Disseminated intravascular coagulation (DIC) occurs in the presence of severe underlying disease, such as severe infection, trauma, hematologic malignancy, and solid tumor (1). Several lines of evidence indicate that DIC increases the risk of death (2-4). In DIC, excessive activation of coagulation results in fibrin formation in systemic microvessels, which leads to multiple organ dysfunction syndrome (MODS). Widespread coagulation consumes platelets and coagulation proteins, thereby inducing bleeding to varying degrees (1).
The pathogenic mechanism of DIC in patients with severe infection involves excessive activation of the coagulation cascade mediated by proinflammatory cytokines and other mediators that are upregulated by endotoxin and other bacterial products (1). The inflammatory and procoagulant responses to infection are closely related, and uncontrollable activation of these reactions leads to MODS (5-7). In the pathogenesis of MODS in patients with DIC, vascular endothelial damage plays an important role (8).
In recent studies on severe sepsis, treatment with activated protein C (APC) (9) or antithrombin (AT) (10) in patients with complications of DIC has reduced mortality rates, suggesting the possibility that antithrombotic strategies improve patient prognosis (11, 12). Treatment with anticoagulants, such as heparin and APC, is recommended in newly published guidelines for the management of DIC (13).
Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface that is an important player in the natural anticoagulant and anti-inflammatory system. When excess thrombin is generated in circulating blood, thrombin binds to TM, leading to the inactivation of the procoagulant activity of thrombin. The thrombin-TM complex then activates protein C (PC) to form APC, which in turn cleaves and inactivates factors Va and VIIIa. As a result, TM acts as a negative feedback regulator of coagulation activation (14). In addition to the anticoagulant property, APC has anti-inflammatory and cytoprotective properties (15, 16). Thrombomodulin directly regulates inflammation through its lectin-like domain independently of PC activation (17-22).
Thrombomodulin α (TM-α) is a recombinant human soluble TM possessing an extracellular domain that includes the active site (23). Thrombomodulin α serves as a negative feedback regulator of blood coagulation (24, 25). In a rat model, TM-α blocked LPS-induced inflammatory response and increased survival rate (26, 27). Furthermore, in a rat model of crush injury, the effects of TM-α and volume resuscitation were demonstrated (28). We have reported the clinical effects of TM-α on DIC in patients with hematologic malignancy and/or infection and its superiority to heparin for the resolution of DIC (29).
To support our hypothesis that TM-α could be effective in the treatment of infection-induced DIC, we conducted a probing study by a retrospective sub-population analysis of the results of our previous study (29), focusing on the patients having infection.
MATERIALS AND METHODS
Study design and patient population
Subjects in the present study comprised a subset of those in the phase 3 clinical trial reported by Saito et al. (29). The trial involved a prospective double-blind study of patients with DIC caused by hematologic malignancy and/or infection, with unfractionated heparin used as a comparator. The subset of patients with infection was extracted from that study. Patients with hematologic malignancy, aplastic anemia, and megakaryocytopenia due to cancer chemotherapy were excluded.
In the phase 3 study, the Japanese Ministry of Health, Labour, and Welfare DIC diagnostic criteria (JMHW criteria) (30) were used to include patients and to assess efficacy (29). Details of other inclusion/exclusion criteria have been described previously (29). Patients were assigned to either the TM-α group or the heparin group, and the study drug used was blinded using the double-dummy method. Either TM-α (0.06 mg·kg−1, intravenously infused for 30 min once daily) or unfractionated heparin (8 U·kg−1·h−1, intravenously infused continuously) was administered for 6 consecutive days. During the study drug infusion period, use of anticoagulants (including synthetic protease inhibitors), antiplatelet agents, and fibrinolytic agents was prohibited. However, use of blood products (excluding AT), catheter heparinization (≤1,000 U·d−1), and urokinase (≤10,000 U per administration) was allowed. During the study drug infusion period, hemodialysis for renal failure and blood purification therapy were contraindicated. The primary end point was the rate of DIC resolution, as assessed by JMHW criteria at 7 days after the start of study drug infusion (or at the time of discontinuing infusion). The 28-day all-cause mortality rate was assessed as a secondary end point.
