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Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

Sepsis is a life-threatening condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome, SIRS). Histones are essential for packing DNA into the cell nucleus and also play role in regulating gene expression but the new study (Jun Xu et al, Nature Medicine 15. 1318, 2009) have shown histones released into blood stream at the onset of sepsis can have devastating effects (endothelial damage and organ failure etc). However, plasma levels of histone are unclear in septic patients with DIC. Therefore, We developed a sandwich ELISA to quantify serum or plasma histone H3 levels and measured plasma levels of histone H3 in septic patients with DIC (n=30). DIC was diagnosed on the Japanese Ministry of Health and Welfare (JMHW) DIC diagnostic criteria. The thrombin antithrombin complexes (TAT) levels were higher in septic patients with DIC as reported by others. The working range of this ELISA was 3-1000 ng/ml. In healthy volunteers, plasma levels of histone (H3) were not detectable. We detected high levels of extracellular hisotone (H3) in the plasma of all septic patients with DIC by sandwich ELISA. Mean plasma levels of hisotone (H3) were 70.5 ± 63.2 (12.9-336.5) ng/ml in septic patients with DIC. However, plasma levels of extracellular histone (H3) were negative in acute promyelocytic leukemia (APL)-induced DIC (n=5). Also, Plasma levels of histone were 10 ng/ml or less by sandwich ELISA in septic patients without DIC (n=8). These results suggest that assess of extracellular hisotones in the plasma may be helpful in the making the diagnosis in septic patients with DIC. Also, it appears that extracellular hisotones in the plasma may contribute to develop DIC in septic patients. Disclosures No relevant conflicts of interest to declare.

Abstract 1167 Evaluation of the overall antithrombotic activity of dabigatran in combination with antiplatelet agents is difficult because plasma-based clotting for dabigatran, and platelet aggregometry in anticoagulated blood are two separate tests which do not reflect physiological interactions between soluble factors and platelets. The use of a flow chamber could be more suitable in evaluating a flow-dependent platelet activation and coagulation responses. The aim of the current study was to comparatively evaluate antithrombotic effects of dabigatran in combination with dual antiplatelet therapy (aspirin plus P2Y12 blockade) using the microchip-based flow chamber (T-TAS, Fujimori Kogyo, Japan)(1), and thrombin generation (TG) assay (Thrombinoscope, Maastricht, the Netherlands)(2). After the local ethics committee approval, blood samples were obtained from consented 5 healthy volunteers in the tubes containing 3.2% sodium citrate. Whole blood samples were mixed with dabigatran (250, 500, 1000 nM), aspirin (100 nM) plus ARC-66096 (P2Y12 inhibitor, 1000 nM) at 25¡C for 10 min. Corn trypsin inhibitor (50 μg/ml) was used to prevent contact activation. The whole blood sample was perfused in the capillary pre-coated with collagen and thromboplastin at the shear rate of 240 or 600 s−1. The process of thrombus formation was monitored by flow pressure increases inside the capillary; (i) lag time before it reaches 10 kPa (T10), (ii) occlusion time (OT) is the lag time before it reaches 80 kPa as thrombus completely occludes the capillary, and (iii) AUC30 is an area under the flow pressure curve (under 80 kPa) after 30 min of perfusion. For TG assay, platelet-rich plasma (platelet count 150 × 103/μl) was prepared from citrated whole blood. TG was triggered by adding 20 μl of CaCl2-fluorogenic substrate buffer to 80 μl of the sample mixed with tissue factor (1 pM) in each well. The lag time (min), and peak thrombin concentration (nM) were evaluated. In the flow chamber, dabigatran inhibited white thrombus formation in a concentration dependent manner at shear rates of 240 and 600 s−1(Fig. 1). At 500 nM of dabigatran, OT was prolonged by ∼2-fold from the (non-treated) control at both shear rates. The combination of aspirin and AR-C66096 only weakly suppressed thrombus formation, but it enhanced the antithrombotic efficacy of dabigatran at both shear rates (Fig. 1). In TG measurements using platelet-rich plasma, dabigatran at 500 nM prolonged the by 3.17-fold, and reduced the peak by 57.6% compared to the untreated control (Table 1). Aspirin and AR-C66096 weakly prolonged the lag time without affecting the peak height. There were relatively small changes in these parameters when antiplatelet agents were combined with dabigatran (Table 1). Our results suggest that combined antithrombotic effects of dabigatran, aspirin, and P2Y12inhibition can be demonstrated in the whole blood using the flow chamber system compared without additional plasma preparation required for TG assay. The re-calcified whole blood was perfused at the shear rate of 240 s−1 or 600 s−1. Asp/AR-C=aspirin and AR-C66096 Table 1. Lag time (min) Peak (nM) Native Asp/AR-C Native Asp/AR-C Control 6.8 ± 0.8 9.4 ± 3.2 92.1 ± 23.7 91.2 ± 29.5 Dabi 250 nM 18.6 ± 5.4 21.1 ± 4.5 69.3 ± 20.6 52.2 ± 13.6 Dabi 500 nM 21.6 ± 5.3 26.2 ± 10.2 53.0 ± 5.8 47.8 ± 9.1 Dabi 1000 nM 30.2 ± 5.6 35.1 ± 6.3 23.0 ± 6.9 22.0 ± 8.4 Dabi=dabigatran, Native=no antiplatelet agents, Asp/AR-C=aspirin and AR-C66096 Disclosures: Hosokawa: Fujimori Kogyo: Employment. Ohnishi:Fujimori Kogyo: Employment. Sameshima:Fujimori Kogyo: Employment.

