ORIGINAL ARTICLE
Systemic matrix metalloproteinase-8 and tissue inhibitor of metalloproteinases-1 levels in severe sepsis-associated coagulopathy M. Sivula1, J. Hästbacka1, A. Kuitunen2, R. Lassila3, T. Tervahartiala4, T. Sorsa4,5 and V. Pettilä1 1
Intensive Care Units, Division of Anaesthesia and Intensive Care Medicine, Helsinki University Central Hospital, Helsinki, Finland Intensive Care Unit, Division of Anaesthesia and Intensive Care Medicine, Tampere University Central Hospital, Tampere, Finland 3 Coagulation Disorders Unit, Department of Haematology, Helsinki University Central Hospital, Helsinki, Finland 4 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland 5 Division of Periodontology, Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden 2
Correspondence M. Sivula, Intensive Care Units, Jorvi Hospital, Helsinki University Central Hospital, PO Box 800, Espoo, HUS 00029, Finland E-mail: mirka.sivula@hus.fi Conflicts of interest The authors confirm that there are no conflicts of interest. Submitted 16 July 2014; accepted 11 September 2014; submission 9 September 2014. Citation Sivula M, Hästbacka J, Kuitunen A, Lassila R, Tervahartiala T, Sorsa T, Pettilä V. Systemic matrix metalloproteinase-8 and tissue inhibitor of metalloproteinases-1 levels in severe sepsis-associated coagulopathy. Acta Anaesthesiologica Scandinavica 2014 doi: 10.1111/aas.12423
Background: Matrix metalloproteinase-8 (MMP-8) and tissue inhibitor of metalloproteinases-1 (TIMP-1) have recently been suggested to be involved in coagulation process. Our objectives were to observe systemic MMP-8 and TIMP-1 levels in patients with severe sepsis with or without disseminated intravascular coagulation (DIC) and to study their relationship with coagulation markers over time. Methods: Our prospective pilot study included 22 patients with severe sepsis, nine (41%) of whom had overt DIC. We analysed MMP-8 and TIMP-1 serum concentrations by time-resolved immunofluorometric and enzyme-linked immunosorbent assays, respectively, on days 1, 2, 4 and 7 after the intensive care unit admission. Traditional coagulation tests were taken at the same time points. The results were compared between patients with and without DIC. Blood samples from 10 healthy volunteers were used to demonstrate normal levels. Results: Both patient groups had elevated levels of MMP-8 and TIMP-1 as compared with healthy controls. TIMP-1 concentration was almost twofold in DIC patients compared with those without DIC on the first 2 days. MMP-8 was elevated only on day 2. TIMP-1 correlated positively with the severity of coagulation disturbance and with disease severity scores. MMP-8 correlated negatively only with platelet count. Conclusion: In this first human study, we could show that TIMP-1 is elevated in the early phase of sepsis-induced overt DIC, and it correlates both with degree of coagulopathy and disease severity. These findings suggest that TIMP-1 may play a role in the pathogenesis of DIC in septic patients.
Severe sepsis and septic shock result from excessive activation of inflammatory response to infectious stimuli, leading to endothelial dysfunction, coagulation activity and haemostatic imbalance, circulatory shock, and ultimately
multi-organ dysfunction.1 More than 30% of patients with severe sepsis develop overt disseminated intravascular coagulation (DIC) over the first few days of severe sepsis.2 DIC is characterised by the activation of coagulation trigActa Anaesthesiologica Scandinavica 59 (2015) 176–184
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gered by exposure of excessive tissue factor (TF), which leads to a widespread intravascular fibrin deposition and clot formation. In DIC, natural anticoagulant system is downgraded due to impaired synthesis, degradation and increased consumption of tissue factor pathway inhibitor (TFPI), activated protein C (aPC) and antithrombin (AT). Fibrinolysis is also impaired partly because of increase in fibrinolysis inhibitors.3 DIC may compromise the circulation of organ systems, thus contributing to the development of multi-organ failure.4 Overt DIC has been shown to roughly double the mortality of severe sepsis patients.2 Recently, increasing interest has been focused on matrix metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitor of metalloproteinases, TIMPs). MMPs form a structurally related group of zinc-dependent proteolytic enzymes that, in addition to their first recognised function, degradation of extracellular matrix (ECM), also cleave cellular surface proteins and other non-matrix substrates. MMPs have been linked to coagulation in many chronic and acute inflammatory states in complex ways.5–11 In clinical studies, high levels of MMP-8 and TIMP-1 have been shown to associate with higher mortality among severe sepsis patients.12–15 Recently, in a murine sepsis model, TIMP-1 was shown to increase in the early phase of septic DIC.16 Although MMP-8 has not been linked to septic coagulation disorders so far, it has been shown to degrade TFPI,9 which can be crucial in DIC. Accordingly, we hypothesised that MMP-8 and TIMP-1 levels may associate with severe sepsisassociated DIC. We aimed to investigate the time courses of MMP-8 and TIMP-1 in this patient group, and evaluate their association with established coagulation parameters and markers of disease severity.
Methods We performed this prospective pilot study in a tertiary multidisciplinary intensive care unit (ICU) of Helsinki University Central Hospital between January 2002 and November 2003. The ethics committee of Helsinki University Central Hospital Department of Surgery approved the
study protocol (Protocol Number HUS 171/E6/01, 2001-03-30). All patients admitted to the ICU were screened for severe sepsis or septic shock. We included 22 adult patients with criteria of severe sepsis or septic shock fulfilled within 48 h prior to admission to the ICU, and obtained informed consent from all the participating patients or their next of kin. The diagnosis of severe sepsis was based on the following criteria: a confirmed or suspected infection and at least two of the four systemic inflammatory response syndrome criteria [body temperature > 38°C or < 36°C, heart rate > 90/min, respiratory rate > 20/min or arterial partial pressure of carbon dioxide < 32 mmHg or need for mechanical ventilation, and white blood cell (WBC) count > 12,000 cells/μl or < 4000/μl],17 and organ dysfunction and hypoperfusion or hypotension after adequate fluid resuscitation. Exclusion criteria were pharmacological anticoagulant therapy within 1 week prior to the admission, pregnancy, haematological malignancy and chronic liver disease. Serum samples for MMP-8 and TIMP-1 control levels were obtained from 10 healthy adults once. We recorded routine clinical data and severity scores, including the Acute Physiology and Chronic Health Evaluation II and daily Sequential Organ Failure Assessment (SOFA) scores, use of blood components, low molecular weight heparins (LMWHs) and corticosteroids in the computerised ICU database. We calculated SOFA scores with and without points given for platelet count to assess the association with other organ failures. The patients received subcutaneous LMWH with a fixed dose once or twice a day routinely unless clinically contraindicated. A written treatment protocol of the ICU guided blood component transfusions. We calculated the International Society on Thrombosis and Haemostasis (ISTH) score for overt DIC daily using lowest blood platelet count, plasma prothrombin time (PT) ratio, fibrinogen concentration and highest plasma D-dimer as described earlier.18 Patients with a score of 5 or higher on any day had diagnosis of overt DIC. We took serum samples for MMP-8 and TIMP-1 analyses on admission (day 1) and on days 2, 4 and 7. We also collected blood samples for the coagulation tests, WBC count and C-reactive
