The pathogenesis of systemic inflammatory response syndrome (SIRS) results from the increased release of pro-inflammatory cytokines and inflammatory mediators during an immunological response. Exacerbation of systemic inflammation often progresses to multiple organ failure (MOF) leading to a lethal outcome (1). Thus, SIRS is an important condition to address in critically ill patients. However, the pathogenesis of SIRS, such as that following trauma and sepsis, has not been thoroughly clarified.
Microparticles (MPs) are small membrane-bound vesicles that are released from various cells upon activation such as monocytes, platelets, erythrocytes, neutrophils, and endothelial cells. MPs play multiple roles in pathology and are important for maintaining homeostasis (2). MPs have been recognized as inflammatory mediators in various inflammatory diseases (3). We previously reported that endothelial and platelet MPs increase and could play an important role as inflammatory mediators in the pathogenesis of SIRS resulting from trauma or sepsis (4, 5). Immune cells such as monocytes have been shown to play important roles as immunological response cells in the development of SIRS (6, 7). The activated monocytes synthesize and express almost all of the tissue factor (TF) in the blood cells (8, 9), which manifests not only as a hypercoagulable but also as an inflammatory state (10). Expression of cell-specific surface antigens such as TF on MPs has recently become a focus of current research. Several reports have shown that TF on MPs has its own specific bioactivity, and TF-positive MPs are associated with both inflammation and coagulation (11–13).
CD13 is a cell-surface ectoenzyme expressed on several granulocytes but mainly monocytes (14, 15). TF, which is synthesized and expressed almost only on activated monocytes (13) and CD13 double-positive MPs (TF+/CD13+MPs), is predominantly produced from the activated monocytes. The objective of this study was to evaluate the practical importance of TF+/CD13+MPs in the pathogenesis of SIRS early after trauma or sepsis.
PATIENTS AND METHODS
This prospective study was conducted at the Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, from November 2012 to February 2015. Trauma patients with SIRS suffering from blunt or penetrating injury and patients with severe sepsis defined as SIRS combined with an infectious episode and dysfunction of at least one organ as adapted from the American College of Chest Physicians/Society of Critical Care Medicine consensus conference (16) and age greater than 18 years were included. Patients with burn injuries or who had surgery before consideration were excluded. Healthy volunteers with no previous history during the same period provided blood samples as controls. This study followed the principles of the Declaration of Helsinki and was approved by the institutional review board of Osaka University Hospital. Written informed consent was obtained from all participants or their close relatives.
Blood samples were collected from the trauma patients within 24 h after injury and from the severe sepsis patients within 24 h of the diagnosis to be used as initial biomarkers. TF+/CD13+MPs in the collection tubes containing trisodium citrate (Insepack II-ST; Kyokuto Pharmaceutical Industrial Co, Ltd, Tokyo, Japan) were evaluated by flow cytometry immediately after blood sampling. Serum and plasma samples for measuring concentrations of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), soluble TF (sTF), and plasminogen activator inhibitor-1 (PAI-1) were separated and stored at −40°C until the time of the assay.
Analyses of TF+/CD13+MPs
TF+/CD13+MPs in the blood were measured by flow cytometry (BD FACSCanto II; BD Biosciences, San Jose, Calif) and counted by the method we recently reported (17) but with some modifications. In brief, anti-CD146 antibody was changed to anti-CD13 antibody, and the anti-CD141 and anti-CD201 antibodies were removed from the protocol. Blood samples were centrifuged for 5 min at 1,800 × g to remove residual cell debris. Platelet-poor plasma was used for the analysis as previously reported (18, 19). The cell-free supernatant fluid was removed, and the platelet-poor plasma was obtained using a Trucount tube (BD Biosciences) in which 50 μL of platelet-poor plasma and 50 μL of a 5-fold dilution of Binding Buffer (BD Biosciences) and 20 μL of heparin (Novo-Heparin, 5,000 units/5 mL for injection; Mochida Pharmaceutical Co, Tokyo, Japan) were mixed. Then, 2.5 μL each of annexin V, anti-CD13 antibody, and anti-CD142 (TF) antibody (all from BD Biosciences) were added and vortexed. Subsequently, the sample solution was incubated at room temperature for 30 min in the dark. The antigen–antibody reactions were suppressed with the addition of 500 μL of a 10-fold dilution of Binding Buffer (BD Biosciences). Before the analysis, the flow cytometer was calibrated with the use of BD Cytometer Setup and Tracking Beads (BD Biosciences). Measurement of MP diameter was calibrated using reference standard beads (2.00-μm-diameter Fluoresbrite YG carboxylate microspheres; Polysciences Inc, Warrington, Pa). Measurement of the numbers of TF+/CD13+MPs was determined with an automated cell counter (XN9000; Sysmex Corporation, Kobe, Japan) for 5 min.
