Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism

Abstract

Introduction: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States.

Methods: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice.

Results: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism.

Conclusions: Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Figure 1. Histone H3 levels, as measured by ELISA, in plasma of patients with sepsis and DIC.

Plasma histone H3 levels were significantly higher in non-survivors (n = 16, minimum = 0, maximum = 1464.4, median = 15.5) compared with healthy volunteers (n = 15, minimum = 0, maximum = 0) as analyzed by Steel-Dwass test.

Figure 2. Extracellular histones cause fatal thromboembolism in mice.

(A) The lethal effect of extracellular histones. Mice were intravenously injected with histones (0-80 µg/g, n = 7-12 per group), and survival was analyzed. (B) Histone-induced thrombocytopenia. Numbers of platelets (PLT), red blood cells (RBC), and white blood cells (WBC) in blood 10 min after infusion with histones (0-95 µg/g, n = 3-7 per group, mean ± S.D.) are shown. Data are presented as percentage of the vehicle group (0 µg/g histones). (C) Distribution of DyLight488-labeled platelets and Alexa-Fluor 594-labeled fibrin(ogen) in lung tissue 10 min after infusion with vehicle or 75 µg/g histones. Nuclei were stained with DAPI. Representative images of n = 4. Scale bar = 100 µm. * P < 0.05 and ** P < 0.01 compared with the vehicle group.

Figure 3. Extracellular histones cause acute right-sided heart failure and ventricular arrest.

(A) ECG in mice intravenously injected with vehicle or histones (80 µg/g). The voltage between the right limb and the feet (lead II) was recorded. Data presented are representative of three independent experiments. (B) Still images from Movies S1-S3. Transthoracic echocardiography was performed in mice before and after intravenous injection of histones. Extracellular histones caused dilatation of the right ventricle (RV) and displacement of the interventricular septum toward the left ventricle (LV).

Figure 4. Extracellular histones cause consumptive coagulopathy.

Plasma fibrinogen (A), APTT (B), and PT (C) of mice 10 minutes after injection with vehicle or histones (20-80 µg/g, n = 4-10). ** P < 0.01 compared with the vehicle group.

Figure 5. Recombinant thrombomodulin (rTM) suppresses the activity of extracellular histones.

(A) Binding assays with a quartz crystal microbalance (QCM) twin sensor system. Two channels of a sensor chip were coated with either histones (1 mg/ml) or BSA (1 mg/ml). The sensor chip was placed into the NAPiCOS Auto and then perfused with rTM (1 mg/ml). The interaction between molecules was recognized as the change in frequency of a quartz crystal resonator. (B) Inhibition of histone H4-mediated platelet aggregation by rTM. Washed platelets were stimulated with histone H4 (5 µg/ml) preincubated with rTM (5-25 µg/ml) or BSA (25 µg/ml). rTM, but not BSA, inhibited histone-induced platelet aggregation (n = 3-7 per group, mean ± S.D.). (C) Inhibition of histone H3-induced platelet aggregation by rTM. Washed platelets were stimulated with histone H3 (25 µg/ml) or collagen (1.44 µg/ml) in the presence or absence of rTM (15 µg/ml). rTM inhibited histone-induced platelet aggregation, but not collagen-induced platelet aggregation. Representative data of three independent experiments are shown. ** P < 0.01 compared with the histone-alone group.

Figure 6. Recombinant thrombomodulin (rTM) protects mice against histone-induced fatal thrombosis.

(A) The protective effect of rTM on histone-induced thrombocytopenia. Pretreatment with rTM (40 or 80 µg/g) 30 min before histone injection (40 µg/g) prevented histone-induced thrombocytopenia in mice (n = 3-4 per group, mean ± S.D.). ** P < 0.01 compared with the histone-alone group. (B) The protective effect of rTM on histone-induced VWF-rich thrombus formation. Arrowheads indicate intravascular thrombi. Pretreatment with rTM (110 µg/g) 30 min before histone injection (75 µg/g) suppressed histone-induced VWF-rich thrombus formation. Representative images of n = 5 (histone-alone) and n = 4 (histones + rTM). Scale bar = 20 µm. (C) The protective effect of rTM on histone-induced right-sided heart failure. Right ventricular diastolic dimension was measured before and 10, 20, and 30 minutes after histone injection using conventional M-mode echocardiography. Arrowheads indicate right ventricles. Pretreatment with rTM suppressed histone-induced dilatation of the right ventricle. RV: right ventricle. LV: left ventricle. Representative images of n = 4 are shown. Scale bar = 5 mm. (D) Quantitation of (C). Pretreatment with rTM (110 µg/g) 30 minutes before histone injection (75 µg/g) suppressed histone-induced dilatation of the right ventricle. The data of histone-alone group in 30 minutes are lacking because none of the mice in this group survived for up to 30 minutes after histone injection. ** P < 0.01 compared with the histone-alone group. (E) The protective effect of rTM on histone-induced lethality. Mice were treated with a single injection of 110 µg/g of rTM 30 minutes before histone injection (75 or 90 µg/g) or double injection of 110 µg/g of rTM 30 minutes before and 90 minutes after histone injection (90 µg/g). rTM therapy significantly improved survival of histone-injected mice (n = 8-21 per group, P < 0.01 compared with mice injected with histone alone).