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protein (CRP) on admission, and then daily in the morning until day 7 simultaneously with MMP-8 and TIMP-1 samples, and if clinically indicated. All samples were drawn from non-heparinised arterial lines after discarding the first 5 ml of blood. WBC and CRP were measured by the routine hospital laboratory. Serum samples for MMP-8 and TIMP-1 analyses were centrifuged at 2000g for 10 min, and the supernatants were stored at −80°C until analysis, which we performed in May 2006. We analysed MMP-8 and TIMP-1 concentrations in random order, blinded to the severity of coagulation disorder. We measured MMP-8 serum concentration by time-resolved immunofluorometric assay as described previously19,20 by using monoclonal MMP-8 specific antibodies 8708 and 8706 (Medix Biochemica, Kauniainen, Finland) for catching and tracing, respectively. We used europium chelate for labelling the tracer antibody. We carried out TIMP-1 analysis by using commercially available enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Biotrak ELISA System; Amersham Biosciences, Buckinghamshire, UK). We report the concentrations as ng/ml. Detection limits for MMP-8 and TIMP-1 are 0.032 ng/ml and 1.25 ng/ml, respectively. The inter-assay coefficient of variation (CV%) for MMP-8 and TIMP-1 are 4.1 and 13.5 (n = 12) and intra-assay CV% 2.5 and 10.1 (n = 12), respectively. We used citrate tubes with final concentration of 3.8% for the coagulation plasma samples and ethylenediaminetetraacetic acid tubes for the haematological test samples. The analytical methods were Nycotest PT (Owren-buffered, Axis-Shield PoC AS, Oslo, Norway) for PT, immunoturbidimetric assay Tina-quant® (Roche Diagnostics, Mannheim, Germany) for D-dimer concentration, Multifibren U (Siemens Healthcare Diagnostics, Erlangen, Germany) for fibrinogen, chromogenic Berichrom® Antithrombin III (A) (Siemens Healthcare Diagnostics) for AT activity, APTT Actin FSL® (Siemens Healthcare Diagnostics) for activated partial thromboplastin time (APTT), Enzygnost® TAT Micro (Siemens Healthcare Diagnostics) for thrombin-antithrombin complex (TAT), Enzygnost® F1 + 2 (monoclonal) (Siemens Healthcare Diagnostics) for prothrombin fragment 1 + 2 (F1 + 2), Berichrom® Protein C (Siemens Healthcare Diagnostics) for protein C
(PC), Pathromtin SL (Siemens Healthcare Diagnostics) and factor VIII deficient plasma for factor VIII activity (FVIII:C), and blood cell counter with direct current detection method for blood platelet count. Due to lack of previous studies on MMP-8 and TIMP concentrations and their distributions in patients with overt DIC, we could not perform sample size calculations for this pilot study. As the prevalence of overt DIC is approximately 30% in patients with severe sepsis in our ICU based on data collected for our previous study,18 the sample size for overt DIC group was targeted to exceed six, and thus the whole sample of at least 20 patients. We present all data as number of patients with percentage or median with interquartile range (IQR) or 95% confidence interval. According to Shapiro–Wilk test, the distribution of the data was non-normal, and we chose non-parametric tests for statistical analyses. We compared the differences between groups in categorical variables using two-tailed Fisher’s exact test, and in continuous data in two or several groups using Mann– Whitney U-test and Kruskal–Wallis test, respectively. We assessed differences in continuous parameters MMP-8 and TIMP-1 between time points with Friedman’s ANOVA, and report the results with chi-square (Χ2) with degrees of freedom (df) and P value. We calculated nonparametric Spearman’s rank correlation coefficient with two-tailed significance for MMP-8, TIMP-1 and blood coagulation parameters on days 1 and 2, and for MMP-8, TIMP-1 and disease severity scores and inflammation parameters on days 1, 2, 4 and 7. We considered differences between groups statistically significant if P value was less than 0.05, and we corrected P value for multiple comparisons as needed. In correlation analyses, we considered P value less than 0.01 statistically significant. We performed statistical analyses with IBM® SPSS® Statistics 20.0 for Mac OS (IBM Corp., Armonk, NY, USA). Results Twenty-two patients with severe sepsis or septic shock were included in the study. Demographic data are summarised in Table 1. None of the patients had used known MMP inhibitors, tetracyclines or bisphosphonates21 prior to admission to the ICU. Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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Table 1 Characteristics of the study groups. All patients n = 22 Sex (male/female) Age Positive blood culture Pathogens Streptococcus pneumoniae Streptococcus pyogenes Miscellaneous APACHE II SOFA day1 Mortality on day 28
Severe sepsis without overt DIC n = 13
Severe sepsis with overt DIC n = 9
P value
13/9 55 (46–64) 12/22
7/6 53 (46–64) 7/13
6/3 56 (47–71) 5/9
0.674 0.471 1.000
6/12 2/12 4/12 17.5 (15–20) 10.5 (6–12) 6/22 (27%)
17 (13–20) 7.5 (6–12) 2/13 (15%)
20 (15–22) 12 (10–14) 4/9 (44%)
0.305 0.069 0.178
Data as number of patients (percentage) or median (interquartile range). DIC, disseminated intravascular coagulation; APACHE II, Acute Physiology and Chronic Health Evaluation II; SOFA, Sequential Organ Failure Assessment.
DIC No DIC Controls
2000
n=8
1500
n = 10
n=9
n = 11
n=9
n = 11
n = 10
500
n=9
P = 0.020*
1000 n = 13
MMP-8 (ng/ml)
Nine patients (41%) fulfilled the ISTH criteria for overt DIC during the first 7 days at the ICU, seven of them on admission, one on day 2 and one on day 3. Medians of DIC score were 6 (IQR 5–6.5) and 3 (IQR 2–3.5) in patients with and without overt DIC, respectively. Fourteen patients (7/9 with overt DIC and 7/13 without) received corticosteroids based on clinical decision-making during the study period (P = 0.38). All but one patient received subcutaneous LMWH thromboprophylaxis. Five patients (1/9 with and 4/13 without overt DIC, P = 0.36) were given recombinant human aPC from day 1 after the first blood samples were taken. More patients with overt DIC received platelet and fresh frozen plasma transfusions as compared with those without DIC (78% vs. 15%, P = 0.007; and 56% vs. 8%, P = 0.023, respectively). The MMP-8 and TIMP-1 serum concentrations over time in patients with and without DIC are presented in Figs 1 and 2, respectively. A significant change in levels of both MMP-8 and TIMP-1 was seen in DIC patients [Χ2 (df 3) = 20.25, P < 0.001 and Χ2 (df 3) = 17.67, P < 0.001, respectively], and in non-DIC patients during the study period [Χ2 (df 3) = 10.47, P = 0.01 and Χ2 (df 3) = 12.75, P = 0.002, respectively]. Both MMP-8 and TIMP-1 concentrations were elevated in patients with severe sepsis as compared with concentrations in healthy controls (P < 0.001 for MMP-8 and TIMP-1 on each day). When the patients with overt DIC were compared with those without, MMP-8 was elevated only on day 2 [702 (IQR 540–1042) ng/ml vs. 246 (58– 550) ng/ml, P = 0.020]. TIMP-1 was significantly
0 1
2
4
7
Day Fig. 1. MMP-8 serum concentrations at different time points in patients with severe sepsis with or without overt disseminated intravascular coagulation. Concentrations (ng/ml) are expressed as median with 95% confidence intervals. *P < 0.05 between patients with and without overt DIC. MMP-8, matrix metalloproteinase-8; DIC, disseminated intravascular coagulation.
higher in overt DIC patients on admission [1034 (IQR 793–1102) ng/ml vs. 542 (IQR 449–836) ng/ ml, P = 0.014)] and day 2 [788 (IQR 601– 1074) ng/ml vs. 447 (IQR 357–545) ng/ml, P = 0.006)], after which it decreased to the level of non-DIC patients. Coagulation test results, WBC count and plasma CRP during the first 2 days are presented in Table 2. Later during the course of the study, platelet count (days 4 and 7, P < 0.001 for both), AT (day 4, P = 0.016) and PC (day 4, P = 0.012) remained reduced, and D-dimer (days 4 and 7,
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P = 0.014* P = 0.006**
n=9
1000
4
7
n = 10
400
n=9 n = 10
n = 11
600
n=9 n = 10
800 n = 13
TIMP-1 (ng/ml)
DIC No DIC Controls
n=9
1200
200 0 1
2 Day
Fig. 2. TIMP-1 serum concentrations at different time points in patients with severe sepsis with or without overt disseminated intravascular coagulation. Concentrations (ng/ml) are expressed as median with 95% confidence intervals. *P < 0.05 between patients with and without overt DIC; **P < 0.01 between patients with and without overt DIC. TIMP-1, tissue inhibitor of metalloproteinases -1; DIC, disseminated intravascular coagulation.
