Associations Among Coagulation-Related Variables, Resolution of Disseminated Intravascular Coagulation (DIC), and Mortality in Patients with Sepsis-Induced DIC Treated with Recombinant Human Soluble Thrombomodulin: A Retrospective Observational Study

Background: We evaluated associations among coagulation-related variables, resolution of disseminated intravascular coagulation (DIC), and mortality in patients with sepsis-induced DIC treated with recombinant human soluble thrombomodulin (rTM). Methods: We retrospectively investigated patients with sepsis-induced DIC treated with rTM. Changes in coagulation-related variables before and after treatment with rTM were examined. Further, associations between coagulation-related variables and DIC resolution were evaluated. Results: A total of 123 patients were included. The platelet count, prothrombin international normalized ratio (PT-INR), and brin/brinogen degradation products (FDP) signicantly (p < 0.001) improved after rTM administration in survivors (n = 98), but not in nonsurvivors (n = 25). However, the DIC score signicantly (p < 0.001) reduced not only in survivors but also in nonsurvivors. PT-INR before rTM was signicantly (p = 0.0029) lower in patients attaining than not attaining DIC resolution (n = 87 and 36, respectively). The 28-day mortality was signicantly lower in patients attaining than not attaining DIC resolution (11.5% vs. 41.7 %, p = 0.0001). Conclusions: The DIC score signicantly reduced after rTM in both survivors and nonsurvivors. rTM might play an important role in improving DIC, especially when treatment with rTM is initiated in the early phase of DIC.


Background
Sepsis is the most common cause of disseminated intravascular coagulation (DIC), which leads to organ dysfunction through intravascular thrombosis [1,2]. In ammation deregulates the coagulation cascade and leads to various coagulopathies including DIC in septic patients [2,3,4]. The endothelium plays a key role in all major pathways involved in the cross-talk between in ammation and coagulation [4]. The essential therapy in patients with sepsis is treatment of underlying infection using appropriate antibiotics and source controls. However, DIC can persist or develop even after the initiation of appropriate therapy.
In such situations, supportive therapy to manage DIC and to modulate cross-talk between in ammation and coagulation may reduce morbidity and mortality in patients with sepsis [4].
Thrombomodulin (TM), which is localized in the vascular endothelium, acts as an anticoagulant. After it binds to thrombin, the thrombin-TM complex activates protein C to produce activated protein C, which inactivates factors Va and VIIIa, thereby inhibiting formation of thrombin (IIa) [5,6]. Furthermore, TM attenuates in ammation through various mechanisms [7,8]. TM directly interferes with complement activation via the classical and lectin pathways and modulates innate immunity [9]. TM binds highmobility group-B1 DNA-binding protein (HMGB1) and suppresses in ammation [10], subsequently promoting degradation of HMGB1 [11]. Since HMGB1 is a late cytokine mediator of endotoxin lethality and systemic in ammation, TM may protect septic patients also by decreasing HMGB1 [12].
A phase III randomized controlled trial (RCT) comparing e cacies of recombinant human soluble TM (rTM) and heparin in patients with DIC showed that rTM improved DIC and relieved bleeding symptoms more effectively than heparin [13]. Accordingly, rTM was approved as an agent for treatment of DIC in 2008 in Japan, and has since been used clinically. A multicenter propensity score analysis of sepsisinduced DIC reported that rTM reduced in-hospital mortality in patients requiring ventilation [14]. In addition, a multicenter retrospective study comparing 452 propensity score-matched pairs of septic patients with DIC revealed that rTM signi cantly reduced in-hospital mortality [15]. Although the Sepsis Coagulopathy Asahi Recombinant LE Thrombomodulin (SCARLET) trial performed as a multicenter RCT found that rTM did not signi cantly reduce 28-day mortality compared with a placebo in patients with sepsis-associated coagulopathy [16], a post-hoc analysis of this trial revealed that the subgroup of rTMtreated patients with higher baseline levels of prothrombin fragment 1.2 and thrombin-antithrombin complex had lower mortality than placebo-treated patients [17]. Therefore, rTM may play an important role in improving coagulopathy and mortality in septic patients with DIC.
The aim of the present study was to evaluate associations among in ammation-or coagulation-related variables, resolution of DIC, and mortality in patients with sepsis-induced DIC treated with rTM.