TF+/CD13+MPs were defined as events detected by annexin V+/CD13+/CD142+ with a diameter of 0.1 to 2.0 μm based on previous reports (20, 21). High-intensity signals caused by antibody aggregation were excluded from the flow cytometry analysis. The platelet counts of the platelet-poor plasma were very low (less than measurement sensitivity, 5 × 103/μL), and the platelet fraction was completely removed with the use of anti-CD13 antibody and anti-CD142 antibody, which do not express on platelets. The formation of MPs was evaluated as the number of TF+/CD13+MPs (Fig. 1).
Analyses of serum levels of TNF-α and IL-6 and plasma levels of sTF and PAI-1
The serum concentrations of TNF-α and IL-6 and plasma concentrations of sTF and PAI-1 were measured with an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, Minn). Frozen samples were thawed and then processed according to the manufacturer's instructions. Absorbance was measured with a microplate reader (SH-9000Lab; Corona Electric Co, Ltd, Ibaraki, Japan). The minimum detectable dose was less than 0.5 pg/mL for TNF-α, 0.70 pg/mL for IL-6, 0.16 pg/mL for sTF, and 0.014 ng/mL for PAI-1.
Definitions of severity and diagnosis of disseminated intravascular coagulation
The Injury Severity Score (ISS) was assessed in trauma patients, and the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the Sequential Organ Failure Assessment (SOFA) score were assessed in both the trauma and severe sepsis patients at the time of enrollment. ISS is the foremost test and gold standard for assessing injury severity. ISS was derived from the Abbreviated Injury Scale score, which was assigned for injuries such as head, face, chest, abdomen, and extremities (including pelvis) (22). The APACHE II score assesses the severity of illness in critically ill patients based on routine physiologic measurements, age, and previous health status and is used to predict the outcome of the critical illness (23). The SOFA score allows calculation of the dysfunction of six organ systems, comprising the respiratory, coagulation, hepatic, cardiovascular, renal and neurologic systems, and the severity of the dysfunction (24).
As defined by the International Society of Thrombosis and Haemostasis (ISTH), disseminated intravascular coagulation (DIC) is a clinical condition characterized by systemic coagulation disorder with loss of localization arising from different causes. It can cause damage to the microvasculature and may produce organ dysfunction (25). In this study, the ISTH overt DIC diagnostic algorithm was used because it has high specificity to ensure an accurate diagnosis of DIC (26). The level of fibrinogen degradation products was used for fibrin-related markers in the ISTH overt DIC criteria and was defined as no increase (0–9 mg/L), moderate increase (10–24 mg/L), or strong increase (>25 mg/L) (27).
Continuous variables are expressed as the group median with interquartile range. Steel test was used to assess multiple comparisons using the post hoc Wilcoxon signed-rank test with Bonferroni correction. Correlations between TF+/CD13+MPs and other markers were evaluated with scatter plots and Spearman rank-correlation coefficients. A receiver operating characteristic (ROC) curve was used to evaluate the usefulness of TF+/CD13+MPs as a diagnostic biomarker in critical patients. A value of P <0.05 was considered to indicate statistical significance. Statistical analyses were performed with JMP 11.1.1 for Windows (SAS Institute Inc, Cary, NC).
