Measurement of plasma histone H3 levels

All experiments involving human blood conformed to the provisions of the Declaration of Helsinki and were approved by the Ethics Committee of Kagoshima University. Written informed consent for participation in this study was obtained from all individuals. Diagnoses of sepsis and DIC were made according to the guidelines of the Society of Critical Care Medicine Consensus Conference Committee and the diagnostic criteria established by the Japanese Association for Acute Medicine criteria (JAAM DIC criteria), respectively. Plasma samples were collected from 15 healthy volunteers and 26 patients with sepsis and DIC. Polystyrene microtiter plates were coated with anti-human histone H3 polyclonal antibody (Abcam, Cambridge, United Kingdom) and were incubated overnight at 2-8°C. The plates were washed three times, and the remaining binding sites were blocked by PBS containing 1% bovine serum albumin (BSA). The plates were washed again then incubated with the calibrator and samples for 24 hours at room temperature. The plates were washed again, and incubated with anti-human histone H3 peroxidase-conjugated polyclonal antibody (Abcam) for 2 hours at room temperature. After another washing step, the chromogenic substrate 3,3’,5,5’-tetra-methylbenzidine (Dojindo Laboratories, Kumamoto, Japan) was added to each well. The reaction was terminated by the addition of 0.35 mol/L Na ₂SO₄, and the absorbance at 450 nm was read using a microplate luminescence reader (Bio-Rad, Hercules, CA).

Mice

Experiments involving animals were approved by the Institutional Animal Care and Use Committee of Kagoshima University, Kagoshima, Japan. Male C57BL/6 mice (9 to 13 weeks old, Kyudo, Fukuoka, Japan) received a single tail vein injection of purified unfractionated calf thymus histones (20-95 µg/g, Sigma-Aldrich, St Louis, MO), which contained little or no endotoxin (1.25 ± 0.2 pg/mg histones). The purity of the histones was further evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining (Figure S1A in File S1), and the identity of each band was confirmed by western blotting. Survival was monitored for up to 96 hours. Blood and lung samples were collected from volatile-anesthetized mice 10 minutes after histone injection. In some experiments, recombinant human soluble thrombomodulin (rTM; Asahi Kasei Pharma Corporation, Tokyo, Japan) was administered as a bolus injection into the tail vein 30 minutes before histone injection. The amino acid composition of soluble thrombomodulin has been described previously [25]. Platelets were depleted by intravenous application of anti-GP Ib antibodies (2 µg/g, Emfret Analytics, Eibelstadt, Germany) 2 hours before the histone injection (80 µg/g).

Electrocardiographic and echocardiographic analyses

Mice were anesthetized via an intraperitoneal injection of ketamine (100 µg/g) and xylazine (10 µg/g). Electrocardiogram (ECG) measurement was performed using a Powerlab/8S multi-channel digital data recorder (A D Instruments, Sydney, Australia), and the voltage between the right limb and the feet (lead II) was recorded. Transthoracic echocardiography was performed using an Acuson Sequoia Ultrasound System (Siemens Medical Systems, Mountain View, CA) in mice anesthetized with an intraperitoneal injection of pentobarbital (50 µg/g). Right ventricular diastolic dimension was measured in the standard parasternal long axis view using conventional M-mode echocardiography.

Blood tests

Blood samples were collected from volatile-anesthetized mice 10 minutes after histone injection, and coagulation was prevented by addition of one-tenth volume of either 3.13% sodium citrate or EDTA. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured by standard methods using a KC1 Delta automatic coagulometer with an electromechanical clot detection instrument (Trinity Biotech, Bray, Ireland). Complete blood counts were performed with EDTA-anti-coagulated blood, using an automated counting device XE-5000 (Sysmex Corporation, Kobe, Japan). Basic biochemical examinations were performed using a LABOSPECT 008 biochemical analyzer (Hitachi, Tokyo, Japan).

Immunofluorescent analyses

For in vivo labeling of platelets, mice were injected intravenously with DyLight 488-conjugated monoclonal antibodies against GP Ibβ (0.15 µg/g, Emfret Analytics) 30 minutes before histone injection. Lung samples were collected 10 minutes after histone injection (75 µg/g), fixed in 4% paraformaldehyde, equilibrated in 10-30% sucrose, and frozen in optimal cutting temperature compound (Sakura Finetech, Tokyo, Japan). Frozen sections (4 µm) were probed with anti-fibrinogen/fibrin IgG (Dako Cytomation, Glostrup, Denmark) followed by Alexa-Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and the sections were analyzed using an LSM700 confocal laser microscope (Zeiss, Oberkochen, Germany).

