New methodological approaches for assessing thrombus formation in cardiovascular disease

Antiplatelet therapy is the mainstay preventive strategy for cardiovascular diseases, and dual antiplatelet therapy comprising aspirin and a P2Y12 inhibitor is the standard treatment for patients who underwent percutaneous coronary intervention. The Total Thrombus‑Formation Analysis System (T‑TAS) is a microchip flow‑chamber system developed to evaluate overall thrombus formation under flow conditions, which is reportedly able to assess single and combined antithrombotic therapy. Here, we focus on this new system, T‑TAS, and review its characteristics together with those of the conventional systems available for evaluation of antithrombotic therapies for cardiovascular diseases

HMGB1 Promotes Intraoral Palatal Wound Healing through RAGE-Dependent Mechanisms

High mobility group box 1 (HMGB1) is tightly connected to the process of tissue organization upon tissue injury. Here we show that HMGB1 controls epithelium and connective tissue regeneration both in vivo and in vitro during palatal wound healing. Heterozygous HMGB1 (Hmgb1+/−) mice and Wild-type (WT) mice were subjected to palatal injury. Maxillary tissues were stained with Mallory Azan or immunostained with anti-HMGB1, anti-proliferating cell nuclear antigen (PCNA), anti-nuclear factor-κB (NF-κB) p50 and anti-vascular endothelial growth factor (VEGF) antibodies. Palatal gingival explants were cultured with recombinant HMGB1 (rHMGB1) co-treated with siRNA targeting receptor for advanced glycation end products (RAGEs) for cell migration and PCNA expression analysis. Measurement of the wound area showed differences between Hmgb1+/− and WT mice on Day 3 after wounding. Mallory Azan staining showed densely packed of collagen fibers in WT mice, whereas in Hmgb1+/− mice weave-like pattern of low density collagen bundles were present. At three and seven days post-surgery, PCNA, NF-κB p50 and VEGF positive keratinocytes of WT mice were greater than that of Hmgb1+/− mice. Knockdown of RAGE prevents the effect of rHMGB1-induced cell migration and PCNA expression in gingival cell cultures. The data suggest that HMGB1/RAGE axis has crucial roles in palatal wound healing

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi

Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

<div><p>Introduction</p><p>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.</p><p>Methods</p><p>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 <i>in vitro</i> and in mice.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

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

<p>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.</p

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

<p>(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. ** <i>P</i> < 0.01 compared with the histone-alone group.</p

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

<p>(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).</p

Extracellular histones cause consumptive coagulopathy.

<p>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). ** <i>P</i> < 0.01 compared with the vehicle group.</p