Thrombomodulin Levels Research Articles

Increased serum thrombomodulin level is associated with disease severity and mortality in pediatric sepsis.

BackgroundEndothelial dysfunction plays an important role in the pathophysiology of sepsis. As previously reported, the serum thrombomodulin is elevated in diseases associated with endothelial injury.ObjectiveThe aim of this study was to investigate the association of serum thrombomodulin level in different pediatric sepsis syndromes and evaluate the relationship with disease severity and mortality.MethodsWe prospectively collected cases of sepsis treated in a pediatric intensive care unit from June 2012 to July 2015 at Chang Gung Children’s Hospital in Taoyuan, Taiwan. Clinical characteristics and serum thrombomodulin levels were analyzed.ResultsIncreased serum thrombomodulin levels on days 1 and 3 of the diagnosis of sepsis were found in different pediatric sepsis syndromes. Patients with septic shock had significantly increased serum thrombomodulin levels on days 1 and 3 [day 1: median, 6.9 mU/ml (interquartile range (IQR): 5.8–12.8) and day 3: median, 5.8 mU/ml (IQR: 4.6–10.8)] compared to healthy controls [median, 3.4 mU/ml (IQR: 2.3–4.2)] (p = <0.001 and 0.001, respectively) and those with sepsis [day 1: median, 2.9 mU/ml (IQR: 1.8–4.7) and day 3: median, 3 mU/ml (IQR: 1.5–3.5)] and severe sepsis [day 1: median, 3.3 mU/ml (IQR: 1.3–8.6) and day 3: median, 4.4 mU/ml (IQR: 0.5–6)] (p = <0.001 and 0.001, respectively). There was also a significant positive correlation between serum thrombomodulin level on day 1 and day 1 PRISM-II, PELOD, P-MOD and DIC scores. The patients who died had significantly higher serum thrombomodulin levels on days 1 and 3 [day 1: median, 9.9 mU/ml (IQR: 6.2–15.6) and day 3: median, 10.4 mU/ml (IQR: 9.2–11.7)] than the survivors [day 1; median, 4.4 mU/ml (IQR: 2.2–7.5) and day 3: [median, 3.5 mU/ml (IQR: 1.6–5.7)] (p = 0.046 and 0.012, respectively).ConclusionIncreased serum thrombomodulin levels were found in different pediatric sepsis syndromes and correlated with disease severity and mortality.

Decreased Expression of Thrombomodulin in Endothelial Cells by Fibroblast Growth Factor-23/α-Klotho.

Chronic kidney disease (CKD) has been known to be a state of excessive fibroblast growth factor-23 (FGF23) and α-Klotho deficiency. Patients undergoing hemodialysis have an increased mortality risk associated with cardiovascular disease and endothelial dysfunction. The mechanism responsible for the relationship of FGF23 to endothelial damage in these patients has been unclear. On the other hands, increasing evidences have demonstrated that thrombomodulin (TM) plays an important role in the endothelial barrier. Here, we report the suppression of membrane TM, in a dose-dependent manner, in human umbilical vein endothelial cells after FGF23 and FGF23/α-Klotho stimulation. In addition, the levels of soluble TM, which reflect endothelial cell injury, were much higher in cell supernatants after FGF23 and FGF23/α-Klotho stimulation than in the control supernatant. This study indicates a possible mechanism by which excessive levels of FGF23 are involved in endothelial TM disruption, which has been implicated as a potential cardiovascular risk factor in patients with CKD, especially in HD patients.