Immunohistochemical analyses

Intravascular von Willebrand factor (VWF)-rich thrombi were stained according to a previously described method [26]. Briefly, lung specimens were probed with rabbit polyclonal anti-VWF antibody (CHEMICON International Inc., Temecula, CA) followed by Histofine simple stain mouse MAX-PO (R) (Nichirei, Tokyo, Japan). Peroxidase activity was visualized using diaminobenzidine (DakoCytomation, Carpinteria, CA), and the slides were lightly counterstained with Lillie-Meyer’s hematoxylin (Wako, Osaka, Japan). Photographs were taken using a Zeiss Axiophot microscope with an AxioCam MRc5 camera equipped with AxioVision Release 4.6 software (Carl Zeiss, Oberkochen, Germany).

In vitro platelet aggregation assays

Blood samples were taken from four healthy volunteers, who had not taken any medications that might affect platelet function or coagulation in the preceding 2 weeks. Washed platelets resuspended in Tyrode-HEPES buffer (pH 7.35) or platelet-rich plasma anticoagulated with either sodium citrate or hirudin were stimulated with recombinant human histone H3 or H4 (New England Biolabs, Ipswich, MA), or with collagen. In some experiments, rTM was pre-incubated with histone H3 or H4, or with collagen, for 30 minutes. Platelet aggregation was optically monitored by a light transmission aggregometer (MCM Hema Tracer 313M; SSR Engineering, Tokyo, Japan).

Binding assays with a quartz crystal microbalance (QCM) twin sensor system

The interaction between histones and rTM was assessed using a NAPiCOS Auto QCM twin sensor system (Nihon Dempa Kogyo Co., Tokyo, Japan) according to a previously described method [27]. Briefly, one channel of a sensor chip was coated with histones (1 mg/ml). The second channel on the same sensor chip was coated with BSA (1 mg/ml, Sigma-Aldrich, St Louis, MO) and served as a reference. The sensor chip was washed five times with phosphate-buffered saline (PBS; pH 7.4), placed into the NAPiCOS Auto instrument, perfused with PBS until a stable baseline was obtained, and then perfused with rTM (1 mg/ml). The interaction between molecules was recognized as the change in frequency of a quartz crystal resonator. All experiments were carried out at 25°C with a flow rate of 5 µl/min.

Immunoblotting

Serum histone H3 levels were determined by immunoblotting analysis using anti-histone H3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with reference to standard curves of recombinant histone H3 (0-10 µg/ml) diluted in normal mouse serum.

Statistical analyses

Data are presented as mean ± SD. Statistical analyses were performed using Dunnett’s test unless otherwise stated. Survival rate was analyzed by the Kaplan-Meier method. A probability of < 0.05 was considered significant.

Histone H3 levels in plasma of patients with sepsis and DIC

Recent studies have shown that extracellular histones play an important role in the pathogenesis of sepsis, ischemia-reperfusion injury, and deep vein thrombosis in mice [18,20,28,29]. To examine the possible participation of extracellular histones in the pathogenesis of human sepsis, we studied 15 healthy volunteers and 26 patients with sepsis and DIC. As shown in Figure 1, histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC, especially in non-survivors. These data support the previous finding that extracellular histones are mediators of death in sepsis [18].

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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.

https://doi.org/10.1371/journal.pone.0075961.g001

Extracellular histones trigger platelet aggregation, leading to fatal thromboembolism in mice

To explore how extracellular histones impact disease conditions, we injected mice intravenously with purified histones. Injection of 20-80 µg/g of histones resulted in serum histone H3 levels of 1-10 µg/ml (Figure S1B in File S1), which is comparable to those seen in mice and human patients with sepsis (Figure 1) [18,24]. Half of the mice died after being injected with 60 µg/g of histones, and all mice died after injection with 80 µg/g of histones (Figure 2A). Histones caused thrombocytopenia, but not erythropenia, in a dose-dependent manner, and completely depleted circulating platelets when larger doses were injected (Figure 2B). Neither platelets nor platelet aggregates were observed in blood smears from histone-injected mice (Figure S2 in File S1). Instead, fragmented red blood cells were observed, suggesting that histone-induced thrombosis may disturb the microcirculation [6]. To investigate the effect of histones on platelets, we labeled platelets in vivo with DyLight488-conjugated immunoglobulin against murine platelet GP Ibβ, which does not interfere with platelet adhesion and aggregation. In response to histones, DyLight488-labeled platelets aggregated and diffusely obstructed pulmonary microvessels (Figure 2C). In addition, fibrin was deposited around platelets (Figure 2C), consistent with previous reports showing that extracellular histones activate coagulation via a platelet polyphosphate-dependent mechanism [30]. Histone H3 and H4 were sufficient to induce platelet aggregation in vitro (Figure S3A in File S1), suggesting that platelet aggregation might be associated with histone-induced thrombocytopenia and thromboembolism. Histone-induced platelet aggregation was attenuated in the presence of citrate (Figure S3B in File S1). This may suggest either that calcium ions are important in histone-induced platelet aggregation or that citrate directly inhibits histone activity.