Anandamide(ANA) and 2-arachidonoylglycerol(2-AG), two lipid mediators existing in the central nervous system and various peripheral tissues, have been shown to exert their diverse biological effects through cannabinoid receptors CB1 and CB2, and are therefore defined as endogenous cannabinoids. Recently, ANA and 2-AG have been shown to be generated by macrophages and platelets, respectively, by the stimulation with lipopolysaccharide(LPS) in vitro and in vivo in rats. We showed the endocannabinoids are increased in septic shock patients. Furthermore, the increased endocannabinoids are efficiently adsorbed onto polymyxin B immobilized column resulting the improvement of hypotensive shock. On the other hand it has been showed that activated protein C (APC) can ameliorate endotoxin induced-shock and -disseminated intravascular coagulation in experimental animals and patients, although the mechanisms governing its action are unknown. In the present study we found that APC inhibited the generation of ANA and 2-AG in vitro by lipopolysaccharide (LPS)-activated macrophages and platelets, respectively. Furthermore, APC significantly decreased the levels of ANA and 2-AG in the sera from rats preinfused with APC followed by LPS infusion. Consistent with the decrease of them, diminishment of hypotension and an increase in survival rate were confirmed in the APC pretreatment group. These findings demonstrate that APC can inhibit the generation of ANA and 2-AG in vitro and in vivo, and thereby suppress their induced hypotension. This action of APC may explain its efficiency in the treatment of septic shock in humans and experimental animals.

High Mobility Group Box-1 (HMGB1) protein, released from the most of necrotic cells and activated macrophages, has been identified as a novel cytokine through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor(TLR)-2 and -4. The HMGB1-RAGE and TLRs-2, -4 interactions contribute to cellular migration and the production of proinflammatory cytokines, and participate in pathomechanisms in tumor growth and invasion, in which an angiogenesis development is an important aspect. We here show that HMGB1 stimulates the expression of Vascular Endothelial Growth Factor (VEGF), the most potent angiogenic factor in tumors, through the HMGB1-RAGE, but neither TLR-2 nor TLR-4 in macrophage-lineage cells in vitro. The mechanism of VEGF production is mediated through the Akt pathway, which is linked to tumor growth and invasion. Furthermore, HMGB1 induced angiogenesis was also observed in an in vivo rabbit corneal assay (Fig). These results suggest that HMGB1, released from the tumor-associated macrophages, may act as a key cytokine in the development of angiogenesis by producing VEGF in tumor growth and invasion. Thus the tumor-associated HMGB1/RAGE system may contribute to our understanding of the mechanism of cancer cell escape from macrophage-associated acceleration of inflammation.

