 13 septic shock patients (all deceased) as compared with 13 SS patients (all surviving) (35). However, such a relationship between APC levels and mortality was not found in another previous series of patients with SS (6). Such a difference might reflect a more pronounced compensatory mechanism in response to a more severe insult, but such an interpretation remains rather hypothetical.

The present study has several limitations. The fact that our biological study was monocentric and limited to 60 critical care patients precludes generalization to the whole spectrum of patients with OF. However, this design was dictated by the constraints of multiple specific measurements, and specifically by the need to immediately isolate monocytes from blood for quantitative flow cytometry. Ideally, confirmation studies on larger populations at multiple sites are warranted. Another limitation of the study, which is explained by the above-mentioned constraints, is that serial biological sampling was not performed. This point is important for the following reasons:

1. The time course of biological parameters could add further prognostic information to initial measurements, as was shown in other series of patients with SS (35, 36).
   
2. In the PROWESS study, there was no significant interaction between baseline biomarkers and treatment by APC (36). However, an effect of the treatment on the measurements of the same biomarkers during the period of infusion has clearly been demonstrated (36).
   
3. Although less likely, one cannot formally exclude that significant differences between patients with SS and those with OF could have occurred later in the time course of biological parameters.
   
4. Our study, performed at the onset of OF, does not allow us to draw any firm conclusions regarding the pathways activated during the initial insult, before the occurrence of OF.
   

As a result, it would be inexact to conclude from our data that the whole spectrum of illness is similar in patients with SS and patients with OF. Finally, the fact that blood was only collected within the same 4-hour period each day precludes analysis of cyclic variations throughout a 24-hour period. However, to our knowledge, such variations in thrombosis and hemostasis have only been described for fibrinolytic activity, and specifically for plasminogen activator inhibitor (PAI)-1 (37). Such variations has not been described for any of the biological markers measured in our study.

In conclusion, this study suggests that the pathogenesis of OF is characterized by an overwhelming systemic inflammatory response and by exacerbated coagulation activation, independent of the triggering event. Our extensive study of clotting factors confirms that the PC system is still operational in patients with SS, despite decreased PC and PS levels. As a consequence, this could suggest a potential benefit of PC treatment, instead of APC treatment, for patients with SS. However, such an approach can only be suggested and should be evaluated on a large-scale basis before any implementation in clinical practice. We also demonstrate, for the first time, that APC is also generated in nonseptic critical care patients, and that TM overexpression on the monocyte surface may participate in PC activation in septic and nonseptic patients. Finally, further studies will be necessary to determine if inflammation exacerbation in patients with nonseptic OF is followed by immunoparalysis, as described during sepsis.

	FOOTNOTES

 
Supported in part by a research grant from the Société de Réanimation de Langue Francaise, by the Leducq Fondation, and by INSERM.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200611-1692OC on August 2, 2007

Conflict of Interest Statement: D.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.H.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.D.-A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.T.E. has participated as a speaker in scientific meetings organized and financed in part by Lilly, Bayer, and Asahi. J.-Y.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.-L.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 23, 2006; accepted in final form July 31, 2007

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