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Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes

Gould, Travis J.; Lysov, Zakhar; Swystun, Laura L.; Dwivedi, Dhruva J.; Zarychanski, Ryan; Fox-Robichaud, Alison E.; Liaw, Patricia C. on behalf of the Canadian Critical Care Translational Biology Group

doi: 10.1097/SHK.0000000000000680
Clinical Aspects
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Objectives: Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells.

Methods/Results: Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH).

Conclusions: Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

*Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada

Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario, Canada

Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada

§George and Fay Yee Center for Healthcare Innovation, Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

||Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Address reprint requests to Patricia C. Liaw, PhD, McMaster University and the Thrombosis and Atherosclerosis Research Institute (TaARI), 237 Barton St. East, Room C5-107 (5th floor), Hamilton, ON L8L 2X2, Canada. E-mail: patricia.liaw@taari.ca

Received 6 May, 2016

Revised 20 May, 2016

Accepted 22 June, 2016

This research was supported by grants-in-aid from the Canadian Institutes for Health Research (MOP-136878, and MOP-123313).

TJG and ZL performed the experiments. TJG and PCL wrote the paper. ZL, LLS, DJD, AEF-R, RZ, and PCL edited the paper. TJG, LLS, and PCL designed the research study.

The authors report no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.shockjournal.com).

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INTRODUCTION

Sepsis is a devastating clinical condition characterized by a systemic inflammatory response to infection, with concomitant dysregulated pathological thrombus formation (1). Although sepsis is triggered by the release of microorganisms and/or microbial toxins into the circulation, the presence of infection alone cannot explain the complex pathophysiology observed in sepsis patients. While the rapid administration of appropriate antibiotics is essential, eradication of an infectious pathogen alone is not sufficient for sepsis treatment (1,2). As a result, sepsis remains the leading cause of morbidity and mortality in non-coronary intensive care units with mortality rates ranging from 18% to 30% (3).

Several studies have suggested that the pathological derangement of coagulation in septic patients is the result of aberrant overexpression of tissue factor (TF) by circulating cells (4,5). TF is the primary initiator of the extrinsic pathway of blood coagulation. Plasma levels of soluble TF have been demonstrated to be elevated in septic patients with an associated increase in concentrations of procoagulant biomarkers (6), while impairment of the TF pathway has been shown to diminish lethality in animal models (4,5). Circulating monocytes are considered to be the major source of intravascular TF (7), and it has been shown that monocytes possess the ability to modulate levels of functional surface TF in response to proinflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1β, and tumor necrosis factor (TNF-α) (8–10). Recently, intensive research has focused on investigating novel biological targets that are expressed or exposed in sepsis patients in an attempt to better understand the pathogenic mechanisms underlying this disorder. Histones are highly cationic nuclear proteins that form hetero-octamers consisting of two copies each of histone subunits H2A, H2B, H3, and H4 and are localized to the nucleus under normal conditions (11). However, in several proinflammatory disease states, histones can be released into the extracellular space, and high plasma concentrations of histones have been detected in sepsis patients who correlate with disease severity (12,13). Histones can be released passively into the circulation by necrotic or apoptotic cells (14), or actively secreted during the formation of neutrophil extracellular traps that are comprised of chromatin, histones, and antimicrobial molecules (15).

Extracellular histones have been shown to contribute to microvascular thrombosis and subsequent organ failure in experimental sepsis models. Extracellular histones are cytotoxic to endothelial cells (16). Histones activate platelets via Toll-like receptors (TLRs) to induce thrombocytopenia in vivo(17). Histones also induce a procoagulant phenotype in red blood cells through the externalization of phosphatidylserine (PS) (18). Administration of neutralizing antibodies against histones resulted in diminished fibrin deposition and vascular occlusion during systemic infection, highlighting the importance of histones in infection-associated thrombosis (19). The activity of circulating histones can also be modified by several anticoagulant mechanisms such as activated protein C which proteolytically cleaves histones (13), as well as thrombomodulin (TM) (20), and heparin (21) which neutralize their procoagulant activities.