Study design and subjects
Prior to this retrospective observational study, the study protocol was approved by the Ethics Committee of Juntendo University Hospital (approval number 19-045), with a waiver of patients' written informed consent.
We retrospectively studied patients who were diagnosed with sepsis-induced DIC and treated with rTM in the intensive care unit at Juntendo University Hospital between April, 2015 and December, 2019. Sepsis was de ned according to the Sepsis-3 criteria [18]. DIC was diagnosed based on a DIC score ≥ 4 points on the Japanese Association for Acute Medicine (JAAM) DIC score [19], which is the sum of the points (graded 0 to 3) for the following four components: systemic in ammatory response syndrome criteria ≥ 3 is scored as 1; a platelet count ≥ 80 and < 120 ×10 9 /L or a > 30% decrease within 24 h is scored as 1, a platelet count < 80 ×10 9 /L or a > 50% decrease within 24 h is scored as 3; a prothrombin international normalized ratio (PT-INR) ≥ 1.2 is scored as 1; a brin/ brinogen degradation products (FDP) level ≥ 10 and < 25 µg/mL is scored as 1, and a FDP level ≥ 25 µg/mL is scored as 3.
rTM at a dose of 380 U/kg was intravenously infused for 30 min once a day, which was usually continued for 6 days or until improvement of DIC was achieved. In patients with severe renal dysfunction, the dose was reduced to 130 U/kg, considering its elimination by the kidneys.

Data collection
Data on patients' clinical characteristics and laboratory tests data were collected from medical records, including age, sex, the outcome (28-day mortality), the Acute Physiology and Chronic Health Evaluation II (APACHE II) score [20], the Sequential Organ Failure Assessment (SOFA) score [21], the DIC score [19], the platelet count, PT-INR values, levels of FDP and C-reactive protein (CRP), surgical interventions, sites of infection, infecting microorganisms, documented bacteremia, and the dose and duration of rTM administration.
Resolution of DIC was de ned as the DIC score ≤ 3 on the day after the last treatment with rTM [22]. To assess the e cacy of rTM, coagulation-and in ammation-related variables before and after treatment with rTM were examined and compared between survivors and nonsurvivors. In addition, these variables before rTM were compared between patients attaining and not attaining DIC resolution.

Statistical analysis
Qualitative data are expressed as numbers (percentages) and were analyzed using the chi-square test.
Quantitative data are expressed as medians [interquartile ranges: IQRs]. Changes in variables over time were analyzed with the Wilcoxon signed-rank test. Variables were compared between dichotomized groups with the Mann-Whitney U test. A p value less than 0.05 was considered statistically signi cant.

Patient characteristics
A total of 123 patients were diagnosed with sepsis-induced DIC and treated with rTM. Baseline clinical characteristics in these patients are shown in Table 1. The 28-day mortality rate in the total cohort was 20.6 % (25/123). APACHE II and SOFA scores were signi cantly lower in survivors than in nonsurvivors (p < 0.01). However, other variables, including age, sex, requirements of surgery, sites of infection, infecting microorganisms including antibiotic-resistant bacteria, or documented bacteremia, were not different between survivors and nonsurvivors.