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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.

https://doi.org/10.1371/journal.pone.0075961.g002

Next, we performed electrocardiographic and echocardiographic monitoring to evaluate the potentially fatal role of histone-induced thromboembolism. Histone injection resulted in a tall, peaked P-wave (Figure 3A), dilatation of the right ventricle (Figure 3B), and displacement of the interventricular septum towards the left ventricle (Movies S1 and S2), suggesting that histone-induced thromboembolism is not a minor event but a severe disorder that results in acute pulmonary hypertension and right-sided heart failure. Furthermore, the right-sided heart failure was followed by ventricular arrest (Figure 3A and Movie S3) within 30 min, which is consistent with the time course of histone-induced death (Figure 2A). These findings indicate that pulmonary thromboembolism was closely associated with histone-induced death.

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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).

https://doi.org/10.1371/journal.pone.0075961.g003

To further examine the causal relationship between platelet-rich thrombi and histone-induced death, we removed platelets from the circulation before injecting histones. As described in previous reports [31,32], anti-GPIbα antibodies reduced the number of platelets in the circulating blood by almost 95%. These platelet-depleted mice were protected from death in the first 30 min after histone challenge (Figure S4 in File S1). However, the protective effect of platelet depletion disappeared over time, indicating that extracellular histones mediate death via several distinct mechanisms, where platelet-mediated microvessel occlusion plays an important role in the early phase death and other factors may be responsible for the later phase death. Taken together, our data indicate that extracellular histones trigger platelet-rich thrombus formation in pulmonary microvessels, resulting in acute right-sided heart failure and ventricular arrest, and this mechanism is responsible for histone-induced death in the early phase in mice.

Extracellular histones cause consumptive coagulopathy in mice

We examined the effects of extracellular histones on coagulation pathways. In line with previous reports [18,30], histone injection resulted in fibrin deposition around platelet-rich thrombi (Figure 2C). Fibrinogen levels were decreased when mice were injected with histones at a dose of 40 µg/g or higher (Figure 4A), while APTT and PT were prolonged (Figure 4B-C). Interestingly, APTT was reduced when mice were injected with 20 µg/g histones (Figure 4B). These results suggest that coagulation factors might be primed in the presence of a relatively small amount of histones, but might be fully activated and severely depleted in the presence of an excess of histones.

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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.

https://doi.org/10.1371/journal.pone.0075961.g004

Recombinant thrombomodulin (rTM) protects mice against histone-induced fatal thromboembolism

Histone-injected mice displayed signs of DIC, including thrombocytopenia (Figure 2B), prolonged PT (Figure 4C), decreased fibrinogen (Figure 4A), fibrin deposition in capillaries (Figure 2C), and bleeding (Figure S5A in File S1), which meet the diagnostic criteria for overt DIC [33]. rTM, a newly approved drug for DIC in Japan [34,35], is known to exert anti-DIC effects through promoting protein C activation; however, little is known about its effect on extracellular histones. As suggested by a previous report [36], rTM bound to histones (Figure 5A). rTM inhibited histone H3- and H4-mediated platelet aggregation, but not collagen-mediated platelet aggregation (Figure 5B-C), indicating that rTM inhibits the activity of histones, rather than platelet aggregation itself. In vivo, a single injection of rTM protected mice against histone-induced thrombocytopenia (Figure 6A), VWF-rich intravascular thrombus formation (Figure 6B), dilatation of the right ventricle (Figure 6C-D), pulmonary hemorrhage (Figure S5A in File S1), and death (Figure 6E). Thus, rTM can suppress the activity of extracellular histones both in vitro and in vivo, and this may contribute to the effectiveness of rTM against thrombotic disorders.

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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.

https://doi.org/10.1371/journal.pone.0075961.g005

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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).

https://doi.org/10.1371/journal.pone.0075961.g006