Although the ability of extracellular histones to modulate the procoagulant activities of several circulating cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is currently unknown. In this study, we examined the effects of histones on TF expression and activity on peripheral blood monocytes and a human monocytic cell line (THP-1). We also determined the total procoagulant potential of histone-exposed monocytes and THP-1 cells in a plasma system using calibrated automated thrombin generation, and evaluated the ability of extracellular histones in plasmas obtained from septic patients to enhance the procoagulant activity of monocytes.

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PATIENTS AND METHODS

Materials

Human FVIIa and FX were from Haematologic Technologies (Essex Junction, Vt). Recombinant human histones H1, H2A, H2B, H3, and H4 were purchased from New England Biolabs (Toronto, ON). Blocking mAbs against human TLR2 (clone T2.5), TLR4 (HTA125), and isotype control (IgG2a) were all purchased from eBiosciences. Inhibitory TF mAb (HTF-1) was purchased from BD Biosciences (Mississauga, ON). Monoclonal inhibitory antibodies against histone H3 (MHIS1947) and histone H4 (MHIS1952) were generously provided by Dr. Charles Esmon (Oklahoma Medical Research Foundation, Oklahoma City, Okla). Heparin was purchased from Leo Pharma (Thornhill, ON). RPMI 1640 growth medium and penicillin-streptomycin were from Invitrogen (Carlsbad, Calif). C-reactive protein from human plasma was purchased from Sigma-Alrich (Oakville, ON). Factor Xa chromogenic substrate S-2765 was purchased from DiaPharma (West Chester, Ohio). Endotoxin-free PBS was purchased from Cedarlane (Burlington, ON).

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Human sample collection

Patients and plasma samples

Frozen plasma samples were obtained from two available biobanks containing samples from patients with severe sepsis. The first biobank was obtained as part of a prospective cohort study (DYNAMICS Study, ClinicalTrials.gov identifier: NCT01355042). The patients were recruited between September 2010 and January 2013 from tertiary care ICUs from nine centers across Canada. Patients with severe sepsis were identified using the inclusion and exclusion criteria previously described by Dwivedi et al (22). All samples selected for the present experiments were obtained from patients with refractory hypotension requiring the institution of ongoing use of vasopressor agents and did not receive heparin for thromboprophylaxis. The study was approved by the Research Ethics Boards of all participating centers.

The second biobank utilized was obtained as part of a randomized double-blind clinical trial (HALO study, ClinicaTrials.gov identifier: HALO NCT01648036). The patients were recruited between August 2012 and December 2013 from tertiary care ICUs from nine centers across Canada. Patients with severe sepsis and septic shock were identified using the inclusion and exclusion criteria previously employed by Zarychanski et al (23) as part of a retrospective, propensity matched cohort study. Patients were randomized to receive a continuous IV infusion of UFH 18 IU/kg/h or to usual care consisting of subcutaneous dalteparin at 5,000 IU daily. Patient samples utilized in the present study were exclusively from the UFH intervention arm. The study was approved by the Research Ethics Boards of all participating centers.

For both biobanks, patient blood samples, which were collected within 24 h of meeting the inclusion criteria for severe sepsis, were processed within 2 h. Briefly, 9 mL of blood was withdrawn from an indwelling catheter and transferred into 15 mL polypropylene tube containing 0.5 mL of 0.105 M buffered trisodium citrate and 100 mM benzamidine (pH 5.4). After centrifugation at 1,500 × g for 10 min at 20°C, platelet poor plasma was harvested and stored in 200 μL aliquots at −80°C until used. All plasmas selected for study were collected on the second day following ICU admission, allowing for 24 h of UFH infusion prior to blood collection.

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Plasma samples from healthy controls

Plasma samples were obtained via venipuncture from five healthy adult volunteers who were not receiving any medication at the time of blood collection. There was no attempt to match controls with cases. The blood was processed as described above, and plasma was stored in aliquots at −80°C until used.