Plasma level of apelin and carotid atherosclerosis in maintenance hemodialysis patients

To determine the plasma level of apelin in patients of maintenance hemodialysis (MHD) and to explore the relationship between apelin level and carotid atherosclerosis (AS) in MHD patients. Methods: A total of 92 MHD patients and 36 sex- and age-matched healthy subjects were enrolled in this study. The plasma level of apelin was evaluated by radiation immunoassay; serum endothelial injury markers including thrombomodulin, von Willebrand factor (vWF), and CD146, and inflammatory factors including high sensitive C-reactive protein (hsCRP), IL-6 and TNF-α were determined by ELISA. Common Carotid arteries intima media thickness (CCA-IMT), cross-sectional calculated intima-media area (cIM area) area and atherosclerotic plaque were measured by non-invasive high-resolution B-mode ultrasonography. Results: The plasma levels of apelin was significantly decreased in MHD patients compared with healthy subjects (P<0.01), accompanied with elevated plasma levels of thrombomodulin, vWF, CD146, hsCRP, IL-6 and TNF-α (all P<0.01). The plasma levels of apelinin in MHD patients with carotid artery plaques were obviously lower than those without plaques [(43.16±10.12) pg/mL vs (61.43±16.25) pg/mL, P<0.01]. Plasma level of apelin was inversely related with CCA-IMT and cIM area (r=-0.355 and r=-0.297 respectively, all P<0.01). Multiple stepwise regression analysis showed that plasma level of apelin was an independent risk factor for CCA-IMT and cIM area. Conclusion: The plasma apelin in MHD patients might take part in vascular endothelial injury and the progress of atherosclerosis. It plays an important role in the initiation and development of uremia associated atherosclerosis through elevating inflammatory factors including hsCRP, IL-6 and TNF-α levels.

Evaluation of Plasma Thrombomodulin in Patients with Coronary Slow Flow

Background: It has been reported that coronary slow flow (CSF) is associated with acute myocardial infarction, ventricular tachycardia, ventricular fibrillation, and even sudden cardiac death. Although studies concerning the etiopathogenesis of CSF are scarce, diffuse atherosclerosis and endothelial dysfunction are thought to play important roles. It has been suggested that a high plasma thrombomodulin (TM) level seems to play an important role in the pathogenesis of atherosclerosis and endothelial dysfunction. Objectives: We hypothesized that a high plasma TM level might be associated with CSF and aimed to research the relationship between plasma TM level and CSF. Methods: Fifty-two CSF patients with angiographically proven CSF and 44 cases with normal coronary flow were included in this study. Coronary flow velocity was determined by the thrombolysis in myocardial infarction (TIMI) frame count method. Plasma TM levels were measured in all the study subjects. Results: Plasma TM levels were significantly higher in the CSF group compared to the control group (3.9 ± 0.5 vs. 3.6 ± 0.3 ng/mL, p = 0.01). There was a positive relationship (r = 0.31, p = 0.002) between plasma TM level and mean TIMI frame count (TFC). Factors associated with mean TFC were plasma TM level (β = 0.206, p = 0.038) and red cell distribution width (β = 0.088, p = 0.009) in multiple linear regression analysis. Conclusions: Patients with CSF have a higher plasma TM level, and this may play an important role in the pathogenesis of CSF. An elevated plasma TM level may be a predictor of CSF. Future studies are needed to confirm these results.

Depleted nitric oxide and prostaglandin E2 levels are correlated with endothelial dysfunction in β-thalassemia/HbE patients

Mechanisms of vascular disorders in β-thalassemia/HbE patients remain poorly understood. In the present study, we aimed to determine the presence of endothelial dysfunction and its association with altered vascular mediators in this population. Forty-three β-thalassemia/HbE patients without clinically documented vascular symptoms and 43 age-sex-matched healthy controls were enrolled. Endothelial function was assessed using flow-mediated dilatation (FMD) before and after administration of nitroglycerine (NTG). β-Thalassemia/HbE patients showed a significant endothelial dysfunction using FMD. The percentage change in the brachial artery diameter before NTG was significantly lower in the thalassemia group compared to the control (5.0±5.9 vs. 9.0±4.0%, p<0.01) while no significant differences after NTG (18.4±8.3 vs. 17.8±6.3%, p=0.71). Plasma nitric oxide metabolites (NO x ) and prostaglandin E2 (PGE2) levels were significantly decreased in β-thalassemia/HbE (117.2±27.3 vs. 135.8±11.3µmol/L, p<0.01) and (701.9±676.0 vs. 1374.7±716.5pg/mL, p<0.01), respectively, while a significant elevation in soluble thrombomodulin levels in β-thalassemia/HbE (3587.7±1310.0 vs. 3093.9±583.8pg/mL, p=0.028). NO x and PGE2 levels were significantly correlated with FMD (r=0.27, p=0.025) and (r=0.35, p=0.003), respectively. These findings suggest roles for endothelial mediators and a new mechanism underlying endothelial dysfunction in β-thalassemia/HbE patients.