During the study period, 24 trauma patients (47.0 [37.0–63.0] years), 25 severe sepsis patients (69 [58.0–78.0] years), and 23 healthy controls (65.0 [36.0–73.0] years) were included (Table 1). The trauma patients comprised 19 men and 5 women. ISS and the APACHE II and SOFA scores of the trauma patients were 25.0 (16.3–32.8), 11.0 (5.0–15.0), and 2.5 (2.0–4.0), respectively. The causes of trauma were motor vehicle collision (n = 12), fall (n = 11), and penetrating wound (n = 1). The locations of injury were the head (n = 7), face (n = 3), neck (n = 3), thorax (n = 9), abdomen (n = 4), and extremities (n = 7). The severe sepsis patients comprised 15 men and 10 women. The APACHE II, SOFA, and ISTH DIC scores of the sepsis patients were 20.0 (15.0–24.0), 7.0 (3.0–11.0), and 3.0 (2.0–4.0), respectively. The sources of severe sepsis were chest (n = 11), abdomen (n = 3), soft tissue (n = 3), urinary (n = 6), and others (n = 2). None of patients with trauma died. Four patients with severe sepsis died from MOF. The cause of severe sepsis was bacterial infection in all cases. Fifteen gram-positive and 12 gram-negative bacteria were isolated, including 2 mixed bacterial infections. Three positive blood cultures and 24 other site cultures (sputum, pleural fluid, ascites, urine, pus) were identified. There were no significant differences between the trauma patients, severe sepsis patients, and healthy controls in regard to age and sex.
Numbers of TF+/CD13+MPs
In the overall analysis of the study participants, the numbers of TF+/CD13+MPs were significantly increased in the trauma patients compared with those in the healthy controls (Fig. 2). Similarly, there was a significant difference between the severe sepsis patients and the healthy controls (Fig. 2).
Correlations between TF+/CD13+MPs and severity of illness in the trauma and severe sepsis patients
We assessed the correlation between the number of TF+/CD13+MPs and the disease severity scoring systems (ISS, APACHE II, and SOFA) in the trauma and severe sepsis patients. In the trauma patients, significant correlations were found between the number of TF+/CD13+MPs and the ISS and the APACHE II score, but no significant correlations were found between the number of TF+/CD13+MPs and the SOFA score (Fig. 3, A–C). In the severe sepsis patients, significant correlations were found between the number of TF+/CD13+MPs and the APACHE II score, but no significant correlations were found between the number of TF+/CD13+MPs and the SOFA score (Fig. 4, A and B).
Correlations between TF+/CD13+MPs and ISTH DIC score in the severe sepsis patients
To investigate the association between the number of TF+/CD13+MPs and systemic coagulant activity, the ISTH DIC score was evaluated in the severe sepsis patients. In these patients, the numbers of TF+/CD13+MPs increased significantly with the increase of the ISTH DIC score (Fig. 4C).
Correlations between TF+/CD13+MPs and biochemical parameters in the trauma and severe sepsis patients
To assess the association between the number of TF+/CD13+MPs and systemic inflammatory markers, the levels of TNF-α and IL6 were measured. Significant correlations were found between the number of TF+/CD13+MPs and IL6 levels (Figs. 3 and 4) but not between the number of TF+/CD13+MPs and TNF-α levels (P = 0.766, rho = 0.064; P = 0.361, rho = 0.195; respectively) in both the trauma and severe sepsis patients. To study the relation between the number of TF+/CD13+MPs and coagulative factor, the levels of sTF were measured. There were no significant correlations between the number of TF+/CD13+MPs and sTF (P = 0.102, rho = −0.342; P = 0.991, rho = −0.002; respectively) in both the trauma and severe sepsis patients. To assess the systemic endothelial damage, the levels of PAI-1 were evaluated. The number of TF+/CD13+MPs showed a tendency to correlate with PAI-1 levels in the trauma patients (Fig. 3), but there was no statistically significant correlation between the number of TF+/CD13+MPs and PAI-1 levels in the severe sepsis patients (Fig. 4).
Cut-off values of TF+/CD13+MPs for the diagnosis of critical patients with trauma and severe sepsis
The patients were divided into the following two groups on the basis of expected mortality: critical patients with trauma (ISS ≥25) and non-critical patients (ISS ≤24) (28), and critical patients with severe sepsis (APACHE II score ≥25, SOFA score ≥12) and non-critical patients (APACHE II score ≤24, SOFA score ≤11) (29, 30). In the model using ISS to diagnose the critical patients with trauma, the cut-off value of TF+/CD13+MPs was 9245/mL blood with an area under the curve (AUC) of 0.82. In the model using the APACHE II and SOFA scores to diagnose critical patients with severe sepsis, the cut-off values of TF+/CD13+MPs were 9245/mL blood with an AUC of 0.73 and 11,916/mL blood with an AUC of 0.83, respectively (Table 2).