Effects of rTM on coagulation-and in ammation-related variables
In the total cohort, the dose and duration of rTM administration were 380 [130-380] U/kg/day and 5 [3][4][5][6]  Coagulation-and in ammation-related variables before and after rTM administration are shown in Table 2. Before treatment with rTM, the platelet count, FDP levels, the DIC score, or CRP levels were not signi cantly different between survivors and nonsurvivors, and only PT-INR was signi cantly lower in survivors than in nonsurvivors (p = 0.0395).
After treatment with rTM, the platelet count increased signi cantly (p < 0.0001) while PT-INR values, FDP levels, CRP levels, and the DIC score decreased signi cantly (p < 0.0001) in the total cohort, and also in survivors. On the other hand, in nonsurvivors, the platelet count, PT-INR values, or FDP levels did not change signi cantly, although the DIC score and CRP levels decreased signi cantly (p < 0.001), albeit to lesser extents compared with survivors. After treatment with rTM, the platelet count was signi cantly higher while PT-INR values, FDP levels, and CRP levels, and the DIC score were signi cantly lower in survivors than in nonsurvivors (p = 0.0038 for FDP, and p < 0.0001 for others). The DIC resolution rate was signi cantly higher in survivors than in nonsurvivors (77/98 [78.6 %] vs. 10/25 [40.0 %], p < 0.001).
Comparisons of variables before treatment with rTM between patients attaining DIC resolution (n = 87) and those not attaining DIC resolution (n = 36) are shown in Table 3. The APACHE II, SOFA, and DIC scores, and PT-INR values were signi cantly lower while the platelet count was signi cantly higher in patients attaining than not attaining DIC resolution (p < 0.01, respectively). Other variables, including demographic data or levels of FDP and CRP were not different between these patients. The 28-day mortality was signi cantly lower in patients attaining than not attaining DIC resolution ( There were no major rTM-related signi cant adverse events, including bleeding, in any patients.