Background: Thrombomodulin is a thrombin receptor on the endothelial cell surface that plays an important role in the regulation of intravascular coagulation. Recombinant human soluble thrombomodulin (ART-123) is composed of the active, extracellular domain of thrombomodulin. ART-123 has been shown to have a wider safety margin than other anticoagulants and to have a favorable antithrombotic profile with less bleeding in animal and in vitro experiments. ART-123 is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. Objective: We conducted a multicenter, double-blind, randomized parallel-group trial to compare the efficacy and safety of ART-123 to those of heparin for the treatment of disseminated intravascular coagulation (DIC) associated with infection or hematologic malignancy. Methods: DIC was diagnosed according to the diagnostic criteria established by the Japanese Ministry of Health and Welfare. DIC patients were assigned to receive ART-123 (0.06 mg/kg for 30 min, once a day) or heparin sodium (8 units/kg/h for 24 h) for six days using a double dummy method. The primary efficacy endpoint was DIC resolution rate (rate of recovery from DIC). The secondary endpoints included clinical course of bleeding symptoms, and mortality at 28 days. Results: 234 DIC patients were randomized (117 in each arm). DIC resolution rate was 66.1% for the ART-123 group and 49.9% for the heparin group (difference 16.2%; 95% CI 3.3–29.1), thus demonstrating that ART-123 is significantly superior to heparin for the improvement of DIC. Patients in the ART-123 group also showed more marked improvement in clinical course of bleeding symptoms (p=0.0271) and the disappearance rate of bleeding symptoms in the ART-123 group was higher than that in the heparin group (35.2% vs 20.9%; difference 14.3%). The incidence of bleeding-related adverse events up to 7 days after the start of administration was lower in the ART-123 group than in the heparin group (43.1% vs 56.5%; difference; −13.4%; p=0.0487). The mortality rate at 28 days in the ART-123 group was 22.0% vs 25.5% in the heparin group (difference −3.4%; p=0.5396). Although no significant differences were seen, 28-day mortality for the ART-123 group was slightly lower. The mortality rate of patients with DIC secondary to infection in the ART-123 and heparin groups was 28.0% (14/50) and 34.6% (18/52), respectively, indicating a 6.6% lower mortality in the ART-123 group. Conclusion: When compared with low-dose heparin, ART-123 more significantly improves DIC and alleviates bleeding symptoms in DIC patients. The incidence of bleeding-related adverse events is significantly lower with ART-123 than with heparin. Because of its safety and efficacy, ART-123 appears to be a first-line agent in the management of DIC.

High Mobility Group Box chromosomal protein 1 (HMGB1) is a nuclear DNA-binding protein acting as a proinflammatory cytokine when released in the extracellular space from necrotic cells, activated macrophages and dendritic cells. HMGB1 acts on a specific receptor, RAGE (receptor for advanced glycation end-products), and induces prolonged inflammation, organ failure, septicaemia and death. The aim of the study was to determine the diagnostic value of plasma HMGB1 concentration and its role in the development of organ failure in patients with disseminated intravascular coagulation (DIC). Plasma HMGB-1 levels were measured in patients with suspected DIC by ELISA and their relationships with DIC, organ failure and clinical outcome were determined. The study group included 201 patients suspected of having DIC (79 females and 122 males, age; 52.1±16.5 years, mean±SD). The study group represented all such patients admitted to the Second Department of Internal Medicine and Intensive Care Unit (ICU) of Mie University Hospital between January 1, 2002 and December 31, 2003. The underlying diseases were infectious diseases (n=40), hematopoietic diseases (n=58), solid cancers (n=16), trauma (n=21), aneurysm (n=12), autoimmune diseases (n=8), liver diseases (n=7), post-operation (n=32) and other diseases (n=7). Results: Plasma HMGB1 was below the detection limit in normal subjects, but moderately elevated in patients with infectious diseases (4.54 ± 8.18 ng/ml, mean±SD), malignancies (2.15 ± 5.34 ng/ml), and traumas (6.47 ± 13.13 ng/ml). DIC was associated with significantly high plasma HMGB1 (14.05 ± 12.56 ng/ml) in these patients. The highest HMGBI levels were in patients with organ failure (8.29 ± 10.99 ng/ml) and non-survivors (16.58 ± 11.01 ng/ml). HMGB1 plasma levels correlated with the DIC score and sepsis-related organ failure assessment (SOFA) score. Plasma levels of HMGB1 were significantly higher in patients with PAI-I of >50 ng/ml (3.66 ± 6.36 ng/ml) than in those with PAI-I 1.5 (3.04 ± 5,56 ng/ml) and those with less than 1.5 pg/ml (2.44 ± 5.36 ng/ml). Our data suggest that HMGB-1 is a potentially suitable prognostic marker of OF or DIC.