The phase 3 study was conducted in compliance with good clinical practice and ethical principles of the Declaration of Helsinki. Prior approval was obtained from the ethics review boards of all participating institutions. Written informed consent was obtained from all patients (or acceptable representatives).
DIC diagnosis and assessment
In addition to the JMHW criteria used to diagnose and assess DIC in the phase 3 study, the new DIC diagnostic criteria for critically ill patients defined by the Japanese Association for Acute Medicine (JAAM criteria) (31) were also used. Disseminated intravascular coagulation resolution rates were assessed using both JMHW and JAAM criteria at 7 days after the start of study drug infusion (or at the time of discontinuing infusion). According to the JAAM criteria (31) and shown in Table 1, DIC is defined as a total score of 4 or greater. As the JAAM criteria were not yet available at the start of the phase 3 study, some items of systemic inflammatory response syndrome (SIRS) were not recorded in the patients analyzed in the present study. Therefore, SIRS items were excluded in the present analysis, and having DIC was defined by a score of 4 or greater, and DIC was considered resolved if the score reached 3 or less.
Platelet counts, fibrin/fibrinogen degradation products, and AT in plasma were measured before and after the study drug infusion.
Fibrin/fibrinogen degradation products were evaluated by the rate of change: value at the end of study drug administration (or withdrawal) divided by value at baseline. Platelet counts and AT were evaluated by absolute difference: value at the end of study drug administration (or withdrawal) minus value at baseline. Medians and 95% confidence of intervals (CIs) of difference between the two groups were calculated. If 95% CI does not cross 0, difference between the two groups was judged to be statistically significant.
Regarding baseline demographic data, categorical variances were expressed as frequencies and percentage values and compared by chi-square test. The difference of the mortality rate between the patients in whom DIC resolved and did not resolve was compared by Fisher's exact test.
Patients and demographics
Of the 227 full-analysis-set patients in the phase 3 study, 147 patients were identified as having a noninfectious comorbidity leading to severe thrombocytopenia. These 147 patients were excluded, and the remaining 80 patients served as subjects in the present sub-population analysis.
Table 2 shows baseline demographic data. There were no marked differences in baseline data other than age between the TM-α and heparin groups. All patients were concurrently administered with antibiotics, except for two patients in the TM-α group (data not shown).
The 28-day survival curves for the TM-α and heparin treatment groups are shown in Figure 1. The 28-day mortality rate for the heparin group was 31.6% (12/38) compared with 21.4% (9/42) for the TM-α group, representing an absolute difference of 10.2% (95% CI of difference, −9.1% to 29.4%).
In patients with AT levels of less than 50% at the start of study drug infusion, the mortality rate was not higher in either TM-α or heparin treatment compared with patients with AT levels of 50% or greater (Table 3). For PC levels of less than 20% at the start of study drug infusion, the mortality rate was not higher in TM-α treatment compared with that for patients with PC levels of 20% or greater (Table 3).
DIC resolution assessed by JMHW criteria and JAAM criteria
Disseminated intravascular coagulation resolution rate was assessed using both JMHW and JAAM criteria (Table 4). At the start of study drug infusion, two patients (both in the heparin group) did not satisfy the JAAM criteria (<4 points). Disseminated intravascular coagulation resolution could not be determined with JAAM criteria because of missing coagulation tests in two patients (both in the TM-α group). Disseminated intravascular coagulation resolution rates for the remaining patients at 7 days after the start of study drug infusion (or at the time of discontinuing infusion) are shown in Table 4. The difference in resolution rates between groups as determined by the JAAM criteria resembled that determined by the JMHW criteria.
