Effects of Dabigatran and Rivaroxaban on Thrombus Formation under Flow Conditions.

<p>The effects of dabigatran and rivaroxaban on thrombus formation in the absence (<b>panels A and B</b>) and presence of aspirin/AR-C66096 (<b>panels C and D</b>) were evaluated using T-TAS. Thrombus formation inside LTP (low thromboplastin) microchips (<b>panels A and C</b>) and HTP (high thromboplastin) microchips (<b>panels B and D</b>). Data (n = 5) are shown as mean values. Shaded boxes indicate the values for T₁₀ and/or OT exceeding 30 min, and the frequency of such measurements is indicated above each box. AUC₃₀, area under the curve for 30 min; OT, occlusion time; T₁₀, time to 10 kPa.</p

Effects of Dabigatran and Rivaroxaban on TG in PRP.

<p>The inhibitory effects of dabigatran (Dabi) and rivaroxaban (Riva) on thrombin generation (TG) in platelet-rich plasma in the absence (<b>panel A</b>) and presence of 100 µM aspirin (Asp) together with 1 µM AR-C66096 (AR-C) (<b>panel B</b>) were calculated using Thrombinoscope™. Typical TG patterns obtained in one of the experiments are shown.</p

Effects of Dabigatran and Rivaroxaban on TG in PPP (high tissue factor).

<p>TG, thrombin generation triggered by 5 pM recombinant tissue factor; PPP, platelet-poor plasma; Dabi, dabigatran; Riva, rivaroxaban; LT, lag time; TTP, time to peak of TG; PH, peak height of TG; ETP, endogenous thrombin generation potential.</p><p>Data are shown as the mean ± SD. * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001 <i>vs.</i> control.</p>§<p>indicates that, since TG curves did not come down to the baseline within 60 min, ETP values were calculated by setting the start tail at 60min.</p

Effects of Dabigatran and Rivaroxaban on TG in PPP (low tissue factor).

<p>TG, thrombin generation triggered by 1 pM recombinant tissue factor; PPP, platelet-poor plasma; Dabi, dabigatran; Riva, rivaroxaban; LT, lag time; TTP, time to peak of TG; PH, peak height of TG; ETP, endogenous thrombin generation potential.</p><p>Data are shown as the mean ± SD. * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001 <i>vs.</i> control.</p>§<p>indicates that, since TG curves did not come down to the baseline within 60 min, ETP values were calculated by setting the start tail at 60min.</p

Effects of Dabigatran and Rivaroxaban on TG in PPP.

<p>The inhibitory effects of dabigatran (Dabi) and rivaroxaban (Riva) on thrombin generation (TG) in platelet-poor plasma were evaluated using the Thrombinoscope™. <b>Panel A:</b> TG triggered by 1 pM recombinant tissue factor, and <b>Panel B:</b> TG triggered by 5 pM recombinant tissue factor. Typical TG patterns reflecting distinctive efficacies of dabigatran and rivaroxaban are shown.</p

HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

<div><p>Rationale</p><p>Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.</p><p>Objectives</p><p>To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.</p><p>Methods</p><p>Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.</p><p>Results</p><p>HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.</p><p>Conclusions</p><p>Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.</p></div

Anti-HMGB1 antibody dampens lung inflammation.

<p>BALF samples were collected from MCT-induced PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge. The number of leukocytes (A) and the concentrations of endothelin-1 (B), TNF-α, MCP-1, and IL-1β (C) in BALF were measured (n = 6–7 per group). All data are expressed as mean ± SEM. *<i>P</i><0.05 and **<i>P</i><0.01.</p

HMGB1 is translocated from the nucleus to the cytoplasm of periarterial infiltrating leukocytes, alveolar macrophages, and bronchial epithelial cells in MCT-injected rats.

<p>Lung samples were collected from rats 1 week after vehicle-injection (A, D, H) or MCT-injection (B, C, E–G, I). (C) and (F) are magnified views of the insets shown in (B) and (E), respectively. The localization of HMGB1 was assessed by immunohistochemistry. Nuclear protein HMGB1 was translocated to the cytoplasm of periarterial infiltrating cells, alveolar macrophages (arrowheads), and bronchial epithelial cells (arrows) in MCT-induced PAH rats. Representative images of n = 5–6. Scale bars represent 50 µm (A–E, H, I) and 20 µm (F, G).</p

Nuclear protein HMGB1 is released to the bronchoalveolar space during the early stage of PAH.

<p>(A–B) BALF samples were collected from MCT- or vehicle-injected rats at the indicated time points. The levels of HMGB1, MCP-1, and TNF-α (A), and the number of leukocytes (B) in BALF were measured. (C) Serum samples were collected from MCT- or vehicle-injected rats, and the levels of HMGB1 and MCP-1 were measured (n = 6–13 per group). All data are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001.</p

Anti-HMGB1 antibody prevents wall thickening of pulmonary arterioles in MCT-injected rats.

<p>(A) H&E staining (upper panels) and Elastica van Gieson staining (lower panels) of pulmonary arteries of MCT-induced PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge. Representative images of n = 6. Scale bars = 20 µm. (B) Pulmonary artery wall thickness were measured in the PAH rats treated with anti-HMGB1 IgY or control IgY at 3 weeks after MCT challenge (n = 6 per group).The external diameter and medial wall thickness of the pulmonary arteries were measured in 20 muscular arteries (ranging in size from 25–100 µm in external diameter) on Elastica van Gieson–stained sections. For each artery, medial wall thickness was expressed as follows: % wall thickness = [(medial thickness×2)/external diameter]×100. All data are expressed as mean ± SEM. **<i>P</i><0.01.</p