This is the first study, to our knowledge, to evaluate the association between TF+/CD13+MPs and the pathogenesis of early SIRS following trauma and sepsis. The aim of this study was to investigate the usefulness of TF+/CD13+MPs as a biomarker for inflammation and severity in the early pathogenesis of SIRS following trauma and sepsis. The numbers of TF+/CD13+MPs significantly increased in both trauma and severe sepsis. This suggests that activated monocytes upregulate the expression of TF and produce TF+/CD13+MPs in the acute phase of trauma and severe sepsis.
In this study, the numbers of TF+/CD13+MPs significantly correlated with ISS in the trauma patients. ISS is a quantitative measure of anatomic tissue injury severity that provides an overall score for patients with multiple injuries. Previously, Park et al. (31) showed that annexin V-positive MPs correlated significantly with ISS in trauma patients, suggesting that there might be an association between MPs and tissue injury severity. Tissue injury leads to the release of damage-associated molecular patterns (DAMPs). Levels of DAMPs are related to the extent of tissue injury (32). Pattern recognition receptors such as Toll-like receptors on immune cells including monocytes respond to DAMPs and activate the immune cells. Our data suggest that monocyte activation is enhanced in accordance with the severity of tissue injury, leading to the expression of TF and production of TF+/CD13+MPs in the acute phase of trauma.
Our data demonstrated that the numbers of TF+/CD13+MPs significantly correlated with the levels of IL-6, a systemic inflammatory marker, and the APACHE II score, which reflects physiological severity in trauma patients. The numbers of TF+/CD13+MPs also significantly correlated with the IL-6 levels and APACHE II score in severe sepsis patients. These findings indicate that activated monocytes upregulate the expression of TF and promote the generation of TF+/CD13+MPs, thus causing the acute systemic inflammation and exacerbating the physiological pathogenesis in the acute phase of trauma and severe sepsis.
In our study, the numbers of TF+/CD13+MPs correlated significantly with the ISTH DIC score in the severe sepsis patients. Sepsis-induced DIC is basically triggered by the release of blood-borne TF, which is present in three forms referred to as MP-borne TF, sTF, and cell-borne TF (33). It was reported that TF dependent on MPs shows significantly higher procoagulant activity than TF independent of MPs in vitro(11). Furthermore, we recently reported that TF-positive endothelial MPs are associated with the pathogenesis of sepsis-induced DIC (17). Therefore, our results suggest that the excessive production of TF+/CD13+MPs might cause harmful microthrombi leading to the progression of sepsis-induced DIC.
Our study clearly demonstrated that TF+/CD13+MPs correlated with severity in the acute phase of trauma and severe sepsis in the study patients. Both the ROC analysis using the ISS for critical patients with trauma and the SOFA score for critical patients with severe sepsis showed high diagnostic values (AUC >0.8). This suggests that the measurement of TF+/CD13+MPs might be useful as a biomarker for the assessment of disease severity in the pathogenesis of early SIRS following trauma and severe sepsis. Interestingly, recent articles reported CD13 to be a specific adhesion molecule involved in monocyte-endothelial cell adhesion, suggesting that TF+/CD13+MPs could be used to evaluate monocyte-endothelial cell adhesion in the early phase of SIRS following trauma and sepsis. CD13 is also expressed on renal tubular cells (34), indicating that increased numbers of TF+/CD13+MPs might be used as a biomarker of acute renal injury in the early phase of SIRS following trauma and sepsis.
Because the inclusion criteria for severe sepsis in this study are different from the latest criteria defined by Sepsis-3 (35), we reanalyzed our data using the Sepsis-3 criteria. The results showed almost the same tendency as those of this study using the previous criteria (see Supplemental Digital Contents Table 1, http://links.lww.com/SHK/A487, which shows patient characteristics), (see Supplemental Digital Content Fig. 1, http://links.lww.com/SHK/A488, which shows the numbers of TF+/CD13+MPs), (see Supplemental Digital Content Fig. 2, http://links.lww.com/SHK/A489, which shows the correlations between TF+/CD13+MPs and severities and biochemical parameters) (see Supplemental Digital Contents Table 2, http://links.lww.com/SHK/A487, which shows cut-off values of TF+/CD13+MPs for the diagnosis of critical patients).
As limitations of this study, the numbers of patients and controls were relatively small, and the data were collected at a single institution. Also, because the mortality rates were low (0% and 16% in the trauma and severe sepsis patients, respectively), prognostic factors could not be analyzed in this study. Second, we evaluated the change in TF+/CD13+MPs at only one time point, and there was no follow-up evaluation to assess the chronological changes of TF+/CD13+MPs. Third, the specific bioactivity of TF+/CD13+MPs was not elucidated in this study. Thus, further studies are needed to assess the association between TF+/CD13+MPs and the pathogenesis of trauma and severe sepsis.