Discussion
In the present study, we investigated mortality and DIC resolution in patients with sepsis-induced DIC who were treated with rTM. Comparisons between survivors and nonsurvivors showed that mortality was signi cantly associated with the APCHE II as well as SOFA score and PT-INR values before treatment with rTM, whereas it was not associated with the DIC score, the platelet count, or levels of FDP and CRP. After treatment with rTM, the DIC score and CRP levels signi cantly decreased in the total cohort, and not only in survivors but also in non-survivors. However, decreases in the DIC score and CRP levels were signi cantly less in nonsurvivors. Further, although the platelet count, PT-INR values, and FDP levels improved signi cantly after treatment with rTM in the total cohort, and also in survivors, they did not improve signi cantly in nonsurvivors. The DIC resolution rate was signi cantly higher in survivors than in nonsurvivors (78.6 % vs. 40.0 %). Comparisons between patients attaining and not attaining DIC resolution showed that DIC resolution was signi cantly associated with APACH II, SOFA, and DIC scores, the platelet count, and PT-INR values before treatment with rTM, but not with FDP or CRP levels. The mortality was signi cantly lower in patients attaining than not attaining DIC resolution (11.5 % vs. 41.7 %).
Reportedly, endothelial expressions of TM and protein C receptors were signi cantly reduced in patients with sepsis [23]. Accordingly, replacement with rTM is expected to compensate for such downregulations of the endothelial thrombomodulin-protein C receptor pathway in septic patients. Although the primary anticoagulant effect of rTM is mediated by activated protein C, plasma activated protein C levels did not increase after administration of rTM in septic patients with DIC [24]. These results suggest that activated protein C acts only locally and does not circulate systemically in response to rTM administration. Accordingly, the risk of bleeding associated with rTM could be signi cantly lower, compared with heparin [13]. Indeed, we did not experience any signi cant adverse events related to rTM, including bleeding, in the present study.
TM, which is localized in the vascular endothelium, integrates biological pathways related to coagulation, innate immunity, and in ammation, and TM thus has anti-in ammatory as well as anticoagulation effects [5][6][7][8]10]. In particular, the N-terminal lectin-like domain of rTM attenuates in ammation through inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, and degradation of HMGB 1 [8,9]. The recombinant lectin-like domain of rTM binds to Gram-negative bacteria and lipopolysaccharides, enhances their elimination from the body, and protects the host by suppressing in ammatory responses [25]. In the present study, CRP, which is one of the most commonly used biomarkers of infection and in ammation [26], signi cantly decreased after treatment with rTM not only in survivors and but also in nonsurvivors. These results suggested that rTM, in addition to antibiotics, played important roles in ameliorating in ammation.
In the present study, severity of sepsis indicated by APACHE II and SOFA scores before treatment with rTM was more signi cantly associated with mortality, compared with severity of coagulopathy indicated by the DIC score. Among coagulation-related variables examined before rTM, only PT-INR values were only marginally associated with mortality. After treatment with rTM, however, all coagulation-related variables improved signi cantly in survivors, but not in nonsurvivors. These results suggested that not only favorable responses of sepsis to therapies with antibiotics and rTM, but also favorable responses of DIC to them were crucial for improving the patients' outcome.
Since the background of sepsis is highly heterogeneous, it is impossible to nd one treatment to t all situations. Therefore, clinicians have to select appropriate patients who are well suited to any supportive therapy including rTM. Using transcriptomics, a previous study classi ed bacterial sepsis into three subtypes, including "In ammopathic", "Adaptive", and "Coagulopathic" subtypes, among which the "Coagulopathic" subtype with more signi cant coagulopathy was associated with higher mortality, suggesting that the early development of hypercoagulability in sepsis is associated with higher mortality [27]. Reportedly, rTM could effectively suppress hypercoagulability indicated by increased levels of prothrombin fragment 1.2 and thrombin-antithrombin complex, compared with a placebo [28]. Further, rTM-treated patients who had higher baseline levels of prothrombin fragment 1.2 and thrombinantithrombin complex had lower mortality than placebo-treated patients, suggesting that rTM is effective in reducing mortality in patients with hypercoagulability [17]. On the other hand, because the anticoagulant effects of rTM are dependent on thrombin activity in circulation, the ability of rTM to activate protein C and thus, the anticoagulant effect of rTM are expected to be reduced after thrombin depletion [5,6]. The present study showed that patients with milder sepsis and milder coagulopathy before treatment with rTM were signi cantly more likely to attain DIC resolution, and patients who attained DIC resolution were associated with signi cantly less mortality. In this regard, diagnosing DIC in early phases of sepsis and starting treatment of DIC with rTM before depletion of coagulation factors might be crucial for inducing DIC resolution and reducing mortality in patients with sepsis-induced DIC.
This study had several limitations. First, only a small number of patients were included because of the study conducted at a single institute. Second, the data were retrospectively collected. Third, there was no control group. Because rTM therapy has been recognized as a standard treatment for sepsis-induced DIC in Japan, ethics consideration prevented us from allocating patients to a group without rTM. Alternatively, we compared coagulation-and in ammation-related variables before and after treatment with rTM. Further, we compared these variables between survivors and nonsurvivors, and between patients attaining and not attaining DIC resolution. Clearly, further studies are required to verify the e cacy of rTM in patients with sepsis-induced DIC.

Conclusions
In the present study, the DIC score signi cantly reduced after rTM administration in the total cohort, and not only in survivors but also in nonsurvivors. After treatment with rTM, the platelet count, PT-INR values, and FDP levels signi cantly improved in survivors, but not in nonsurvivors. The platelet count before treatment with rTM was signi cantly higher while PT-INR values and the DIC score before treatment were signi cantly lower in patients attaining than not attaining DIC resolution. Patients attaining DIC resolution was associated with lower mortality. Therefore, rTM may play an important role in improving DIC in septic patients, especially when treatment with rTM is initiated in the early phase of DIC, although further studies are needed to verify its e cacy in patients with sepsis-induced DIC. The study protocol was approved by the Ethics Committee of Juntendo University Hospital (approval number 19-045), with a waiver of patients' written informed consent.

Consent for publication
Not applicable Table 2 DIC score, platelet count, coagulation biomarkers, and CRP level before and after rTM administration in patients with sepsis-induced DIC Table 3 Comparison of various variables before rTM administration between patients attaining DIC resolution and those not attaining DIC resolution