P = 0.003 and P = 0.017, respectively) was elevated in patients with overt DIC. WBC did not differ between groups at any time point. CRP was higher in patients with DIC only on day 4. Data for the days 4 and 7 are not shown. Correlation coefficients with respective P values among MMP-8, TIMP-1, blood coagulation parameters, disease severity scores and inflammation parameters for days 1 and 2 are expressed in Table 3. Later during the study period, no correlation was seen between either MMP-8 or TIMP-1, and coagulation parameters at selected significance level. No correlations were detected between MMP-8 and disease severity, CRP or WBC count at any time point. Discussion Our study showed that concentrations of both MMP-8 and TIMP-1 were highly elevated in patients with severe sepsis during the study period of 7 days as compared with healthy controls. Furthermore, patients who developed sepsis-induced overt DIC had higher MMP-8 and TIMP-1 levels than patients without DIC.
Our study demonstrated that MMP-8 levels were markedly elevated in severe sepsis as compared with those of healthy controls during the study period of 7 days, but values in patients with DIC exceeded those without DIC only on day 2. MMP-8, also known as collagenase-2 or neutrophil collagenase, is a proteinase normally stored in specific granules in neutrophils, and to a lesser extent in activated macrophages and endothelial cells.22,23 Besides its ability to cleave collagen in ECM, it also degrades several other substrates, such as chemokines and serine protease inhibitors,24 and serves as a promoter of neutrophil chemotaxis at the site of inflammation. In inflammatory or infectious states, MMP-8 pro-enzyme is released from the cellular granules to the site of inflammation and activated. Several proinflammatory mediators, such as tumour necrosis factor-α, interleukin-1β and interleukin-6, are potent MMP-8 synthesis inducers as well. Studies on MMP-8 deficient knockout mice have suggested that in early sepsis, MMP-8 has mostly deleterious effects,24–26 but it may be beneficial later in the healing phase of the disease.23,27 However, in clinical studies, MMP-8 levels either have or have not correlated with poor prognosis.13,26,28,29 We could not trace any correlation with disease severity in septic patients with or without DIC. We detected a negative correlation between MMP-8 and platelet count in the first days, but not between MMP-8 and the global coagulation parameters. Decrease in platelet count in overt DIC reflects the consumption of platelets due to widespread microthrombosis. MMP-8 seems to interact with coagulation by cleaving TFPI, which has a crucial inactivating role on both FVIIa/TF complex and FXa. Cunningham and colleagues have reported that MMP-8 rapidly cleaves TFPI and progressively decreases its anticoagulant activity in vitro.9 Based on these findings, it has been suggested that MMP-8 contributes to the local procoagulant environment, and the effect depends on MMP-8 concentration. However, it is unknown whether the binding of MMP-8 with TFPI really occurs and which are the dose responses of MMP-8 on TFPI inactivation in vivo. MMP-8 has been shown to degrade also fibrinogen in vitro. MMP-digested fibrinogen had a dramatically impaired clotting ability.10 This cleaved fibrinogen was unable to contribute to platelet Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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Table 2 Blood test results on the first 2 days after the intensive care unit admission.
Platelet count (150–360 × 109) Prothrombin time ratio (70–130%) D-dimer (< 0.5 mg/l) Fibrinogen (1.7–4.0 g/l) Antithrombin (84–108%) TAT (< 4.1 μg/l) F1 + 2 (< 1.1 nmol/l) PC (74–141%) FVIII (52–148%) APTT (23–33 s) White blood cells (3.4–8.2 × 10E9/l) CRP (< 3 mg/l)
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Severe sepsis without overt DIC
Severe sepsis with overt DIC
P value
131 (80–179) 126 (80–181) 67 (52–77) 71 (47–85) 1.9 (1.0–4.2) 2.2 (1.0–5.0) 7.0 (5.9–10.6) 7.2 (6.3–9.9) 56 (41–64) 60 (50–75) 5.4 (3.4–7.7) 4.0 (2.5–5.0) 1.07 (0.52–2.01) 0.67 (0.26–1.31) 59 (44–74) 65 (41–77) 211 (187–237) 235 (181–280) 36 (33–41) 37.5 (35–41) 12.7 (3.0–20.2) 18.1 (9.7–19.9) 310 (233–397) 192 (148–347)
50 (34–81) 40 (24–46) 34 (24–53) 46 (34–60) 6.5 (4.8–41.5) 8.1 (5.3–47.5) 5.6 (4.8–7.5) 5.4 (4.2–6.7) 30 (21–43) 52 (30–70) 16.0 (5.1–64.8) 6.4 (4.5–24.8) 2.03 (0.73–3.61) 1.06 (0.51–2.03) 36 (33–37) 33 (26–45) 178 (75–229) 149 (97–199) 38.5 (36–62) 49.5 (39–64) 10.3 (2.2–22.2) 16.5 (13.6–24.5) 280 (170–311) 347 (215–372)
0.004** < 0.001** 0.006** 0.041* 0.001** 0.009** 0.185 0.019* 0.002** 0.345 0.051 0.009** 0.164 0.169 0.009** 0.006** 0.235 0.036* 0.129 0.022* 0.948 0.695 0.324 0.126
Data as median (interquartile range). Reference values for each parameter are reported in the left column. *P < 0.05: severe sepsis with vs. without overt DIC. **P < 0.01: severe sepsis with vs. without overt DIC. DIC, disseminated intravascular coagulation; TAT, thrombin-antithrombin complex; F1 + 2, prothrombin fragment 1 + 2; PC, protein C; FVIII, factor VIII; APTT, activated partial thromboplastin time, CRP; C-reactive protein.
aggregation. Taken together, these results suggest that MMP-8 may have both pro- and anticoagulant action in DIC depending on the substrate and mediators of the interplay between inflammation and coagulation. Therefore, no causality can be concluded from any correlation in this multifactorial phenomenon. Furthermore, the analytical method used in our study does not differentiate between the pro-enzymatic and active forms of MMP-8. Our study showed that in severe sepsis, patients with overt DIC TIMP-1 was strongly elevated early, on the first 2 days after the admission, as compared with those without overt DIC. Thereafter, TIMP-1 started to decline to the level of the patients without DIC. TIMP-1 is an endogenous regulator of MMP activity, and it has been shown to inhibit all MMP subtypes.30 Therefore, TIMP-1 is involved in regulating the degradation of ECM, and it has been shown to have additional non-MMP-related functions. An independent
positive correlation with fibrinogen levels was seen in healthy adults, suggesting that TIMP-1 is prothrombotic.31 TIMP-1 is expressed in both soluble and cell surface-bound form in many mammalian cell types, including circulating platelets. The time pattern of TIMP-1 concentration in our study resembles closely the results of a previous study in septic patients.14 Interestingly, in patients with severe sepsis, we could show that TIMP-1 correlated inversely with platelet count and PT ratio, and positively with marker of increased thrombin formation and fibrin turnover: D-dimer. This is in line with the results from two previous studies with septic patients.15,32 TIMP-1 was recently studied in an animal model of septic DIC. Under standardised circumstances, an increase in TIMP-1 level was seen as early as 2 h after the onset of sepsis in mice that later developed DIC, and the level remained elevated until 72 h.16 Despite the heterogeneous patient material, the increase of
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Table 3 Correlations between MMP-8 and TIMP-1 with coagulation parameters, disease severity scores and inflammation parameters on days 1 and 2. MMP-8
MMP-8
TIMP-1
Day 1 (r)
Day 2 (r)
NA
NA
Day 1 (r)
0.660** P < 0.001 Platelet count −0.483 −0.626* −0.514 P = 0.026 P = 0.003 P = 0.014 Prothrombin −0.087 −0.080 −0.760** time ratio P = 0.709 P = 0.745 P < 0.001 D-dimer 0.307 0.253 0.384 P = 0.175 P = 0.297 P = 0.078 Fibrinogen 0.211 −0.035 −0.472 P = 0.373 P = 0.882 P = 0.031 Antithrombin −0.307 −0.016 −0.276 P = 0.176 P = 0.949 P = 0.213 0.519 TAT 0.168 0.323 P = 0.468 P = 0.164 P = 0.013 Fragment 1 + 2 0.147 0.060 0.300 P = 0.526 p = 0.801 P = 0.175 PC −0.197 −0.228 −0.533 P = 0.406 P = 0.347 P = 0.013 SOFA 0.363 0.238 0.634* P = 0.127 P = 0.392 P = 0.003 SOFA without 0.254 0.111 0.612* platelets P = 0.294 P = 0.693 P = 0.004 APACHE II 0.207 0.048 0.489 P = 0.396 P = 0.850 P = 0.029 CRP 0.125 0.469 −0.246 P = 0.588 P = 0.037 P = 0.269 White blood −0.236 −0.121 −0.118 cells P = 0.303 P = 0.611 P = 0.601
Day 2 (r) 0.716** P < 0.001 −0.641* P = 0.002 −0.620* P = 0.005 0.663* P = 0.002 −0.372 P = 0.106 0.215 P = 0.376 0.553 P = 0.011 0.550 P = 0.012 −0.586* P = 0.008 0.634* P = 0.010 0.440 P = 0.101 0.086 P = 0.735 0.460 P = 0.041 −0.068 P = 0.777
Data are shown as Spearman’s correlation coefficient r with P value and significance. *P < 0.01 and **P < 0.001. MMP-8, matrix metalloproteinase-8; TIMP-1, tissue inhibitor of metalloproteinase -1; TAT, thrombin-antithrombin complex; F1 + 2, prothrombin fragment 1 + 2; PC, protein C; SOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology and Chronic Health Evaluation II; CRP, C-reactive protein; NA, not applicable.