The 28-day mortality rate among patients in whom DIC resolved (according to JAAM criteria) was less than that in whom DIC did not resolve: 8.5% (4/47) and 44.8% (13/29), respectively (P = 0.0004, Fisher's exact test). In the TM-α treatment group, the 28-day mortality rate among patients in whom DIC resolved was 3.7% (1/27), the rate for those in whom DIC did not resolve was 46.2% (6/13) (P = 0.0026, Fisher's exact test). In the heparin treatment group, the 28-day mortality rate among patients in whom DIC resolved was 15.0% (3/20); the rate for those in whom DIC did not resolve was 43.8% (7/16) (P = 0.0732, Fisher's exact test). Mortality rates were significantly lower for patients in whom DIC resolved than those in whom DIC did not resolve, and this tendency was more pronounced in the TM-α group than in the heparin group.
The absolute difference in AT in the TM-α group was significantly higher than that in the heparin group (Fig. 2; 95% CI did not cross 0). Fibrin/fibrinogen degradation products and platelet counts tended to normalize in the TM-α group, but there was no statistical difference in rate of change and absolute difference between the TM-α and heparin groups (Fig. 2; 95% CI crossed 0).
Bleeding-related adverse events
Study drug infusion was discontinued because of onset of a bleeding-related adverse event (intrapleural bleeding) in one patient in the heparin group but not in any patients in the TM-α group.
For the TM-α group, no patient died due to bleeding-related adverse events. For the heparin group, two patients died due to bleeding-related adverse events (DIC-induced bleeding, n = 1; gastrointestinal bleeding, n = 1).
Disseminated intravascular coagulation is commonly associated with severe infection, and the derangement of coagulation in patients with infection may contribute to organ dysfunction and mortality. Evidence suggests that amelioration of DIC is associated with improved survival (11, 12). Clinical management of DIC may be hampered by the lack of clear diagnostic criteria for this syndrome. Nevertheless, DIC has been diagnosed in Japan using the JMHW criteria for more than 20 years. Recently, the JAAM criteria were proposed for simple and early diagnosis and have been validated in a prospective study (31).
Our phase 3 study on TM-α (29) used the JMHW criteria as an inclusion criteria. The present study attempted to apply the JAAM criteria, which had not been published at the time of the phase 3 study, to evaluate the effects of TM-α. Among patients enrolled using the JMHW criteria, all but two patients were diagnosed as having DIC by the JAAM criteria. Disseminated intravascular coagulation resolution rates by these two diagnostic criteria were similar in the present study patients. Furthermore, the mortality rate on day 28 was lower for patients in whom DIC resolved within 7 days according to JAAM criteria, suggesting that resolution of DIC is associated with lower mortality. As reported by Gando et al. (31), the JAAM diagnostic criteria are useful for predicting prognosis in DIC patients.
As the JAAM criteria had not yet been devised at the start of the phase 3 study on TM-α, complete assessment could not be made using these criteria. Specifically, in the protocol of the phase 3 study, SIRS score and daily platelet counts were not included as evaluation items; therefore, these scores were not included in calculations of total DIC score in the present study. To estimate the influence of SIRS score on analysis of DIC resolution rate, analysis was conducted by defining DIC as a total score of 3 or greater (in other words, all patients were assumed to have point 1 of SIRS items), but results remained the same (data not shown). In the future, evaluation using JAAM criteria as inclusion criteria in a comparative study will be necessary.
The 28-day mortality rate was 31.6% (12/38) for the heparin group and 21.4% (9/42) for the TM-α group, with an intergroup absolute difference of 10.2% (95% CI of difference, −9.1% to 29.4%). The 95% CI crossed 0, and the mortality difference between the two groups was not statistically significant. The present probing study was a retrospective analysis and did not have enough power to detect the statistical significance; this is one limitation of the study. The difference in mortality rate for the entire infectious patient group in the previous trial was 6.6% (TM-α group, 28.0% [14/50]; heparin group, 34.6% [18/52]) (29). The difference between the TM-α and heparin groups was enhanced by selecting patients with DIC caused by infection, that is, removing patients in whom the effects of bone marrow megakaryocytopenia were marked from the previously reported group. In addition, when only patients with overt DIC in the PROWESS study were retrospectively analyzed, the mortality rates for the drotrecogin alfa and placebo groups were 30.5% (n = 233) and 43.0% (n = 221), respectively, with a relative risk reduction of 29.1% (11). Although the statistical significance of the mortality reduction was not obtained, the relative risk reduction for death in the TM-α group as compared with the heparin group was 32.1%, similar to the reduction obtained in the PROWESS study.