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Cell culture

Human acute monocytic leukemia suspension cell line (THP-1) was purchased from American Type Culture Collection (Manassas, Va). Cells were maintained in RMPI 1640 medium supplemented with 10% FBS and 100 U/mL penicillin-streptomycin. Cultures were maintained at a concentration between 2 × 105 and 1 × 106 cells/mL, with medium being added every 3 days. Cells were subcultured by total medium replacement using centrifugation at 4,000 × g every 5 to 6 days and incubated at 37°C in a 5% CO2 humidified incubator.

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Isolation of peripheral human monocytes from whole blood

Peripheral human monocytes were isolated from the whole blood of healthy volunteers by magnetic cell sorting (MACS) as previously described according to manufacturer instructions. Isolated monocytes were resuspended in RPMI 1640 (10% FBS and 100 U/mL penicillin-streptomycin). Cell density of 1 × 106 cells/mL as determined by hemocytometer counting was used for monocyte culture experiments.

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Viability assays

Cell viability was assessed using propidium iodide exclusion. Following incubation with histones, cells were washed twice with endotoxin-free PBS and incubated with propidium iodide (4 μM) for 1 min. Dead or dying cells were identified by flow cytometry based on propidium iodide uptake.

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Flow cytometric analysis of tissue factor expression and phosphatidylserine exposure

Following exposure to histones, cultured blood monocytes or THP-1 cells were washed twice and resuspended with endotoxin-free PBS. Cells were incubated at room temperature in the absence of light with 2 μg/mL of FITC-conjugated anti-human TF antibody for 30 min. Incubation with annexin V-FITC was performed in annexin V-binding buffer according to the manufacturer's instruction. IgG isotype antibodies were used as controls. Cell-bound fluorescence was determined using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif) with 3,500 events counted per sample in duplicates. Data acquisition was performed using CellQuest Pro software.

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Tissue factor activity assays

Tissue factor activity of histone-treated cell types was measured by FVIIa-dependent activation of FX. Briefly, 1 × 106 blood monocytes or THP-1 cells were seeded into a 24-well dish and were exposed to purified histone protein for up to 24 h. Cells were pelleted by centrifugation at 4,000 × g for 3 min, washed twice with endotoxin-free PBS, and were then resuspended in TBS containing 5 nM FVIIa, 150 nM FX, and 0.005 mM CaCl2. Cells were incubated at 37°C for 30 min, at which point cells were once again pelleted and the reaction mixture was removed and placed in a 96-well plate. To determine FXa generation, 0.2 mM (final concentration) of chromogenic substrate S-2765 was added and allowed to incubate at room temperature for 5 min. The reaction was terminated by the addition of acetic acid and absorbance was determined at 405 nm.

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Thrombin generation assays

To prepare platelet-poor plasmas (PPP), peripheral venous blood was collected from healthy volunteers into 3.8% trisodium citrate. PPP was prepared by centrifugation at 1,500 × g for 10 min at room temperature. Blood monocytes or THP-1 cells, either untreated or pretreated for 24 h with 100 μg/mL unfractionated histones, carefully washed three times with endotoxin-free PBS, and were added at a final concentration of 5 × 104 cells to 40 μL aliquots of PPP in wells of a 96-well black Costar plate. Where indicated, cells were pretreated for 30 min with the following inhibitors before being added to plasma: anti-TLR2 (50 μg/mL), anti-TLR4 (50 μg/mL), IgG2a (50 μg/mL), or HTF-1 (10 μg/mL) monoclonal antibodies. Also where indicated, 200 μg/mL each of C-reactive protein (CRP), unfractionated heparin (UFH), or antihistone H3 and H4 inhibitory antibodies were added at the same time as histone or plasma application. Coagulation was initiated by the addition of 15 mM CaCl2 and 1 mM Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) and thrombin generation was monitored using the Technothrombin TGA thrombin generation assay (Technoclone, Vienna, Austria). Thrombin generation profiles were analyzed using Technothrombin TGA software (Technoclone).

