Combination of thrombin-antithrombin complex, plasminogen activator inhibitor-1, and protein C activity for early identification of severe coagulopathy in initial phase of sepsis: a prospective observational study

Sepsis is frequently complicated with coagulopathy [1]. The severity of sepsis-associated coagulopathy is variable, ranging from subclinical abnormalities that are detectable by a mild decrease in platelet count and prolongation of global clotting times, to severe forms of coagulopathy or disseminated intravascular coagulation (DIC) [2]. The incidence of DIC is up to 25 to 50% in patients with sepsis [3].

Septic DIC is characterized by systemic intravascular activation of coagulation, and microvascular endothelial injury with impaired anticoagulation and insufficient fibrinolysis, which leads to widespread thrombosis in microvasculature. In sepsis, DIC has a feature of vascular endothelial dysfunction, as well as being an etiological factor in the failure of other organs: excessive thrombin generation and subsequent fibrin deposition exacerbate inflammation and ischemia, contributing to organ damage [4]. A number of studies have reported that DIC is an independent risk factor for organ dysfunction and mortality in patients with sepsis [2, 3, 5]. DIC might, therefore, be an important therapeutic target in the management of sepsis, and the development of reliable methods for early identification of DIC is a high priority.

However, the early diagnosis of sepsis-associated coagulopathy and evaluation of its severity is still challenging [6]. Currently, the overt DIC criteria of the International Society on Thrombosis and Haemostasis (ISTH) are the diagnostic standard for severe coagulopathy in sepsis [4]. Although the ISTH criteria for overt DIC are simple and widely used, and shown to be associated with organ failure and mortality, they have limited application in the early phase of sepsis to improve outcome [7, 8]. The ISTH overt DIC criteria use global markers, such as prothrombin time (PT) and platelet count for scoring. The coagulation factors and platelets are consumed and decrease over time because of progressive thrombin generation and endothelial injury, thus it takes several days to reveal their abnormalities and fulfill the overt DIC criteria in the course of sepsis [9, 10]. Furthermore, introduction of the concept of pre-DIC, which is considered as the stage prior to overt DIC, has failed to predict disease progression. An ISTH subcommittee defined non-overt DIC as compensated coagulopathy, or pre-stage DIC, for the early diagnosis of overt DIC [4]. However, previous studies have shown that only 10 to 30% of patients with non-overt DIC progressed to overt DIC, although the mortality rates were similar between patients with non-overt and overt DIC [6, 11].

In the past decade, there has been increasing evidence that inflammation and coagulation play pivotal roles in the pathogenesis of sepsis [12, 13]. Pro-inflammatory cytokines produced by the host response against infection stimulate tissue factor expression and lead to activation of coagulation. An activated coagulation system in turn modulates inflammatory activity through specific receptors, such as protease-activated receptors. Considering that excessive crosstalk between inflammation and coagulation is ongoing from the onset of sepsis, severe coagulopathy may have developed early in the course.

The objective of this study was to identify hemostatic biomarkers that can be used for early diagnosis of severe coagulopathy in patients with sepsis. We postulate that severe coagulopathy has already developed in the initial phase of sepsis, and is related to the subsequent fulfillment of the criteria for overt DIC [14]. We, therefore, evaluated the association between plasma biomarkers measured at the time of intensive care unit (ICU) admission and development of overt DIC in the following five days. We also investigated the hemostatic biomarkers as predictors for 28-day mortality.

The main findings of our study were as follows. 1) Coagulopathy developed in the initial phase of sepsis, and the severity of hemostatic biomarker abnormalities on the day of admission was associated with the subsequent development of overt DIC. 2) Among all the studied biomarkers, TAT, PAI-1 and PC had the best discriminative power for the patients who newly developed overt DIC. 3) However, only TAT and PAI-1 on Day 0 were significant predictors of 28-day mortality among the diagnostic biomarkers for the development of overt DIC. In contrast, Day 2 markers had higher predictive power for 28-day mortality compared with Day 0 markers, suggesting that persistence of severe coagulopathy was correlated with mortality.

Inflammation and coagulation constitute two host defense systems with complementary roles against infection [13], which means that an overwhelming systemic inflammatory reaction in sepsis is accompanied by severe coagulopathy, and both may contribute to tissue damage in the early phase of sepsis. In our study, most patients with sepsis exhibited coagulation and fibrinolytic abnormalities at the time of ICU admission, which is consistent with the data from the PROWESS trial [1]. In addition, most hemostatic biomarkers measured on ICU admission were associated with subsequent fulfillment of overt DIC criteria. These results support the hypothesis that coagulopathy is present in the initial phase of sepsis, and the strategy to identify markers of acute ongoing coagulopathy, rather than to detect pre-DIC state, may be necessary for the early diagnosis of septic DIC.