Benefits of anti‑inflammatory therapy in the treatment of ischemia/reperfusion injury in the renal microvascular endothelium of rats with return of spontaneous circulation.

The aim of the present study was to investigate whether prostaglandin E1 (PGE1), target temperature management (TTM) and a combined intervention involving the two would be beneficial as anti‑inflammatory therapies for ischemia/reperfusion (I/R) injury to the renal microvascular endothelium of rats with return of spontaneous circulation (ROSC). In each group of rats with different interventions, following successful cardiopulmonary resuscitation, the levels of thrombomodulin (TM), interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) in the plasma were evaluated. The expression of vascular endothelial (VE)‑cadherin and vascular cell adhesion molecule‑1 (VCAM‑1) mRNA was analyzed in the kidney. Hematoxylin and eosin staining and VE‑cadherin/vascular endothelial growth factor receptor double fluorescent immunohistochemistry staining were also performed. PGE1 improved tubular cell swelling and inflammatory cell infiltration. PGE1 also alleviated VE‑cadherin protein loss in renal microvascular endothelium cells (RMECs), lowered the tubular injury score, decreased VE‑cadherin and VCAM‑1 mRNA expression, and markedly inhibited the release of TM (at 3 time points) and TNF‑α (4 and 8h; P<0.05). In addition to improving the renal tubular injury score and altering the concentration of TNF‑α at 8h, the effect of TTM was the same as PGE1 for the other indicators (P>0.05). The PGE1/TTM combined intervention significantly reduced IL‑6 concentration at 8h (P<0.05). The correlation analysis demonstrated that the peak TM and TNF‑α levels (P<0.001, r=0.809), and IL‑6 levels (P<0.001, r=0.792) were positively associated. PGE1 and TTM had a protective effect against I/R injury to the RMEC, while the PGE1/TTM combined intervention exhibited an increased synergistic effect as an anti‑inflammatory treatment when compared with either of the single interventions.

Carbon monoxide-releasing molecule-2 suppresses thrombomodulin and endothelial protein C receptor expression of human umbilical vein endothelial cells induced by lipopolysaccharide in vitro.

Objective:The aim of this study was to observe the counter-effect of carbon monoxide-releasing molecule-2 (CORM-2) on lipopolysaccharide (LPS)-suppressed thrombomodulin (TM) and endothelial protein C receptor (EPCR) expressions from human umbilical vein endothelial cell (HUVEC), and to reveal its mechanisms.Methods:HUVECs were divided into 5 treatment groups, wherein reagents were added simultaneously. TM and EPCR proteins of the cells and the culture medium levels of soluble TM, soluble EPCR, and matrix metalloproteinase-2 (MMP-2) were detected after administration, whereas mRNA levels of TM and EPCR, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity among groups, were also evaluated.Results:No significant difference was observed in any indicator between CORM-2 and sham groups. Addition of LPS produced drastic increase in MMP-2 expression, NF-κB activity, shedding of TM and EPCR (into the culture medium), as well as remarkable decrease in both mRNA and protein expressions of TM and EPCR, and cell viability. LPS + CORM-2 treatment significantly reduced the increase in MMP-2, NF-κB activity, and TM/EPCR shedding, whereas maintained both mRNA and protein levels of TM and EPCR, and preserved cell viability.Conclusions:CORM-2 protects HUVEC from LPS-induced injury, by way of suppressing NF-κB activity, which downregulates TM and EPCR mRNAs. It also decreases MMP-2 expression and prevents the shedding of TM and EPCR from the surface of endothelial cells, so as to preserve their protective effect.