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Quantification of circulating DNA/histone complexes

DNA-histone complex levels in patient plasma samples were quantified using the Cell Death Detection ELISA Plus kit from Roche Applied Science according to the manufacturer's instructions.

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Statistical analyses

Statistical analysis was performed on experiments with an n = 3 or greater. Values are expressed as means ± standard error. Significance of differences was determined by one-way ANOVA and Tukey pair-wise comparisons or by t tests using SIGMAPLOT Software (San Jose, Calif).

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RESULTS

Histones enhance TF activity, antigen, and phosphatidylserine exposure on THP-1 and human blood monocytes

To study the procoagulant effects of histones on monocytic cells, increasing concentrations (0–200 μg/mL) of unfractionated bovine histones were incubated for 24 h with human acute monocytic leukemia suspension cell line (THP-1) or peripheral blood monocytes immediately after isolation from healthy volunteers. TF activity was determined by the conversion of factor X to factor Xa in the presence of factor VIIa and Ca2+. As shown in Figure 1A, exposure of THP-1 cells to histones upregulated TF activity in a dose-dependent fashion. Similarly, treatment of blood monocytes with increasing concentrations of histones resulted in increased TF activity (Fig. 1B). Both THP-1 cells and monocytes exhibited increased surface TF activity in as little as 6 h following exposure to 100 μg/mL of histones (Fig. 1C). In contrast, no changes in TF activity were observed when THP-1 cells or monocytes were exposed to histones subjected to heat denaturation (data not shown). The cytotoxicity of histones toward monocytes was also determined by propidium iodide exclusion (Supplemental Digital Content, Figure 1, http://links.lww.com/SHK/A410). Monocytes incubated with up to 100 μg/mL of histones for 24 h demonstrated >75% viability, which was not significantly different (P = 0.51) from untreated cells.

Fig. 1

Fig. 1

Increases in tissue factor activity may be a result of increased expression of surface TF, or the result of decryption of latent TF to a more biologically active form in the presence of negatively charged cell surface phospholipid PS (24). Both THP-1 cells and blood monocytes exposed to histones exhibited a ∼3- and ∼5-fold increase in surface TF antigen levels, respectively (Fig. 1D). In addition, incubation with histones resulted in a dose-dependent increase in surface PS for both THP-1 cells and blood monocytes alike (Fig. 1E).

It was previously demonstrated that histone subunits H3 and H4 are the main contributors to histone cytotoxicity toward endothelial cells (13). To determine which histone subunits are responsible for the observed increase in TF activity, we incubated THP-1 and blood monocytes with 100 μg/mL of purified, recombinant histone subunits. While histone subunits H1, H2A, and H2B demonstrated only a modest ability to modulate TF activity, exposure to histone subunits H3 and H4 significantly increased TF activity on both THP-1 cells and monocytes (Fig. 1F). Collectively, these results suggest that the exposure of THP-1 cells and peripheral blood monocytes to histones, particularly subunits H3/H4, increases surface TF activity via increased surface TF antigen and PS exposure.

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The procoagulant effects of histones on THP-1 and blood monocytes are abrogated by C-reactive protein, heparin, and Toll-like receptor blockade

Recently, it was demonstrated that co-administration of C-reactive protein (CRP) or unfractionated heparin (UFH) along with LPS dampens coagulation and reduces mortality in sepsis models (21,25). To investigate whether CRP and/or heparin could diminish the procoagulant effects of histones, TF activity assays were performed on THP-1 cells and blood monocytes exposed to 100 μg/mL unfractionated histones with or without 200 μg/mL each of CRP or heparin for 24 h. As shown in Figure 2A, the presence of either CRP or heparin attenuated TF activity on both cell types. In accordance, we observed reduced levels of both surface TF antigen and PS exposure (Fig. 2B and C, respectively).