TIMP-1 in DIC patients was seen also in our clinical study, although in our study we could not show that an increase in TIMP-1 level precedes changes in traditional coagulation assays. Because TIMP-1 is a universal inhibitor of different MMPs, its increase can reflect changes also in other MMPs. For instance, in the largest clinical study of severe sepsis patients, non-survivors had higher TIMP-1 and lower MMP-9 levels,15 which may associate with inflammatory and prothrombotic or antifibrinolytic state, and contrib-
ute to the multiple organ dysfunction and eventually death. We avoid assuming any causality between TIMP-1 and MMP-8 levels, as the increase in TIMP-1 may be affected by other important mediators as well. To the best of our knowledge, this study was the first to investigate systemic concentrations of MMP-8 and TIMP-1 and their associations with different coagulation markers in patients with severe sepsis and overt DIC. However, our study confronted some limitations. First, the sample size of this pilot study was small. Nevertheless, we could show significant differences between study groups suggesting that both MMP-8 and TIMP-1 may have a role in septic coagulopathy. The results of this pilot study are novel, and to be confirmed they require a further, larger study where the effect of disease severity can be controlled. Second, the phase and the degree of inflammatory reaction likely varied between different patients, although the patients were recruited at the time of admission to the ICU and less than 48 h after the onset of the severe sepsis. This may explain the wide range in the concentrations of both MMP-8 and TIMP-1 observed during the first 2 days of the study. Third, the storage time of the serum samples before MMP-8 and TIMP-1 analyses was up to 4 years. As far as we know, there are no reports on serum MMP-8 and TIMP-1 behaviour over time. Instead, plasma TIMP-1 concentration over storage time has been shown to be very stable.33 However, we know that studied biomarkers may be affected when the assays are performed with samples thawed at different occasions. To avoid this, we have kept our samples in −80 degrees until analysis and made all the assays at the same time. Finally, TIMP-1, but according to our knowledge not MMP-8, has been shown to be present in platelets. Therefore, it may be possible that TIMP-1 can be released from platelets also in vitro, confounding the results. However, a rather negative correlation between TIMP-1 and platelet count was seen, indicating that those patients with lowest platelet count had higher TIMP-1 concentration. Conclusions We conclude that in severe sepsis-associated overt DIC, TIMP-1 is strongly elevated during the first 2 days after admission to the ICU, and it correlates Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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with disease severity scores and activation of coagulation and fibrinolysis. MMP-8 was increased only on day 2 in DIC patients as compared with those without DIC. These findings suggest that TIMP-1 may play a pathogenetic role in sepsis-associated DIC. Acknowledgements The study was financially supported by Helsinki University Hospital EVO Grants T102010070 and V1020AHES3. The technical assistance by Marja Lemponen of analysing TAT and F1 + 2 is greatly acknowledged. References 1. Abraham E, Singer M. Mechanisms of sepsis-induced organ dysfunction. Crit Care Med 2007; 35: 2408–16. 2. Dhainaut J-F, Yan SB, Joyce DE, Pettilä V, Basson B, Brandt JT, Sundin DP, Levi M. Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation. J Thromb Haemost 2004; 2: 1924–33. 3. Levi M, van der Poll T. Inflammation and coagulation. Crit Care Med 2010; 38: S26–34. 4. Gando S. Microvascular thrombosis and multiple organ dysfunction syndrome. Crit Care Med 2010; 38: S35–42. 5. Santos-Martinez MJ, Medina C, Gilmer JF, Radomski MW. Matrix metalloproteinases in platelet function: coming of age. J Thromb Haemost 2008; 6: 514–6. 6. Lijnen HR, Collen D. Matrix metalloproteinase system deficiencies and matrix degradation. Thromb Haemost 1999; 82: 837–45. 7. Pekovich SR, Bock PE, Hoover RL. Thrombin-thrombomodulin activation of protein C facilitates the activation of progelatinase A. FEBS Lett 2001; 494: 129–32. 8. Belaaouaj AA, Li A, Wun TC, Welgus HG, Shapiro SD. Matrix metalloproteinases cleave tissue factor pathway inhibitor. Effects on coagulation. J Biol Chem 2000; 275: 27123–8. 9. Cunningham AC, Hasty KA, Enghild JJ, Mast AE. Structural and functional characterization of tissue factor pathway inhibitor following degradation by matrix metalloproteinase-8. Biochem J 2002; 367: 451–8.
10. Hiller O. Matrix metalloproteinases collagenase-2, macrophage elastase, collagenase-3, and membrane type 1-matrix metalloproteinase Impair clotting by degradation of fibrinogen and factor XII. J Biol Chem 2000; 275: 33008–13. 11. Savov JD, Brass DM, Berman KG, McElvania E, Schwartz DA. Fibrinolysis in LPS-induced chronic airway disease. Am J Physiol Lung Cell Mol Physiol 2003; 285: L940–8. doi: 10.1152/ajplung.00102.2003. 12. Hoffmann U, Hoffmann U, Bertsch T, Hoffmann U, Bertsch T, Dvortsak E, Liebetrau C, Lang S, Liebe V, Huhle G, Borggrefe M, Brueckmann M. Matrix-metalloproteinases and their inhibitors are elevated in severe sepsis: prognostic value of TIMP-1 in severe sepsis. Scand J Infect Dis 2006; 38: 867–72. 13. Lauhio A, Hästbacka J, Pettilä V, Tervahartiala T, Karlsson S, Varpula T, Varpula M, Ruokonen E, Sorsa T, Kolho E. Serum MMP-8, -9 and TIMP-1 in sepsis: high serum levels of MMP-8 and TIMP-1 are associated with fatal outcome in a multicentre, prospective cohort study. Hypothetical impact of tetracyclines. Pharmacol Res 2011; 64: 590–4. 14. Mühl D, Nagy B, Woth G, Falusi B, Bogár L, Weber G, Lantos J. Dynamic changes of matrix metalloproteinases and their tissue inhibitors in severe sepsis. J Crit Care 2011; 26: 550–5. 15. Lorente L, Martín MM, Labarta L, Díaz C, Solé-Violán J, Blanquer J, Orbe J, Rodríguez JA, Jiménez A, Borreguero-León JM, Belmonte F, Medina JC, LLimiñana MC, Ferrer-Agüero JM, Ferreres J, Mora ML, Lubillo S, Sánchez M, Barrios Y, Sierra A, Páramo JA. Matrix metalloproteinase-9, -10, and tissue inhibitor of matrix metalloproteinases-1 blood levels as biomarkers of severity and mortality in sepsis. Crit Care 2009; 13: R158. 16. Song J, Hu D, He C, Wang T, Liu X, Ma L, Lin Z, Chen Z. Novel biomarkers for early prediction of sepsis-induced disseminated intravascular coagulation in a mouse cecal ligation and puncture model. J Inflamm (Lond) 2013; 10: 7. 17. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 1992; 101: 1644–55. 18. Sivula M, Tallgren M, Pettilä V. Modified score for disseminated intravascular coagulation in the critically ill. Intensive Care Med 2005; 31: 1209–14.