Effects of alloantibodies to human leukocyte antigen on endothelial expression and serum levels of thrombomodulin

Rationale Thrombomodulin (TBM) is an anticoagulant and anti-inflammatory transmembrane protein expressed on endothelial cells. Donor-specific alloantibodies, particularly those against human leukocyte antigen (HLA) class II, are associated with microvascular endothelial damage in solid allografts. Objective Our aim was to characterize the effects of anti-HLA antibodies on endothelial expression of TBM, and in particular, the differential effects of anti-HLA class I compared with those of anti-HLA class II. Methods We used human glomerular microvascular endothelial cells to examine TBM expression on anti-HLA-treated cells, and we tested sera from transplant recipients for soluble TBM. Results We found that whereas membrane TBM expression increased in a dose-dependent manner in the presence of anti-HLA class I antibodies, treatment with anti-HLA class II led to minimal TBM expression on the endothelial surface but to a cytosolic accumulation. Platelet adhesion studies confirmed the functional impact of anti-HLA class II. Quantitative densitometry of the membrane lysates further suggested that anti-HLA class II impairs TBM glycosylation. Furthermore, we found a significant association between the presence of circulating anti-HLA class II antibodies in transplant recipients and low serum levels of TBM. Conclusion These results indicate that ligation of anti-HLA class I and II antibodies produces different effects on the endothelial expression of TBM and on serum levels of TBM in transplant recipients. Anti-HLA class II antibodies may be associated with a prothrombotic state, which could explain the higher occurrence of microangiopathic lesions in the allograft and the poor outcomes observed in patients with these alloantibodies.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

Pentraxin 3 as a new indicator of cardiovascular‑related death in patients with advanced chronic kidney disease.

INTRODUCTION Pentraxin3 (PTX3) play an important role in the inflammatory response, taking part in recognizing pathogens and damaged tissues. OBJECTIVES The aim of the study was to assess the relationship between PTX3 levels and all-cause and cardiovascular (CV) mortality in chronic kidney disease (CKD) patients during five-year observation period. PATIENTS AND METHODS The study comprised 78 patients (51 hemodialyzed, 27 predialysis). The examined parameters included PTX3, calcium, phosphate, iPTH, interleukin-6 (IL-6), fibroblast growth factor 23 (FGF-23), osteopontin (OPN), osteoprotegerin (OPG), osteocalcin (OC), osteopontin (OPN), osteoprotegerin (OPG), osteocalcin, tumor necrosis factor receptor II (TNF-R II), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), stromal cell-derived factor α (SDF1α), and thrombomodulin (TM). In a subgroup of 45 patients, fragments of radial artery obtained during creation of hemodialysis access were stained for calcifications. In 51 patients, ultrasonography was performed to assess intima-media thickness (CCA-IMT). RESULTS Median serum concentration of PTX3 was 1.43 (0.74-2.50) ng/ml. Higher concentrations of fibrinogen, CRP, IL-6, TNF-R II, TGFβ1, HGF, OPN, OPG, FGF-23, TM, SDF1α, lower albumin and uric acid levels were observed in patients with PTX3 above the median. During follow-up, 27 patients (35%) died, including 25 due to CV causes. In contrast to CRP, baseline PTX3 predicted CV mortality independently of classical CV risk factors. Also, PTX3 concentrations significantly predicted mortality after adjustment for age, baseline dialysis status, serum OPG and CRP, radial artery calcifications, and CCA-IMT. CONCLUSIONS We postulate that PTX3 might be an early marker of CV mortality in patients with advanced CKD yet before the increase of specific marker for systemic inflammation like hsCRP.