The pathogenesis of DIC is primarily due to excess production of thrombin [20]. In sepsis, anticoagulation impairment and insufficient fibrinolysis also contribute to thrombin generation and fibrin deposition. Anticoagulation pathways such as the antithrombin and protein C systems are impaired because of increased consumption, decreased protein synthesis, extravasation and degradation by several proteolytic enzymes such as neutrophil elastase [21, 22]. The fibrinolytic system is largely suppressed by increased production of PAI-1, which is a principal inhibitor of this system [23, 24]. In our study, increased levels of TAT and PAI-1, and decreased PC activity, were observed at the time of ICU admission and each independently discriminated the patients who developed overt DIC from those who did not. Our findings indicate that activation of coagulation, anticoagulation impairment and insufficient fibrinolysis develop early in the course of sepsis, and these three mechanisms should be evaluated individually for the diagnosis of DIC in patients with sepsis.

In this study, we found that TAT, a marker of thrombin generation, and PAI-1, which is induced by pro-inflammatory cytokines, were highest at baseline and improved when diagnosis of DIC was made in patients who developed overt DIC. These significant trends were obvious in survivors with overt DIC. In non-survivors with overt DIC, elevated levels of TAT and PAI-1 persisted during the study period. Similar trends in those biomarkers were observed in an experimental model of sepsis and in clinical studies [25, 26]. TAT and PAI-1 have short half-lives and they are produced early in the course of septic coagulopathy, while other biomarkers, such as platelets, PT-INR or PC, are the markers of consumption. The differences in those biomarkers over time between survivors and non-survivors indicate that TAT and PAI-1 may well reflect disease progress in septic coagulopathy.

Current criteria for early diagnosis of DIC have some potential limitations. Considering easy implementation, most criteria, including ISTH non-overt DIC and JAAM acute DIC criteria, use readily available coagulation tests for scoring. However, it is clear that global coagulation tests, such as PT and platelet count, primarily reflect the result of consumption and impaired synthesis rather than direct ongoing coagulopathy. Kinasewitz et al. [27] and Dhainaut et al. [28] established a simple diagnostic scoring system for the acute phase of septic coagulopathy, but these systems depend partly on worsening trends of global markers, which take at least two days to identify.

Several hemostatic molecular biomarkers, including AT, PC, TAT, PIC and PAI-1, have also been evaluated in patients with sepsis, but the reported results were inconsistent [1, 24, 25, 28‐30]. Several possible explanations could account for these conflicting results. First, we demonstrated dynamic changes in the biomarkers within a few days in the initial phase of sepsis, which is consistent with previous studies [1, 25]; therefore, the timing of biomarker measurement is important for interpretation of the results. Second, the cutoff value is another factor that influences the diagnostic ability of biomarkers. Oh et al. [6] and Egi et al. [31] evaluated the cutoff value of the lower limit of normal (70%) in AT levels for ISTH non-overt DIC criteria, and showed that the diagnostic ability for overt DIC did not improve by adding AT to non-overt DIC criteria. In our study, AT activity, as well as PC, was decreased below the lower limit of normal, even in most of the patients without overt DIC, and the cutoff value of AT level to discriminate patients with and without overt DIC was much lower (46.1%). Last, most of the previous studies evaluated the impact of hemostatic biomarkers on prognosis in patients with sepsis. We found that some plasma biomarkers at baseline were good predictors for the development of overt DIC, but were less predictive for 28-day mortality compared with Day 2 markers, which indicates that persistence of coagulopathy, rather than just the development of it, influences the prognosis in patients with sepsis. In addition, multiple interactive systemic factors other than coagulopathy would be involved in the pathogenesis of organ failure and the risk of mortality. We, therefore, evaluated diagnostic and prognostic values of biomarkers individually.

There were some potential limitations to our study. First, this was a prospective observational study conducted in a single center with a relatively small population size. Although the overall rate of DIC matched that in previous studies [3, 32], our cohort included fewer pneumonia patients, who often die from respiratory failure rather than multiple organ failure, including DIC. A large validation study is needed to confirm our results. Second, there is no gold standard for diagnosis of or the criteria for intervention in sepsis-associated coagulopathy. We used the ISTH overt DIC criteria as the diagnostic standard, considering coagulopathy that fulfilled these criteria would be severe enough to be eligible for intervention. Third, although our management of sepsis followed the SSCG guidelines, and did not deviate from standard care, prophylactic anticoagulation and interventions, such as blood transfusion as well as AT substitution, may have influenced the levels of hemostatic biomarkers except for baseline profile, and their relationship with the scores of overt DIC. Last, our study lacked explanations about why TAT, PAI-1 and PC were the best diagnostic markers for overt DIC. We found a strong correlation among AT, PC, plasminogen and α₂-PI at baseline. Considering that the same mechanism of consumption might be the main reason for decreased activity of those biomarkers [33], it is unclear why PC had superior diagnostic ability. Of particular interest is the contrast between the diagnostic value of TAT and another thrombin generation marker, SF. One possible explanation is the differences in half-life or mechanisms of clearance, where TAT has a shorter half-life (10 to 15 minutes), compared with SF (several hours). Further study is needed to better understand the processes of these biomarkers, and for the development of new therapeutic strategies in septic DIC.

The results of our study provide evidence that almost half of the patients developed severe coagulopathy in the initial phase of sepsis, which was demonstrated by baseline abnormalities in hemostatic biomarkers and their strong association with subsequent fulfillment of overt DIC criteria. In particular, a single determination of TAT, PAI-1 and PC activity at ICU admission allowed early identification of severe coagulopathy, or DIC, leading to early intervention for patients with sepsis.