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19. Hanemaaijer R, Sorsa T, Konttinen YT, Ding Y, Sutinen M, Visser H, van Hinsbergh VW, Helaakoski T, Kainulainen T, Rönkä H, Tschesche H, Salo T. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem 1997; 272: 31504–9. 20. Hästbacka J, Hynninen M, Kolho E, Pettilä V, Tervahartiala T, Sorsa T, Lauhio A. Collagenase 2/matrix metalloproteinase 8 in critically ill patients with secondary peritonitis. Shock 2007; 27: 145–50. 21. Llavaneras A, Ramamurthy NS, Heikkilä P, Teronen O, Salo T, Rifkin BR, Ryan ME, Golub LM, Sorsa T. A combination of a chemically modified doxycycline and a bisphosphonate synergistically inhibits endotoxin-induced periodontal breakdown in rats. J Periodontol 2001; 72: 1069–77. 22. Herman MP, Sukhova GK, Libby P, Gerdes N, Tang N, Horton DB, Kilbride M, Breitbart RE, Chun M, Schonbeck U. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation 2001; 104: 1899–904. 23. Owen CA, Hu Z, Lopez-Otin C, Shapiro SD. Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant collagenase and serpinase. J Immunol 2004; 172: 7791–803. 24. Vanlint P, Libert C. Matrix metalloproteinase-8: cleavage can be decisive. Cytokine Growth Factor Rev 2006; 17: 217–23. 25. Vandenbroucke RE, Dejonckheere E, Van Lint P, Demeestere D, Van Wonterghem E, Vanlaere I, Puimege L, Van Hauwermeiren F, De Rycke R, McGuire C, Campestre C, Lopez-Otin C, Matthys P, Bayrak A, Libert C. Matrix metalloprotease 8-dependent extracellular matrix cleavage at the blood-CSF barrier contributes to lethality during
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systemic inflammatory diseases. J Neurosci 2012; 32: 9805–16. Solan PD, Dunsmore KE, Denenberg AG, Odoms K, Zingarelli B, Wong HR. A novel role for matrix metalloproteinase-8 in sepsis*. Crit Care Med 2012; 40: 379–87. Gutierrez-Fernandez A, Inada M, Balbin M, Fueyo A, Pitiot AS, Astudillo A, Hirose K, Hirata M, Shapiro SD, Noel A, Werb Z, Krane SM, Lopez-Otin C, Puente XS. Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8). FASEB J 2007; 21: 2580–91. Gäddnäs FP, Sutinen MM, Koskela M, Tervahartiala T, Sorsa T, Salo TA, Laurila JJ, Koivukangas V, Ala-Kokko TI, Oikarinen A. Matrix-metalloproteinase-2, -8 and -9 in serum and skin blister fluid in patients with severe sepsis. Crit Care 2010; 14: R49. Yazdan-Ashoori P, Liaw P, Toltl L, Webb B, Kilmer G, Carter DE, Fraser DD. Elevated plasma matrix metalloproteinases and their tissue inhibitors in patients with severe sepsis. J Crit Care 2011; 26: 556–65. Murphy G. Tissue inhibitors of metalloproteinases. Genome Biol 2011; 12: 233. Aznaouridis K, Vlachopoulos C, Dima I, Vasiliadou C, Ioakeimidis N, Baou K, Stefanadi E, Stefanadis C. Divergent associations of tissue inhibitors of metalloproteinases-1 and -2 with the prothrombotic/fibrinolytic state. Atherosclerosis 2007; 195: 212–5. Lorente L, Martín M, Plasencia F, Solé-Violán J, Blanquer J, Labarta L, Díaz C, Borreguero-León JM, Jiménez A, Páramo JA, Orbe J, Rodríguez JA, Salido E. The 372 T/C genetic polymorphism of TIMP-1 is associated with serum levels of TIMP-1 and survival in patients with severe sepsis. Crit Care 2013; 17: R94. Rouy D, Ernens I, Jeanty C, Wagner DR. Plasma storage at −80°C does not protect matrix metalloproteinase-9 from degradation. Anal Biochem 2005; 338: 294–8.
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Systemic matrix metalloproteinase-8 and tissue inhibitor of metalloproteinases-1 levels in severe sepsis-associated coagulopathy M. Sivula1, J. Hästbacka1, A. Kuitunen2, R. Lassila3, T. Tervahartiala4, T. Sorsa4,5 and V. Pettilä1 1
Intensive Care Units, Division of Anaesthesia and Intensive Care Medicine, Helsinki University Central Hospital, Helsinki, Finland Intensive Care Unit, Division of Anaesthesia and Intensive Care Medicine, Tampere University Central Hospital, Tampere, Finland 3 Coagulation Disorders Unit, Department of Haematology, Helsinki University Central Hospital, Helsinki, Finland 4 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland 5 Division of Periodontology, Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden 2
Correspondence M. Sivula, Intensive Care Units, Jorvi Hospital, Helsinki University Central Hospital, PO Box 800, Espoo, HUS 00029, Finland E-mail: mirka.sivula@hus.fi Conflicts of interest The authors confirm that there are no conflicts of interest. Submitted 16 July 2014; accepted 11 September 2014; submission 9 September 2014. Citation Sivula M, Hästbacka J, Kuitunen A, Lassila R, Tervahartiala T, Sorsa T, Pettilä V. Systemic matrix metalloproteinase-8 and tissue inhibitor of metalloproteinases-1 levels in severe sepsis-associated coagulopathy. Acta Anaesthesiologica Scandinavica 2014 doi: 10.1111/aas.12423
Background: Matrix metalloproteinase-8 (MMP-8) and tissue inhibitor of metalloproteinases-1 (TIMP-1) have recently been suggested to be involved in coagulation process. Our objectives were to observe systemic MMP-8 and TIMP-1 levels in patients with severe sepsis with or without disseminated intravascular coagulation (DIC) and to study their relationship with coagulation markers over time. Methods: Our prospective pilot study included 22 patients with severe sepsis, nine (41%) of whom had overt DIC. We analysed MMP-8 and TIMP-1 serum concentrations by time-resolved immunofluorometric and enzyme-linked immunosorbent assays, respectively, on days 1, 2, 4 and 7 after the intensive care unit admission. Traditional coagulation tests were taken at the same time points. The results were compared between patients with and without DIC. Blood samples from 10 healthy volunteers were used to demonstrate normal levels. Results: Both patient groups had elevated levels of MMP-8 and TIMP-1 as compared with healthy controls. TIMP-1 concentration was almost twofold in DIC patients compared with those without DIC on the first 2 days. MMP-8 was elevated only on day 2. TIMP-1 correlated positively with the severity of coagulation disturbance and with disease severity scores. MMP-8 correlated negatively only with platelet count. Conclusion: In this first human study, we could show that TIMP-1 is elevated in the early phase of sepsis-induced overt DIC, and it correlates both with degree of coagulopathy and disease severity. These findings suggest that TIMP-1 may play a role in the pathogenesis of DIC in septic patients.