Protective effects of molecular hydrogen on steroid-induced osteonecrosis in rabbits via reducing oxidative stress and apoptosis

BackgroundThe objective of this study was to investigate the protective effects of molecular hydrogen, a novel and selective antioxidant, on steroid-induced osteonecrosis (ON) in a rabbit model.MethodsSixty rabbits were randomly divided into two groups (model group and hydrogen group). Osteonecrosis was induced according to an established protocol of steroid-induced ON. Rabbits in the hydrogen group were treated with intraperitoneal injections of molecular hydrogen at 10 ml/kg body weight for seven consecutive days. Plasma levels of total cholesterol, triglycerides, soluble thrombomodulin(sTM), glutathione(GSH) and malondialdehyde(MDA) were measured before and after steroid administration. The presence or absence of ON was examined histopathologically. Oxidative injury and vascular injury were assessed in vivo by immunohistochemical staining of 8-hydoxy-2-deoxyguanosine(8-OHdG) and MDA, and ink artery infusion angiography. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to measure apoptosis.ResultsThe incidence of steroid-induced ON was significantly lower in hydrogen group (28.6%) than that in model group (68.0%). No statistically differences were observed on the levels of total cholesterol and triglycerides. Oxidative injury, vascular injury and apoptosis were attenuated in the hydrogen group compared with those in the model group in vivo.ConclusionsThese results suggested that molecular hydrogen prevents steroid-induced osteonecrosis in rabbits by suppressing oxidative injury, vascular injury and apoptosis.

Protective Effects of Total Flavones of Elaeagnus rhamnoides (L.) A. Nelson against Vascular Endothelial Injury in Blood Stasis Model Rats.

The aim was to evaluate the protective effects of total flavones of Elaeagnus rhamnoides (L.) A. Nelson (TFE) against vascular endothelial injury in blood stasis model rats and explore the potential mechanisms preliminarily. The model of blood stasis rat model with vascular endothelial injury was induced by subcutaneous injection of adrenaline combined with ice-water bath. Whole blood viscosity (WBV), histological examination, and prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) were measured. Meanwhile, the levels of Thromboxane B2 (TXB2), 6-keto-PGF1α, von Willebrand factor (vWF), and thrombomodulin (TM) were detected. In addition, Quantitative Real-Time PCR (qPCR) was performed to identify PI3K, Erk2, Bcl-2, and caspase-3 gene expression. The results showed that TFE can relieve WBV, increase PT and APTT, and decrease FIB content obviously. Moreover, TFE might significantly downregulate the levels of TXB2, vWF, and TM in plasma and upregulate the level of 6-keto-PGF1α in plasma. Expressions of PI3K and Bcl-2 were increased and the expression of caspase-3 was decreased by TFE pretreatment in the rat model. Consequently, the study suggested that TFE may have the potential against vascular endothelial injury in blood stasis model rats induced by a high dose of adrenaline with ice-water bath.

Serum Soluble Thrombomodulin Level on Admission Is a Useful Predictor of Treatment Response in Patients with Acute Pulmonary Thromboembolism

Serum Soluble Thrombomodulin Level on Admission Is a Useful Predictor of Treatment Response in Patients with Acute Pulmonary Thromboembolism

Soluble thrombomodulin in bronchoalveolar lavage fluid is an independent predictor of severe drug-induced lung injury.