Fig. 2

Fig. 2

Histones have been shown to engage with surface TLRs on numerous cell types to induce procoagulant effects (17,18). To determine if histone-TLR interactions were involved in the ability of histones to upregulate TF activity, we repeated the previous experiments using THP-1 and blood monocytes pre-incubated with inhibitory antibodies directed toward TLR-2 and TLR-4. Following exposure to histone, TF activity of these cells was partially attenuated (Fig. 2A). Similarly, blockade of TLR-2 and 4 resulted in a decrease in both surface TF antigen (Fig. 2B) and PS (Fig. 2C). The selected histone inhibitors had no effect on cell surface TF activity alone, as shown in Supplemental Digital Content, Figure 2, http://links.lww.com/SHK/A410. Taken together, these results demonstrate that histones modulate monocyte procoagulant activity through TLR-2 and TLR-4 interactions, and these effects can be attenuated by CRP- or heparin-induced inhibition.

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Histone exposure enhances thrombin generation mediated by THP-1 cells and blood monocytes

Next, we assessed the ability of histone-treated THP-1 cells and blood monocytes to generate thrombin using a calibrated automated thrombin generation assay. THP-1 cells and blood monocytes were exposed to 100 μg/mL of unfractionated bovine histones for 24 h. The histones were then removed following several washes with PBS and the cells were resuspended in platelet-poor plasma which was subsequently recalcified to initiate coagulation. Thrombin generation was measured by cleavage of a fluorogenic substrate at 1-min intervals.

As shown in Figure 3, exposure of platelet-poor plasma to untreated THP-1 cells or monocytes resulted in only modest changes in thrombin generation parameters. However, THP-1 and blood monocytes exposed to histones significantly decreased the lag time (Fig. 3A) and the time to peak thrombin generated (Fig. 3B). The area under the curve (AUC) which indicates the total amount of thrombin generated confirmed the ability of histone-treated cells to enhance thrombin generation (Fig. 3C).

Fig. 3

Fig. 3

To confirm the contributions of TF to the augmented thrombin generation parameters observed, the thrombin generation assays were repeated using histone-treated cells that had been blocked with an inhibitory TF antibody (HTF-1). Inhibition of TF abrogated the procoagulant effects of both histone-treated THP-1 and monocytes and restored lag time, time to peak, and AUC to basal levels (Fig. 3, A–C). In contrast, addition of HTF-1 inhibitory antibody to recalcified plasma alone had no effect (data not shown). Representative thrombin generation curves of histone-exposed monocytes are depicted in Figure 3D. This data suggests that increases in TF activity as a result of histone exposure translate to enhanced thrombin generation in plasma.

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Extracellular DNA-histone complexes are elevated in septic patient plasma and enhance TF activity of blood monocytes

To explore the potential clinical relevance of our in vitro studies, we utilized plasma samples from two severe sepsis biobanks. Plasmas were obtained from patients with severe sepsis (as outlined in Patients and Methods), with one patient subgroup receiving prophylactic UFH while the other patient subgroup received usual care without UFH intervention. Patient characteristics from both groups are summarized in Table 1. Plasma levels of DNA–histone complexes were quantified in both UFH and non-UFH patient plasmas. Plasmas from patients receiving UFH contained significantly lower (P = 0.005) levels of DNA–histone complexes compared with non-UFH patients (Fig. 4A). Levels of DNA–histone complexes from healthy control plasmas were below the lower limit of detection for the assay (data not shown). To determine if elevated DNA–histone complexes could modulate TF activity of monocytes, we incubated peripheral blood monocytes with 75% septic plasma containing citrate and benzamidine diluted with media for 24 h. Compared with control plasma, all septic plasmas enhanced TF activity of monocytes. However, plasmas from patients receiving UFH induced significantly lower TF activity compared with non-UFH patients (Fig. 4B). In a separate set of experiments, monocytes were incubated with both septic plasma and a mixture of 100 μg/mL each of histone H3 and H4 inhibitory antibodies. The addition of inhibitory histone antibodies significantly reduced monocyte TF activity in non-UFH sepsis patients, decreasing TF activity levels to those observed in UFH patients. In contrast, the inclusion of inhibitory histone antibodies had no effect in UFH patient plasma (Fig. 4B). In addition, levels of plasma DNA–histone complexes positively correlated with the degree of TF activity induced by these plasmas. Linear regression analysis demonstrated an r2 = 0.67, with a slope of 17.06 that was significantly different from zero (P <0.0001; Fig. 4C). Taken together, this data suggests that extracellular histones found within the circulation of septic patients may induce a procoagulant phenotype of circulating monocytes, and this effect may be attenuated by inhibiting histone activity.