This study is the first report to show that increased numbers of TF+/CD13+MPs were significantly correlated with disease severity in the acute phase of SIRS in patients with trauma or severe sepsis. Our findings suggest that the evaluation of the number of TF+/CD13+MPs might be useful as a biomarker for severity in patients in the early phase of SIRS following trauma and sepsis.
The authors thank Tomomi Yamada (Department of Clinical Epidemiology and Biostatistics, Osaka University Graduate School of Medicine) for support with the statistical analysis. The authors are grateful to the patients and their families for their trust, and to the healthy controls.
1. Namas R, Ghuma A, Hermus L, Zamora R, Okonkwo DO, Billiar TR, Vodovotz Y. The acute inflammatory response in trauma /hemorrhage and traumatic brain injury: current state and emerging prospects. Libyan J Med
2009; 4 3:97–103.
2. Barteneva NS, Fasler-Kan E, Bernimoulin M, Stern JN, Ponomarev ED, Duckett L, Vorobjev IA. Circulating microparticles: square the circle. BMC Cell Biol
3. Reid V, Webster NR. Role of microparticles in sepsis. Br J Anaesth
2012; 109 4:503–513.
4. Ogura H, Kawasaki T, Tanaka H, Koh T, Tanaka R, Ozeki Y, Hosotsubo H, Kuwagata Y, Shimazu T, Sugimoto H. Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. J Trauma
2001; 50 5:801–809.
5. Ogura H, Tanaka H, Koh T, Fujita K, Fujimi S, Nakamori Y, Hosotsubo H, Kuwagata Y, Shimazu T, Sugimoto H. Enhanced production of endothelial microparticles with increased binding to leukocytes in patients with severe systemic inflammatory response syndrome. J Trauma
2004; 56 4:823–830.
6. Lenz A, Franklin GA, Cheadle WG. Systemic inflammation after trauma. Injury
2007; 38 12:1336–1345.
7. Jaffer U, Wade RG, Gourlay T. Cytokines in the systemic inflammatory response syndrome: a review. HSR Proc Intensive Care Cardiovasc Anesth
2010; 2 3:161–175.
8. Egorina EM, Sovershaev MA, Hansen JB. The role of tissue factor in systemic inflammatory response syndrome. Blood Coagul Fibrinolysis
2011; 22 6:451–456.
9. Østerud B. Tissue factor expression in blood cells. Thromb Res
2010; 125 (suppl 1):S31–S34.
10. Chu AJ. Tissue factor mediates inflammation. Arch Biochem Biophys
2005; 440 2:123–132.
11. Key NS. Analysis of tissue factor positive microparticles. Thromb Res
2010; 125 (suppl 1):S42–S45.
12. Woei-A-Jin FJ, De Kruif MD, Garcia Rodriguez P, Osanto S, Bertina RM. Microparticles expressing tissue factor are concurrently released with markers of inflammation and coagulation during human endotoxemia. J Thromb Haemost
2012; 10 6:1185–1188.
13. Zwicker JI, Trenor CC 3rd, Furie BC, Furie B. Tissue factor-bearing microparticles and thrombus formation. Arterioscler Thromb Vasc Biol
2011; 31 4:728–733.
14. Look AT, Ashmun RA, Shapiro LH, Peiper SC. Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J Clin Invest
1989; 83 4:1299–1307.
15. Ashmun RA, Look AT. Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells. Blood
1990; 75 2:462–469.
16. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G. SCCM/ESICM/ACCP/ATS/SIS. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med
2003; 31 4:1250–1256.
17. Matsumoto H, Yamakawa K, Ogura H, Koh T, Matsumoto N, Shimazu T. Enhanced expression of cell-specific surface antigens on endothelial microparticles in sepsis-induced disseminated intravascular coagulation. Shock
2015; 43 5:443–449.
18. Takeshita J, Mohler ER, Krishnamoorthy P, Moore J, Rogers WT, Zhang L, Gelfand JM, Mehta NN. Endothelial cell-, platelet-, and monocyte/macrophage-derived microparticles are elevated in psoriasis beyond cardiometabolic risk factors. J Am Heart Assoc
2014; 3 1:e000507.