Severe sepsis and septic shock result from excessive activation of inflammatory response to infectious stimuli, leading to endothelial dysfunction, coagulation activity and haemostatic imbalance, circulatory shock, and ultimately
multi-organ dysfunction.1 More than 30% of patients with severe sepsis develop overt disseminated intravascular coagulation (DIC) over the first few days of severe sepsis.2 DIC is characterised by the activation of coagulation trigActa Anaesthesiologica Scandinavica 59 (2015) 176–184
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gered by exposure of excessive tissue factor (TF), which leads to a widespread intravascular fibrin deposition and clot formation. In DIC, natural anticoagulant system is downgraded due to impaired synthesis, degradation and increased consumption of tissue factor pathway inhibitor (TFPI), activated protein C (aPC) and antithrombin (AT). Fibrinolysis is also impaired partly because of increase in fibrinolysis inhibitors.3 DIC may compromise the circulation of organ systems, thus contributing to the development of multi-organ failure.4 Overt DIC has been shown to roughly double the mortality of severe sepsis patients.2 Recently, increasing interest has been focused on matrix metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitor of metalloproteinases, TIMPs). MMPs form a structurally related group of zinc-dependent proteolytic enzymes that, in addition to their first recognised function, degradation of extracellular matrix (ECM), also cleave cellular surface proteins and other non-matrix substrates. MMPs have been linked to coagulation in many chronic and acute inflammatory states in complex ways.5–11 In clinical studies, high levels of MMP-8 and TIMP-1 have been shown to associate with higher mortality among severe sepsis patients.12–15 Recently, in a murine sepsis model, TIMP-1 was shown to increase in the early phase of septic DIC.16 Although MMP-8 has not been linked to septic coagulation disorders so far, it has been shown to degrade TFPI,9 which can be crucial in DIC. Accordingly, we hypothesised that MMP-8 and TIMP-1 levels may associate with severe sepsisassociated DIC. We aimed to investigate the time courses of MMP-8 and TIMP-1 in this patient group, and evaluate their association with established coagulation parameters and markers of disease severity.
Methods We performed this prospective pilot study in a tertiary multidisciplinary intensive care unit (ICU) of Helsinki University Central Hospital between January 2002 and November 2003. The ethics committee of Helsinki University Central Hospital Department of Surgery approved the
study protocol (Protocol Number HUS 171/E6/01, 2001-03-30). All patients admitted to the ICU were screened for severe sepsis or septic shock. We included 22 adult patients with criteria of severe sepsis or septic shock fulfilled within 48 h prior to admission to the ICU, and obtained informed consent from all the participating patients or their next of kin. The diagnosis of severe sepsis was based on the following criteria: a confirmed or suspected infection and at least two of the four systemic inflammatory response syndrome criteria [body temperature > 38°C or < 36°C, heart rate > 90/min, respiratory rate > 20/min or arterial partial pressure of carbon dioxide < 32 mmHg or need for mechanical ventilation, and white blood cell (WBC) count > 12,000 cells/μl or < 4000/μl],17 and organ dysfunction and hypoperfusion or hypotension after adequate fluid resuscitation. Exclusion criteria were pharmacological anticoagulant therapy within 1 week prior to the admission, pregnancy, haematological malignancy and chronic liver disease. Serum samples for MMP-8 and TIMP-1 control levels were obtained from 10 healthy adults once. We recorded routine clinical data and severity scores, including the Acute Physiology and Chronic Health Evaluation II and daily Sequential Organ Failure Assessment (SOFA) scores, use of blood components, low molecular weight heparins (LMWHs) and corticosteroids in the computerised ICU database. We calculated SOFA scores with and without points given for platelet count to assess the association with other organ failures. The patients received subcutaneous LMWH with a fixed dose once or twice a day routinely unless clinically contraindicated. A written treatment protocol of the ICU guided blood component transfusions. We calculated the International Society on Thrombosis and Haemostasis (ISTH) score for overt DIC daily using lowest blood platelet count, plasma prothrombin time (PT) ratio, fibrinogen concentration and highest plasma D-dimer as described earlier.18 Patients with a score of 5 or higher on any day had diagnosis of overt DIC. We took serum samples for MMP-8 and TIMP-1 analyses on admission (day 1) and on days 2, 4 and 7. We also collected blood samples for the coagulation tests, WBC count and C-reactive
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protein (CRP) on admission, and then daily in the morning until day 7 simultaneously with MMP-8 and TIMP-1 samples, and if clinically indicated. All samples were drawn from non-heparinised arterial lines after discarding the first 5 ml of blood. WBC and CRP were measured by the routine hospital laboratory. Serum samples for MMP-8 and TIMP-1 analyses were centrifuged at 2000g for 10 min, and the supernatants were stored at −80°C until analysis, which we performed in May 2006. We analysed MMP-8 and TIMP-1 concentrations in random order, blinded to the severity of coagulation disorder. We measured MMP-8 serum concentration by time-resolved immunofluorometric assay as described previously19,20 by using monoclonal MMP-8 specific antibodies 8708 and 8706 (Medix Biochemica, Kauniainen, Finland) for catching and tracing, respectively. We used europium chelate for labelling the tracer antibody. We carried out TIMP-1 analysis by using commercially available enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Biotrak ELISA System; Amersham Biosciences, Buckinghamshire, UK). We report the concentrations as ng/ml. Detection limits for MMP-8 and TIMP-1 are 0.032 ng/ml and 1.25 ng/ml, respectively. The inter-assay coefficient of variation (CV%) for MMP-8 and TIMP-1 are 4.1 and 13.5 (n = 12) and intra-assay CV% 2.5 and 10.1 (n = 12), respectively. We used citrate tubes with final concentration of 3.8% for the coagulation plasma samples and ethylenediaminetetraacetic acid tubes for the haematological test samples. The analytical methods were Nycotest PT (Owren-buffered, Axis-Shield PoC AS, Oslo, Norway) for PT, immunoturbidimetric assay Tina-quant® (Roche Diagnostics, Mannheim, Germany) for D-dimer concentration, Multifibren U (Siemens Healthcare Diagnostics, Erlangen, Germany) for fibrinogen, chromogenic Berichrom® Antithrombin III (A) (Siemens Healthcare Diagnostics) for AT activity, APTT Actin FSL® (Siemens Healthcare Diagnostics) for activated partial thromboplastin time (APTT), Enzygnost® TAT Micro (Siemens Healthcare Diagnostics) for thrombin-antithrombin complex (TAT), Enzygnost® F1 + 2 (monoclonal) (Siemens Healthcare Diagnostics) for prothrombin fragment 1 + 2 (F1 + 2), Berichrom® Protein C (Siemens Healthcare Diagnostics) for protein C
(PC), Pathromtin SL (Siemens Healthcare Diagnostics) and factor VIII deficient plasma for factor VIII activity (FVIII:C), and blood cell counter with direct current detection method for blood platelet count. Due to lack of previous studies on MMP-8 and TIMP concentrations and their distributions in patients with overt DIC, we could not perform sample size calculations for this pilot study. As the prevalence of overt DIC is approximately 30% in patients with severe sepsis in our ICU based on data collected for our previous study,18 the sample size for overt DIC group was targeted to exceed six, and thus the whole sample of at least 20 patients. We present all data as number of patients with percentage or median with interquartile range (IQR) or 95% confidence interval. According to Shapiro–Wilk test, the distribution of the data was non-normal, and we chose non-parametric tests for statistical analyses. We compared the differences between groups in categorical variables using two-tailed Fisher’s exact test, and in continuous data in two or several groups using Mann– Whitney U-test and Kruskal–Wallis test, respectively. We assessed differences in continuous parameters MMP-8 and TIMP-1 between time points with Friedman’s ANOVA, and report the results with chi-square (Χ2) with degrees of freedom (df) and P value. We calculated nonparametric Spearman’s rank correlation coefficient with two-tailed significance for MMP-8, TIMP-1 and blood coagulation parameters on days 1 and 2, and for MMP-8, TIMP-1 and disease severity scores and inflammation parameters on days 1, 2, 4 and 7. We considered differences between groups statistically significant if P value was less than 0.05, and we corrected P value for multiple comparisons as needed. In correlation analyses, we considered P value less than 0.01 statistically significant. We performed statistical analyses with IBM® SPSS® Statistics 20.0 for Mac OS (IBM Corp., Armonk, NY, USA). Results Twenty-two patients with severe sepsis or septic shock were included in the study. Demographic data are summarised in Table 1. None of the patients had used known MMP inhibitors, tetracyclines or bisphosphonates21 prior to admission to the ICU. Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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Table 1 Characteristics of the study groups. All patients n = 22 Sex (male/female) Age Positive blood culture Pathogens Streptococcus pneumoniae Streptococcus pyogenes Miscellaneous APACHE II SOFA day1 Mortality on day 28
Severe sepsis without overt DIC n = 13
Severe sepsis with overt DIC n = 9
P value
13/9 55 (46–64) 12/22
7/6 53 (46–64) 7/13
6/3 56 (47–71) 5/9
0.674 0.471 1.000
6/12 2/12 4/12 17.5 (15–20) 10.5 (6–12) 6/22 (27%)
17 (13–20) 7.5 (6–12) 2/13 (15%)
20 (15–22) 12 (10–14) 4/9 (44%)
0.305 0.069 0.178
Data as number of patients (percentage) or median (interquartile range). DIC, disseminated intravascular coagulation; APACHE II, Acute Physiology and Chronic Health Evaluation II; SOFA, Sequential Organ Failure Assessment.