Table 1

Table 1

Fig. 4

Fig. 4

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DISCUSSION

The underlying host response to sepsis involves the interplay of complex biological mechanisms and cell types that culminate in severe dysregulation of inflammation and coagulation. To date, over 100 randomized clinical trials have attempted to identify therapies that modulate the septic response and improve patient outcomes; however, none of these potential therapies has been successfully translated into the clinic (26). Therefore, an improved understanding of the molecular basis of sepsis pathophysiology, including interactions between inflammatory, coagulation, and fibrinolytic systems, is needed. Recently, there has been renewed enthusiasm for the identification of novel therapeutic targets in sepsis, and circulating histones have emerged not only as a promising biomarker for assessing disease severity, but also as a potential target for pharmacological intervention.

Circulating monocytes are responsible for modulating hemostasis through multiple mechanisms. Among the leukocytes, monocytes have been shown to be unique in that their cell surface membrane serves as a template for the assembly and function of TF-mediated coagulation (27). While quiescent monocytes display limited procoagulant activity, they possess the ability to synthesize TF de novo and subsequently express it on the cell surface following exposure to bacterial components, proinflammatory cytokines, as well as host-derived ligands (8–10). The present study demonstrates that histones also serve as a potent procoagulant stimulus for monocytes, resulting in the upregulation of TF expression on the cell surface as well as inducing the exposure of the anionic phospholipid PS.

In addition to TF, monocytes also express several anticoagulant factors including endothelial protein C receptor, TM, and tissue factor pathway inhibitor (TFPI) (28). Notably, TF and TFPI are the primary regulators of coagulation initiation on the monocyte surface, and TF/TFPI ratios are essential for modulating the procoagulant activity of monocytes (29). In resting monocytes, levels of TFPI are approximately 2-fold higher than TF, which serve to rapidly neutralize surface TF. However, common proinflammatory agents such as LPS (29) result in a concurrent upregulation of TF and, to a lesser degree, TFPI, potentiating a hemostatic imbalance that favors TF activity. Furthermore, histones have been shown to associate with TM, diminishing its ability to bind thrombin and facilitate protein C activation (30). Similarly, our studies suggest that there is a cumulative shift to a procoagulant phenotype for histone-stimulated monocytes, as evidenced by a shortened thrombin lag phase and a net increase in the total amount of thrombin generated.

Many of the pathobiological effects exerted by histones are regulated by TLR2/4 signaling including the induction of cell death in an animal model of kidney inflammation (31), inducing cytotoxicity during liver injury (17), and stimulating platelet activation and subsequent aggregation (32). More recently, it was demonstrated that histones may induce TF expression on endothelial cells and in a murine macrophage-like cell line, again through TLR2/4 involvement (33), though these studies did not explore the functional significance of elevated TF to modulate coagulation. Furthermore, in murine models of endotoxemia, reciprocal bone marrow transfer studies between normal mice and mice expressing ∼1% human TF demonstrate that the activation of coagulation can be attributed to hematopoietic TF rather than vessel wall-derived TF (34). Therefore, the contribution of histones to activating monocyte TF may be of greater pathophysiological relevance. The present results demonstrate mechanistically that TLR2 and TLR4 are the primary receptors for extracellular histones on human monocytes, and demonstrate that antagonizing the histone-TLR interactions may be an attractive target for modulating the procoagulant effects of histones in vivo.