19. Windeløv NA, Johansson PI, Sørensen AM, Perner A, Wanscher M, Larsen CF, Ostrowski SR, Rasmussen LS. Low level of procoagulant platelet microparticles is associated with impaired coagulation and transfusion requirements in trauma patients. J Trauma Acute Care Surg
2014; 77 5:692–700.
20. Aras O, Shet A, Bach RR, Hysjulien JL, Slungaard A, Hebbel RP, Escolar G, Jilma B, Key NS. Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia. Blood
2004; 103 12:4545–4553.
21. Puddu P, Puddu GM, Cravero E, Muscari S, Muscari A. The involvement of circulating microparticles in inflammation, coagulation and cardiovascular diseases. Can J Cardiol
2010; 26 4:140–145.
22. Baker SP, O’Neill B, Haddon W Jr, Long WB. The injury severity score: a method for describing patients with multiple injuries and evaluating emergency care. J Trauma
1974; 14 3:187–196.
23. Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit Care Med
1985; 13 10:818–829.
24. Janssens U, Dujardin R, Graf J, Lepper W, Ortlepp J, Merx M, Zarse M, Reffelmann T, Hanrath P. Value of SOFA (Sequential Organ Failure Assessment) score and total maximum SOFA score in 812 patients with acute cardiovascular disorders. Critical Care
2001; 5 (suppl 1):225.
25. Taylor FB Jr, Toh CH, Hoots WK, Wada H, Levi M. Scientific Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis, Haemostasis (ISTH). Towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost
2001; 86 5:1327–1330.
26. Takemitsu T, Wada H, Hatada T, Ohmori Y, Ishikura K, Takeda T, Sugiyama T, Yamada N, Maruyama K, Katayama N, et al. Prospective evaluation of three different diagnostic criteria for disseminated intravascular coagulation. Thromb Haemost
2011; 105 1:40–44.
27. Gando S, Saitoh D, Ogura H, Mayumi T, Koseki K, Ikeda T, Ishikura H, Iba T, Ueyama M, Eguchi Y, et al. Japanese Association for Acute Medicine Disseminated Intravascular Coagulation (JAAM DIC) Study Group. Disseminated intravascular coagulation (DIC) diagnosed based on the Japanese Association for Acute Medicine criteria is a dependent continuum to overt DIC in patients with sepsis. Thromb Res
2009; 123 5:715–718.
28. Hwabejire JO, Kaafarani HM, Lee J, Yeh DD, Fagenholz P, King DR, de Moya MA, Velmahos GC. Patterns of injury, outcomes, and predictors of in-hospital and 1-year mortality in nonagenarian and centenarian trauma patients. JAMA Surg
2014; 149 10:1054–1059.
29. Ferreira FL, Bota DP, Bross A, Melot C, Vincent JL. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA
2001; 286 14:1754–1758.
30. Kaukonen KM, Bailey M, Suzuki S, Pilcher D, Bellomo R. Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand, 2000-2012. JAMA
2014; 311 13:1308–1316.
31. Park MS, Owen BA, Ballinger BA, Sarr MG, Schiller HJ, Zietlow SP, Jenkins DH, Ereth MH, Owen WG, Heit JA. Quantification of hypercoagulable state after blunt trauma: microparticle and thrombin generation are increased relative to injury severity, while standard markers are not. Surgery
2012; 151 6:831–836.
32. Cohen MJ, Brohi K, Calfee CS, Rahn P, Chesebro BB, Christiaans SC, Carles M, Howard M, Pittet JF. Early release of high mobility group box nuclear protein 1 after severe trauma in humans: role of injury severity and tissue hypoperfusion. Crit Care
2009; 13 6:R174.
33. Steffel J, Lüscher TF, Tanner FC. Tissue factor in cardiovascular diseases: molecular mechanisms and clinical implications. Circulation
2006; 113 5:722–731.
34. Riemann D, Kehlen A, Langner J. Stimulation of the expression and the enzyme activity of aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 on human renal cell carcinoma cells and renal tubular epithelial cells by T cell-derived cytokines, such as IL-4 and IL-13. Clin Exp Immunol
1995; 100 2:277–283.
35. Shankar-Hari M, Phillips GS, Levy ML, Seymour CW, Liu VX, Deutschman CS, Angus DC, Rubenfeld GD, Singer M. Sepsis Definitions Task Force. Developing a new definition and assessing new clinical criteria for septic shock: for the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA
2016; 315 8:775–787.