Thrombomodulin α exerts an anticoagulant effect by activating PC, whereas heparin exerts anticoagulant activities by facilitating AT activity. Outcomes were, hence, analyzed with respect to PC and AT levels at the start of the study drug infusion. In severe patients with infection, lower AT levels are associated with poorer prognosis (32). However, in the present study, the mortality rate in patients with AT levels of less than 50% at the start of study drug infusion was not higher, in either TM-α treatment or heparin treatment, as compared with patients with AT levels of 50% or greater. The reason for this could not be clarified. During the drug infusion period in the present study, use of anticoagulants (including AT) was prohibited, but no provision was set after the end of the study drug infusion. As there was no restriction on administration of AT preparations after the study drug infusion, AT administration after study drug infusion could have somehow influenced outcomes by day 28.
Antithrombin levels in TM-α group increased significantly compared with the heparin group, despite that AT preparations were not administered during the study drug infusion. The reason for this is unclear, but we speculate that the consumption and the capillary leak of AT were suppressed by TM-α administration.
Thrombomodulin α is an anticoagulant and thus carries the potential to increase the risk of bleeding. In the phase 3 study, the frequency of adverse bleeding events was lower in the TM-α group than in the heparin group (29). Also, in the present subanalysis population, adverse events of bleeding leading to discontinuation of study drug or death seemed to be less frequent in the TM-α group than in the heparin group. The 28-day mortality rate among patients in whom DIC resolved seemed to be less in patients with TM-α treatment than in patients with heparin treatment. This suggests that TM-α treatment has effects not only on DIC but also on other morbidities such as inflammation, although we did not measure the established biochemical markers such as IL-6 in the present study.
In recent years, TM has been shown to inhibit the adhesion of neutrophils to vascular endothelia (17). Thrombomodulin has also been suggested to be an important molecule for various defense mechanisms, including control of the coagulation system and suppression of inflammation (18). High-mobility group B1 DNA-binding protein is known as a lethal mediator during the late inflammation period, and TM reportedly inactivates this protein by binding and enhancing its proteolysis by thrombin (19, 20). Furthermore, the lectin-like domain of TM reportedly binds to LPS and neutralizes LPS-induced inflammatory responses (21). Thrombomodulin is thus known to be involved not only in anticoagulation but also in anti-inflammation and to play a central role in defense mechanisms involving endothelial cells (33). Like natural TM, TM-α possesses anti-inflammatory activity in addition to anticoagulative activity (19, 20). As a result, TM-α therapy may be a useful treatment in DIC patients complicated by severe infection, where inflammatory reactions and coagulation abnormalities occur concurrently to cause the DIC. The present analysis supports this possibility.
A limitation to the present study is the retrospective analysis of subgroups of patients who were not predefined in the original analytical plan and that baseline demographic data and disease characteristics between the two groups were not completely balanced. In addition, because of a limited number of the patients in the subgroups, we were unable to confirm the effects of TM-α on infection-induced DIC patients. However, with an implication of the effectiveness obtained in the present study, a prospective randomized phase 2 trial of 750 patients with sepsis-induced DIC is in progress, and the subsequent verification trial (phase 3) should address the hypothesis that treating DIC in severe infection with TM-α might significantly reduce mortality.
The authors thank all of the investigators and study coordinators who participated in the phase 3 study of TM-α. They are also deeply grateful to the late K. Ohsato, MD, for assisting in the phase 3 trial of TM-α.
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