Antihistone treatments in various animal models of sepsis have been shown to confer protective effects. In particular, the administration of heparin has been shown to reduce the cellular cytotoxicity of histones in vitro and decrease mortality in animal models of LPS-induced endotoxemia and cecal ligation and puncture (21,35). It is hypothesized that since histones are polycationic peptides, negatively charged heparins bind and inactivate circulating histones through high-affinity electrostatic interactions, ultimately attenuating their cytotoxicity (35). In the presence of heparin, we observed diminished procoagulant activity of monocytes exposed to histones. We were able to recapitulate this effect in plasmas obtained from septic patients, whereby plasmas from patients receiving IV administration of UFH induced less procoagulant activity on monocytes compared with plasmas without UFH. In accordance with these results, a recent meta-analysis has shown that heparin therapy may be beneficial to severe sepsis patients without an increased risk for adverse bleeding events (36). Although the presence of heparin in plasma was unable to attenuate TF activity to basal levels, the complex proinflammatory milieu of the septic plasmas utilized for this study likely contain a number of mediators, including LPS and lipoteichoic acid, or proinflammatory cytokines such as IL-6 and TNF-α that may also contribute to monocyte procoagulant activity. The attenuation of TF activity by antihistone antibodies in our plasma experiments demonstrates that pathophysiological levels of circulating histones are sufficient to induce a procoagulant phenotype on monocytes and that our in vitro observations were not simply a phenomena initiated by supraphysiological histone concentrations.

In summary, these studies are the first to examine the effects of histones on monocytic cells, specifically within the context of sepsis. Our findings suggest that histones potentiate a procoagulant phenotype on circulating monocytes, thereby establishing a novel mechanism by which extracellular histones modulate hemostasis and identify histones as a potential therapeutic target in sepsis treatment.

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Acknowledgments

The authors are extremely grateful to Dr Deborah Cook, Ellen McDonald, Nicole Zytaruk, and Bronwyn Cash-Barlow for the recruitment of patients with sepsis in Hamilton, Ontario, Canada.