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC), the latter an enzyme with potent anti-coagulant and anti-inflammatory properties. We have found that the N-terminal, lectin-like domain (D1) of thrombomodulin has unique anti-inflammatory properties. Thrombomodulin, via D1, binds high mobility group-B1 DNA binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing leukocyte activation in vitro, and ultraviolet radiation-induced cutaneous inflammation and lipopolysaccharide-induced lethality in vivo. Our data also demonstrate anti-inflammatory properties of a peptide spanning the D1 domain of TM and suggest its therapeutic potential. These findings highlight a novel mechanism through which an endothelial cofactor, TM, suppresses inflammation; i.e., sequestration of mediators thereby preventing their interaction with cell surface receptors on effector cells in the vasculature. Results: TM binds HMGB1 and prevents expression of pro-inflammatory activity. Our co-culture studies of leukocytes and HUVEC, and results in the cutaneous irritation model suggested that early release of a mediator, such as HMGB1, might contribute importantly to cellular activation in inflammation at later time points. In this context, TM might have the ability to decrease HMGB1-mediated inflammatory events. Binding studies using surface plasmon resonance (SPR), performed to directly assess the interaction of TM and immobilized HMGB1, demonstrated dose-dependent binding in the nanomolar range (Kd ~232 nM). Furthermore, addition of rhs-TM decreased, in a dose-dependent manner, the binding of HMGB1 to RAGE through the its N-terminal domain, but not anti-coagulant domain. TM and the N-terminal-derived TM peptide have anti-inflammatory effects in settings where HMGB1 is a likely key mediator. In HMGB1-mediated skin inflammation model, systemic administration of rhs-TM, its lectin-like domain and sRAGE resulted in a significant blunting of the inflammatory response. In contrast, the effect of anti-coagulant domain, although showing a trend toward decreased ear swelling, did not achieve statistical significance (anticoagulant domain has anti-inflammatory effects in vivo that probably reflect its ability to support thrombin-mediated activation of protein C; the latter does not occur in vitro after inactivation of the protein C zymogen by heat treatment). In view of recent data suggesting a link between HMGB1 released from injured tissue and endotoxin-induced lethality in mice, we also tested whether rhs-TM and its lectin-like domain might also have protective effects in this model. We employed a dose of intraperitoneal (IP) LPS (10 mg/kg) resulting in 100% lethality by 96 hrs. Systemic (IP) treatment of animals with anti-HMGB1 IgY had a protective effect with respect to lethality at 4 days, whereas the same regimen of nonimmune IgY was without effect. Similarly, IP administration of rhs-TM and its N-teminal lectin domain, but not anti-coagulant domain had complete protective effects compared with anti-HMGB1 IgY. Conclusion: Our findings have elucidated an unexpected anti-inflammatory property of TM residing in the D1 domain, namely binding of HMGB1.

High Mobility Group box 1(HMGB1) is an abundant DNA-binding protein that acts as a proinflammatory cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Moreover, an increased HMGB1 in the circulation of septic patients may induce multi-organ failure and lethality. However, very recent observations suggest that the protein also acts as an innate adjuvant, stem cell chemoattractant and growth factor. Thus only systemic and circulatory HMGB1 may induce morbidity and mortality, however, localized HMGB1 may have beneficial effects. Therefore, we serially examined the serum HMGB1 level in patients with various diseases, and also evaluated the significance of the protein. We demonstrate here how HMGB1 is localized and acts as an immune-adjuvant and a repairing factor in damaged tissue. We first established specific ELISA method to measure HMGB1. An increased level of HMGB1 was detected in the serum from patients with sever sepsis, infections, malignancy and so on. However, serum HMGB1 concentrations were fluctuated during the clinical course, and could not be concluded as a lethal mediator as previously reported. Next we investigated the reason of dynamic fluctuations of the protein in the circulation. Based on our findings, we proposed that this fluctuation of HMGB1 concentrations may be mediated by at least following three fashions; 1) proteolytic degradation by plasmin and thrombin, 2) endothelial thrombomodulin(TM) adsorption, and 3) generation of antibody against the protein. We observed that plasmin efficiently degraded HMGB1 into small fragments. However, interestingly the generated fragments of the protein still possess an ability to produce TNFa in macrophages through an undefined pathway. TM binds the protein on its N-terminus lectin-like domain. Binding of HMGB1 to TM resulted in decrement of TM’s cofactor activity to activate protein C by thrombin. HMGB1 bound to TM was gradually degraded by thrombin. These may be a system to localize HMGB1 only in injury sites where TM is down-regulated or disappeared through endothelial-loss. This may exert endothelial defense system against extracellular HMGB1 in severe tissue injury. Another possibility is that the generated antibody against HMGB1 may neutralize the proinflammatory action of the protein. In this context, we found that some of the antibodies against HMGB1 have the characteristics of P-ANCA(perinuclear anti-neutrophil cytoplasmic antibody). This may alter the phenotype of the underlining diseases. In conclusion, we suggest that HMGB1 is not merely a lethal mediator, but a kind of “testament” mediator of cell necrosis or invasive attacks to dendritic cells.