DIC No DIC Controls
2000
n=8
1500
n = 10
n=9
n = 11
n=9
n = 11
n = 10
500
n=9
P = 0.020*
1000 n = 13
MMP-8 (ng/ml)
Nine patients (41%) fulfilled the ISTH criteria for overt DIC during the first 7 days at the ICU, seven of them on admission, one on day 2 and one on day 3. Medians of DIC score were 6 (IQR 5–6.5) and 3 (IQR 2–3.5) in patients with and without overt DIC, respectively. Fourteen patients (7/9 with overt DIC and 7/13 without) received corticosteroids based on clinical decision-making during the study period (P = 0.38). All but one patient received subcutaneous LMWH thromboprophylaxis. Five patients (1/9 with and 4/13 without overt DIC, P = 0.36) were given recombinant human aPC from day 1 after the first blood samples were taken. More patients with overt DIC received platelet and fresh frozen plasma transfusions as compared with those without DIC (78% vs. 15%, P = 0.007; and 56% vs. 8%, P = 0.023, respectively). The MMP-8 and TIMP-1 serum concentrations over time in patients with and without DIC are presented in Figs 1 and 2, respectively. A significant change in levels of both MMP-8 and TIMP-1 was seen in DIC patients [Χ2 (df 3) = 20.25, P < 0.001 and Χ2 (df 3) = 17.67, P < 0.001, respectively], and in non-DIC patients during the study period [Χ2 (df 3) = 10.47, P = 0.01 and Χ2 (df 3) = 12.75, P = 0.002, respectively]. Both MMP-8 and TIMP-1 concentrations were elevated in patients with severe sepsis as compared with concentrations in healthy controls (P < 0.001 for MMP-8 and TIMP-1 on each day). When the patients with overt DIC were compared with those without, MMP-8 was elevated only on day 2 [702 (IQR 540–1042) ng/ml vs. 246 (58– 550) ng/ml, P = 0.020]. TIMP-1 was significantly
0 1
2
4
7
Day Fig. 1. MMP-8 serum concentrations at different time points in patients with severe sepsis with or without overt disseminated intravascular coagulation. Concentrations (ng/ml) are expressed as median with 95% confidence intervals. *P < 0.05 between patients with and without overt DIC. MMP-8, matrix metalloproteinase-8; DIC, disseminated intravascular coagulation.
higher in overt DIC patients on admission [1034 (IQR 793–1102) ng/ml vs. 542 (IQR 449–836) ng/ ml, P = 0.014)] and day 2 [788 (IQR 601– 1074) ng/ml vs. 447 (IQR 357–545) ng/ml, P = 0.006)], after which it decreased to the level of non-DIC patients. Coagulation test results, WBC count and plasma CRP during the first 2 days are presented in Table 2. Later during the course of the study, platelet count (days 4 and 7, P < 0.001 for both), AT (day 4, P = 0.016) and PC (day 4, P = 0.012) remained reduced, and D-dimer (days 4 and 7,
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P = 0.014* P = 0.006**
n=9
1000
4
7
n = 10
400
n=9 n = 10
n = 11
600
n=9 n = 10
800 n = 13
TIMP-1 (ng/ml)
DIC No DIC Controls
n=9
1200
200 0 1
2 Day
Fig. 2. TIMP-1 serum concentrations at different time points in patients with severe sepsis with or without overt disseminated intravascular coagulation. Concentrations (ng/ml) are expressed as median with 95% confidence intervals. *P < 0.05 between patients with and without overt DIC; **P < 0.01 between patients with and without overt DIC. TIMP-1, tissue inhibitor of metalloproteinases -1; DIC, disseminated intravascular coagulation.
P = 0.003 and P = 0.017, respectively) was elevated in patients with overt DIC. WBC did not differ between groups at any time point. CRP was higher in patients with DIC only on day 4. Data for the days 4 and 7 are not shown. Correlation coefficients with respective P values among MMP-8, TIMP-1, blood coagulation parameters, disease severity scores and inflammation parameters for days 1 and 2 are expressed in Table 3. Later during the study period, no correlation was seen between either MMP-8 or TIMP-1, and coagulation parameters at selected significance level. No correlations were detected between MMP-8 and disease severity, CRP or WBC count at any time point. Discussion Our study showed that concentrations of both MMP-8 and TIMP-1 were highly elevated in patients with severe sepsis during the study period of 7 days as compared with healthy controls. Furthermore, patients who developed sepsis-induced overt DIC had higher MMP-8 and TIMP-1 levels than patients without DIC.
Our study demonstrated that MMP-8 levels were markedly elevated in severe sepsis as compared with those of healthy controls during the study period of 7 days, but values in patients with DIC exceeded those without DIC only on day 2. MMP-8, also known as collagenase-2 or neutrophil collagenase, is a proteinase normally stored in specific granules in neutrophils, and to a lesser extent in activated macrophages and endothelial cells.22,23 Besides its ability to cleave collagen in ECM, it also degrades several other substrates, such as chemokines and serine protease inhibitors,24 and serves as a promoter of neutrophil chemotaxis at the site of inflammation. In inflammatory or infectious states, MMP-8 pro-enzyme is released from the cellular granules to the site of inflammation and activated. Several proinflammatory mediators, such as tumour necrosis factor-α, interleukin-1β and interleukin-6, are potent MMP-8 synthesis inducers as well. Studies on MMP-8 deficient knockout mice have suggested that in early sepsis, MMP-8 has mostly deleterious effects,24–26 but it may be beneficial later in the healing phase of the disease.23,27 However, in clinical studies, MMP-8 levels either have or have not correlated with poor prognosis.13,26,28,29 We could not trace any correlation with disease severity in septic patients with or without DIC. We detected a negative correlation between MMP-8 and platelet count in the first days, but not between MMP-8 and the global coagulation parameters. Decrease in platelet count in overt DIC reflects the consumption of platelets due to widespread microthrombosis. MMP-8 seems to interact with coagulation by cleaving TFPI, which has a crucial inactivating role on both FVIIa/TF complex and FXa. Cunningham and colleagues have reported that MMP-8 rapidly cleaves TFPI and progressively decreases its anticoagulant activity in vitro.9 Based on these findings, it has been suggested that MMP-8 contributes to the local procoagulant environment, and the effect depends on MMP-8 concentration. However, it is unknown whether the binding of MMP-8 with TFPI really occurs and which are the dose responses of MMP-8 on TFPI inactivation in vivo. MMP-8 has been shown to degrade also fibrinogen in vitro. MMP-digested fibrinogen had a dramatically impaired clotting ability.10 This cleaved fibrinogen was unable to contribute to platelet Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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Table 2 Blood test results on the first 2 days after the intensive care unit admission.