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REFERENCES

1. Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003; 348 2:138–1350.
2. Wheeler AP, Bernard GR. Treating patients with severe sepsis. N Engl J Med 1999; 340 3:207–214.
3. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001; 29 7:1303–1310.
4. Pawlinski R, Mackman N. Tissue factor, coagulation proteases, and protease-activated receptors in endotoxemia and sepsis. Crit Care Med 2004; 32 (5 Suppl):S293–S297.
5. Semeraro N, Ammollo CT, Semeraro F, Colucci M. Sepsis-associated disseminated intravascular coagulation and thromboembolic disease. Mediterr J Hematol Infect Dis 2010; 2 3:e2010024.
6. Gando S, Nanzaki S, Sasaki S, Kemmotsu O. Significant correlations between tissue factor and thrombin markers in trauma and septic patients with disseminated intravascular coagulation. Thromb Haemost 1998; 79 6:1111–1115.
7. Pawlinski R, Mackman N. Cellular sources of tissue factor in endotoxemia and sepsis. Thromb Res 2010; 125 (S1):S70–S73.
8. Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989; 9 6:2752–2755.
9. Cermak J, Key NS, Bach RR, Balla J, Jacob HS, Vercellotti GM. C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor. Blood 1993; 82 2:513–520.
10. Ito T, Kawahara K, Nakamura T, Yamada S, Nakamura T, Abeyama K, Hashiguchi T, Maruyama I. High-mobility group box 1 protein promotes development of microvascular thrombosis in rats. J Thromb Haemost 2007; 5 1:109–116.
11. Richmond TJ, Davey CA. The structure of DNA in the nucleosome core. Nature 2003; 423 6936:145–150.
12. Zeerleder S, Zwart B, Wuillemin WA, Aarden LA, Groeneveld ABJ, Caliezi C, van Nieuwenhuijze AEM, van Mierlo GJ, Eerenberg AJM, Lämmle B, et al. Elevated nucleosome levels in systemic inflammation and sepsis. Crit Care Med 2003; 31 7:1947–1951.
13. Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, Taylor FB, Esmon NL, Lupu F, Esmon CT. Extracellular histones are major mediators of death in sepsis. Nat Med 2009; 15 11:1318–1321.
14. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 2001; 61 4:1659–1665.
15. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science 2004; 303 5663:1532–1535.
16. Abrams ST, Zhang N, Manson J, Liu T, Dart C, Baluwa F, Wang SS, Brohi K, Kipar A, Yu W, et al. Circulating histones are mediators of trauma-associated lung injury. Am J Respir Crit Care Med 2013; 187 2:160–169.
17. Xu J, Zhang X, Monestier M, Esmon NL, Esmon CT. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J Immunol 2011; 187 5:2626–2631.
18. Semeraro F, Ammollo CT, Esmon NL, Esmon CT. Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells. J Thromb Haemost 2014; 12 10:1697–1702.
19. Massberg S, Grahl L, Bruehl von M-L, Manukyan D, Pfeiler S, Goosmann C, Brinkmann V, Lorenz M, Bidzhekov K, Khandagale AB, et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med 2010; 16 8:887–896.
20. Nakahara M, Ito T, Kawahara K-I, Yamamoto M, Nagasato T, Shrestha B, Yamada S, Miyauchi T, Higuchi K, Takenaka T, et al. Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism. PLoS One 2013; 8 9:e75961.
21. Wildhagen KCAA, Garcia de Frutos P, Reutelingsperger CP, Schrijver R, Areste C, Ortega-Gomez A, Deckers NM, Hemker HC, Soehnlein O, Nicolaes GAF. Non-anticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood 2014; 123 7:1098–1101.
22. Dwivedi DJ, Toltl LJ, Swystun LL, Pogue J, Liaw K-L, Weitz JI, Cook DJ, Fox-Robichaud AE, Liaw PC. Group TCCCTB: prognostic utility and characterization of cell-free DNA in patients with severe sepsis. Crit Care 2012; 16 4:R151–R162.
23. Zarychanski R, Doucette S, Fergusson D, Roberts D, Houston DS, Sharma S, Gulati H, Kumar A. Early intravenous unfractionated heparin and mortality in septic shock. Crit Care Med 2008; 36 11:2973–2979.
24. Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol 2006; 26 3:456–461.
25. Abrams ST, Zhang N, Dart C, Wang SS, Thachil J, Guan Y, Wang G, Toh C-H. Human CRP defends against the toxicity of circulating histones. J Immunol 2013; 191 5:2495–2502.
26. Marshall JC. Why have clinical trials in sepsis failed? Trends Mol Med 2014; 20 4:195–203.
27. McGee MP, Li LC, Hensler M. Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa. J Exp Med 1992; 176 1:27–35.
28. Bouchard BA, Tracy PB. The participation of leukocytes in coagulant reactions. J Thromb Haemost 2003; 1 3:464–469.
29. Basavaraj MG, Gruber FX, Sovershaev M, Appelbom HI, Østerud B, Petersen LC, Hansen J-B. The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes. Thromb Res 2010; 126 5:418–425.
30. Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost 2011; 9 9:1795–1803.
31. Allam R, Scherbaum CR, Darisipudi MN, Mulay SR, Hagele H, Lichtnekert J, Hagemann JH, Rupanagudi KV, Ryu M, Schwarzenberger C, et al. Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol 2012; 23 8:1375–1388.
32. Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon CT. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011; 118 7:1952–1961.
33. Yang X, Li L, Liu J, Lv B, Chen F. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-(B and AP-1. Thromb Res 2016; 137 1:211–218.
34. Pawlinski R, Wang JG, Owens AP, Williams J, Antoniak S, Tencati M, Luther T, Rowley JW, Low EN, Weyrich AS, et al. Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice. Blood 2010; 116 5:806–814.
35. Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, Lohmeyer J, Preissner KT. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012; 7 2:e32366.
36. Wang C, Chi C, Guo L, Wang X, Guo L, Sun J, Sun B, Liu S, Chang X, Li E. Heparin therapy reduces 28-day mortality in adult severe sepsis patients: a systematic review and meta-analysis. Crit Care 2014; 18 5:563–572.
Keywords:

Coagulation; heparin; histone; leukocyte; sepsis

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