Recent investigations have been revealing that platelet can sense shear stress and cold stimulation. These mechanical and physical stimuli alter the platelet functions, including induction of activation and irreversible dysfunction. These data may suggest that platelets, most small-sized cells, can efficiently circulate in the microcirculatory vasculature, and can sense the peripheral physical conditions including temperature. Here we show that human megakaryocytic cell line, MEG-01 and platelets express vanilloid receptor VR1, a receptor for capsaicin. It has been identified that VR1 is linked to thermal sensor, and this can sense hyperthermia around 43 °. Therefore we examined the physiological significance of VR1 expressed on MEG-01 and platelets. First we identified the expression of VR1 on human platelets and MEG-01 by immunoblotting, flowcytometer and RT-PCR, and identified the expression of VR1 on these cells. Hyperthermia stimulation of MEG-01 at 40–43 ° generated a endocannabinoids, 2-arachidonylglycerol(2-AG) and anandamide. Since this effect of hyperthermia stimulation was abolished by the incubation with capsazepine, an antagonistic analogue of capsaicin, it was suggested that the effect of hyperthermia might through VR1. It is well known that 2-AG and anandamide act on their specific receptors, CB1, CB2 and VR1 expressed on various cell-types, and act as an endocannabinoids, and accomplish diverse physiological functions, including vessel dilatation. Hyperthermia stimulation also affected the cell-cycle and induced G1-phase arrest in MEG-01 cells, partially through autocrine manner of generated endocannabinoids by acting of CB1 and CB2 receptors. Moreover prolonged hyperthermia stimulation up to 6 hours induced cellular apoptosis in MEG-01 by releasing cytocrome C and resulting caspase cascade activation. Based on these results, we hypothesize that VR1 on platelets may act as a hyperthermal sensor and generate endocannabinoid, and this may regulate the diverse systems including vessel functions.

Uric acid, a final metabolite of purine metabolism has gained attention for its pathogenic role in the development of hypertension, vascular disease, renal disease, and cardiovascular events. Recent studies have shown that soluble uric acid induces proliferation as well as synthesis of proinflammatory chemokine, monocyte chemoattractant protein 1 in rat vascular smooth muscle cells. It also stimulates human monolayer cells to produce IL-1B. IL-6 and TNF alpha. However, high mobility group box 1(HMGB1), a non histone DNA binding protein which is recently identified as a proinflammatory cytokine and can be released from cells of the macrophage/monocyte lineage by proinflammatory stimuli or by cells undergoing necrotic cell death pathway. In the present study, we hypothesized that soluble uric acid may stimulates monocytes/macrophages to release HMGB1. Here we demonstrate that crystal and endotoxin free uric acid triggers the release of HMGB1 in a time and dose dependant fashion in mouse macrophage cells, Raw 264.7, human leukemic promonocytes(THP-1 cells) as well as in macrophages but not in fibroblast obtained from synovial fluid of rheumatoid arthritis patients. Moreover, translocation of HMGB1 to the cytoplasm before secreted in the extracellular milieu was significantly detected within 6h of Uric acid exposure. No loss of cell viability was observed in our experimental condition as judged by trypan blue exclusion, MTT reduction and LDH release assays, indicating that HMGB1 release was not due to cell death. Uric acid treatment resulted in the activation of p38 mitogen activated protein kinase(MAPK), c-Jun N terminal kinase(JNK) and extracellular regulatroy kinase (ERK1/2). Inhibition of each of p38 MAPK, ERK1/2 and JNK by using potent and selective inhibitors, SB203580, U0126 and SP60012 respectively, almost completely blocked uric acid-induced HMGB1 release. Furthermore, Curcumin, a potent inhibitor of the transcription factor, activator protein 1(AP-1) strongly suppressed HMGB1 release, indicating that these signaling patways are essential for the induction of HMGB1 release in response to uric acid. In conclusion, our data suggest that uric acid may regulate critical proinflammatory pathways at least in part through its action as a monocyte/macrophage derived potent proinflammatory cytokine, HMGB1 release. This may play pathologic role for hyperuricemia-induced morbidities including arthritis, atherosclerosis, renal disease and so on.