Platelet count (150–360 × 109) Prothrombin time ratio (70–130%) D-dimer (< 0.5 mg/l) Fibrinogen (1.7–4.0 g/l) Antithrombin (84–108%) TAT (< 4.1 μg/l) F1 + 2 (< 1.1 nmol/l) PC (74–141%) FVIII (52–148%) APTT (23–33 s) White blood cells (3.4–8.2 × 10E9/l) CRP (< 3 mg/l)
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Severe sepsis without overt DIC
Severe sepsis with overt DIC
P value
131 (80–179) 126 (80–181) 67 (52–77) 71 (47–85) 1.9 (1.0–4.2) 2.2 (1.0–5.0) 7.0 (5.9–10.6) 7.2 (6.3–9.9) 56 (41–64) 60 (50–75) 5.4 (3.4–7.7) 4.0 (2.5–5.0) 1.07 (0.52–2.01) 0.67 (0.26–1.31) 59 (44–74) 65 (41–77) 211 (187–237) 235 (181–280) 36 (33–41) 37.5 (35–41) 12.7 (3.0–20.2) 18.1 (9.7–19.9) 310 (233–397) 192 (148–347)
50 (34–81) 40 (24–46) 34 (24–53) 46 (34–60) 6.5 (4.8–41.5) 8.1 (5.3–47.5) 5.6 (4.8–7.5) 5.4 (4.2–6.7) 30 (21–43) 52 (30–70) 16.0 (5.1–64.8) 6.4 (4.5–24.8) 2.03 (0.73–3.61) 1.06 (0.51–2.03) 36 (33–37) 33 (26–45) 178 (75–229) 149 (97–199) 38.5 (36–62) 49.5 (39–64) 10.3 (2.2–22.2) 16.5 (13.6–24.5) 280 (170–311) 347 (215–372)
0.004** < 0.001** 0.006** 0.041* 0.001** 0.009** 0.185 0.019* 0.002** 0.345 0.051 0.009** 0.164 0.169 0.009** 0.006** 0.235 0.036* 0.129 0.022* 0.948 0.695 0.324 0.126
Data as median (interquartile range). Reference values for each parameter are reported in the left column. *P < 0.05: severe sepsis with vs. without overt DIC. **P < 0.01: severe sepsis with vs. without overt DIC. DIC, disseminated intravascular coagulation; TAT, thrombin-antithrombin complex; F1 + 2, prothrombin fragment 1 + 2; PC, protein C; FVIII, factor VIII; APTT, activated partial thromboplastin time, CRP; C-reactive protein.
aggregation. Taken together, these results suggest that MMP-8 may have both pro- and anticoagulant action in DIC depending on the substrate and mediators of the interplay between inflammation and coagulation. Therefore, no causality can be concluded from any correlation in this multifactorial phenomenon. Furthermore, the analytical method used in our study does not differentiate between the pro-enzymatic and active forms of MMP-8. Our study showed that in severe sepsis, patients with overt DIC TIMP-1 was strongly elevated early, on the first 2 days after the admission, as compared with those without overt DIC. Thereafter, TIMP-1 started to decline to the level of the patients without DIC. TIMP-1 is an endogenous regulator of MMP activity, and it has been shown to inhibit all MMP subtypes.30 Therefore, TIMP-1 is involved in regulating the degradation of ECM, and it has been shown to have additional non-MMP-related functions. An independent
positive correlation with fibrinogen levels was seen in healthy adults, suggesting that TIMP-1 is prothrombotic.31 TIMP-1 is expressed in both soluble and cell surface-bound form in many mammalian cell types, including circulating platelets. The time pattern of TIMP-1 concentration in our study resembles closely the results of a previous study in septic patients.14 Interestingly, in patients with severe sepsis, we could show that TIMP-1 correlated inversely with platelet count and PT ratio, and positively with marker of increased thrombin formation and fibrin turnover: D-dimer. This is in line with the results from two previous studies with septic patients.15,32 TIMP-1 was recently studied in an animal model of septic DIC. Under standardised circumstances, an increase in TIMP-1 level was seen as early as 2 h after the onset of sepsis in mice that later developed DIC, and the level remained elevated until 72 h.16 Despite the heterogeneous patient material, the increase of
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Table 3 Correlations between MMP-8 and TIMP-1 with coagulation parameters, disease severity scores and inflammation parameters on days 1 and 2. MMP-8
MMP-8
TIMP-1
Day 1 (r)
Day 2 (r)
NA
NA
Day 1 (r)
0.660** P < 0.001 Platelet count −0.483 −0.626* −0.514 P = 0.026 P = 0.003 P = 0.014 Prothrombin −0.087 −0.080 −0.760** time ratio P = 0.709 P = 0.745 P < 0.001 D-dimer 0.307 0.253 0.384 P = 0.175 P = 0.297 P = 0.078 Fibrinogen 0.211 −0.035 −0.472 P = 0.373 P = 0.882 P = 0.031 Antithrombin −0.307 −0.016 −0.276 P = 0.176 P = 0.949 P = 0.213 0.519 TAT 0.168 0.323 P = 0.468 P = 0.164 P = 0.013 Fragment 1 + 2 0.147 0.060 0.300 P = 0.526 p = 0.801 P = 0.175 PC −0.197 −0.228 −0.533 P = 0.406 P = 0.347 P = 0.013 SOFA 0.363 0.238 0.634* P = 0.127 P = 0.392 P = 0.003 SOFA without 0.254 0.111 0.612* platelets P = 0.294 P = 0.693 P = 0.004 APACHE II 0.207 0.048 0.489 P = 0.396 P = 0.850 P = 0.029 CRP 0.125 0.469 −0.246 P = 0.588 P = 0.037 P = 0.269 White blood −0.236 −0.121 −0.118 cells P = 0.303 P = 0.611 P = 0.601
Day 2 (r) 0.716** P < 0.001 −0.641* P = 0.002 −0.620* P = 0.005 0.663* P = 0.002 −0.372 P = 0.106 0.215 P = 0.376 0.553 P = 0.011 0.550 P = 0.012 −0.586* P = 0.008 0.634* P = 0.010 0.440 P = 0.101 0.086 P = 0.735 0.460 P = 0.041 −0.068 P = 0.777
Data are shown as Spearman’s correlation coefficient r with P value and significance. *P < 0.01 and **P < 0.001. MMP-8, matrix metalloproteinase-8; TIMP-1, tissue inhibitor of metalloproteinase -1; TAT, thrombin-antithrombin complex; F1 + 2, prothrombin fragment 1 + 2; PC, protein C; SOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology and Chronic Health Evaluation II; CRP, C-reactive protein; NA, not applicable.
TIMP-1 in DIC patients was seen also in our clinical study, although in our study we could not show that an increase in TIMP-1 level precedes changes in traditional coagulation assays. Because TIMP-1 is a universal inhibitor of different MMPs, its increase can reflect changes also in other MMPs. For instance, in the largest clinical study of severe sepsis patients, non-survivors had higher TIMP-1 and lower MMP-9 levels,15 which may associate with inflammatory and prothrombotic or antifibrinolytic state, and contrib-
ute to the multiple organ dysfunction and eventually death. We avoid assuming any causality between TIMP-1 and MMP-8 levels, as the increase in TIMP-1 may be affected by other important mediators as well. To the best of our knowledge, this study was the first to investigate systemic concentrations of MMP-8 and TIMP-1 and their associations with different coagulation markers in patients with severe sepsis and overt DIC. However, our study confronted some limitations. First, the sample size of this pilot study was small. Nevertheless, we could show significant differences between study groups suggesting that both MMP-8 and TIMP-1 may have a role in septic coagulopathy. The results of this pilot study are novel, and to be confirmed they require a further, larger study where the effect of disease severity can be controlled. Second, the phase and the degree of inflammatory reaction likely varied between different patients, although the patients were recruited at the time of admission to the ICU and less than 48 h after the onset of the severe sepsis. This may explain the wide range in the concentrations of both MMP-8 and TIMP-1 observed during the first 2 days of the study. Third, the storage time of the serum samples before MMP-8 and TIMP-1 analyses was up to 4 years. As far as we know, there are no reports on serum MMP-8 and TIMP-1 behaviour over time. Instead, plasma TIMP-1 concentration over storage time has been shown to be very stable.33 However, we know that studied biomarkers may be affected when the assays are performed with samples thawed at different occasions. To avoid this, we have kept our samples in −80 degrees until analysis and made all the assays at the same time. Finally, TIMP-1, but according to our knowledge not MMP-8, has been shown to be present in platelets. Therefore, it may be possible that TIMP-1 can be released from platelets also in vitro, confounding the results. However, a rather negative correlation between TIMP-1 and platelet count was seen, indicating that those patients with lowest platelet count had higher TIMP-1 concentration. Conclusions We conclude that in severe sepsis-associated overt DIC, TIMP-1 is strongly elevated during the first 2 days after admission to the ICU, and it correlates Acta Anaesthesiologica Scandinavica 59 (2015) 176–184
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