Drug-induced lung injury (DLI) can result from a vast number of agents, and sometimes presents findings similar to those of acute respiratory distress syndrome (ARDS). Previous studies have reported that circulating levels of soluble thrombomodulin (TM) reflect endothelial injuries, which play key roles in the development of ARDS. We hypothesized that endothelial injuries are an important aspect of pathogenesis in severe DLI. The primary aim of this study was to examine the associations between soluble TM and disease severity in DLI patients. Of the 2580 patients who underwent a bronchoalveolar lavage (BAL) procedure at Tosei General Hospital between May 2007 and February 2015, we retrospectively analysed the data of 68 DLI patients. Soluble TM in plasma and BAL fluid (BALF), and other biomarkers were included in our analysis. At the time of diagnosis, 39 patients (57%) had respiratory failure (partial pressure of oxygen/inspiratory oxygen fraction ratio, PaO2 /FiO2 ratio < 300). There was a significant negative linear correlation between the PaO2 /FiO2 ratio and soluble TM in BALF (r = -0.448, P < 0.001). In a stepwise multiple regression analysis, soluble TM in BALF and surfactant protein D (SP-D) were the only independent determinants of the PaO2 /FiO2 ratio. Additionally, in a multivariate logistic regression model, soluble TM in BALF (adjusted OR (aOR): 7.48, 95% CI: 1.60-34.98) and SP-D (aOR: 5.31, 95% CI: 1.40-20.15) was an independent predictor of respiratory failure (PaO2 /FiO2 ratio < 300). Soluble TM in BALF is an independent predictor of severe DLI. These findings underscore the importance of pulmonary endothelial injuries in the pathogenesis of severe DLI.

Down-Regulation of Protein C Pathway Associated with Increased Inflammation in Patients with Implanted Ventricular Assist Devices As a Potential Cause of Pump Thrombosis

Introduction: The use of left ventricular assist devices (LVADs) is increasingly common in patients suffering from heart failure. However, the placement of an LVAD comes with risks, including pump thrombosis that occurs in up to 20% of patients. Pump thrombosis constitutes a major cause of death in this patient population, and understanding the mechanism behind this complication will allow its prevention. The protein C pathway is critical in the maintenance of blood hemostasis, aiding in the prevention of hypercoagulability. It is hypothesized that the state of inflammation in LVAD patients dysregulates the protein C pathway, creating a hypercoagulable state with resultant thrombosis. The objective of this project was to characterize changes in the protein C pathway in patients supported by an LVAD who experienced a thrombotic event.Materials and Methods: Citrated plasma sampleswere collected peri-operatively and at varying times post-operatively from 22 patients implanted with a Thoratec HeartMate II LVAD. Plasma samples were stored frozen (-80°C) and batch analyzed for levels of total protein S, free protein S, protein C, C-reactive protein (CRP), soluble endothelial cell protein C receptor (EPCR) and soluble thrombomodulin (TM) by ELISA. Samples near the time of an event and 1-3 months earlier were analyzed. The occurrence of adverse clinical events was determined retrospectively using an internal RedCap database. LVAD-associated thrombosis was characterized as: cerebrovascular accident/transient ischemic attack diagnosed by a neurologist, rise in LDH or plasma free hemoglobin, hemolysis, evidence of pump dysfunction consistent with thrombus identified by pump parameters, echocardiographic or computed tomographic evidence of clot, or surgical pump exchange for thrombus. A comparative group was composed of patients with LVAD-associated bleeding (the other frequent complication in this population) events characterized as: anemia and bleeding determined by a cardiologist.Results: The median level of CRP was higher and levels of total and free protein S were lower in patients with thrombotic events compared to those with bleeding events. Median levels of protein C and TM were comparable in patients with thrombotic or bleeding events. Secondly, for patients with a thrombotic event, levels of total protein S, free protein S, and protein C (but not TM or CRP) were further decreased at time near the event. No changes were observed in any of these parameters preceding or at the time of a bleeding complication. Compared to normal ranges, levels of proteins C and S were consistent with low-dose warfarin therapy (INR 1.5-2.0) given to all LVAD patients; TM levels were similar to normal (2.8 ng/ml). EPCR levels were lower than normal (828 ng/ml) in all groups with no apparent change at time of event. Compared to normal (0.8 µg/ml) CRP levels were very elevated in the thrombosis patients and slightly elevated in the bleeding patients.Conclusions: This data demonstrates excessively high CRP levels in patients who experienced LVAD-associated thrombosis. These patients also had lower total protein S and free protein S levels which decreased further at time of the thrombotic event. In the immunoassays used in this study no difference was observed between the levels of total and free protein S. Whether the up-regulated inflammatory state increases levels of the protein S linked C4b binding protein and whether the function of protein S is inhibited could not be proven here. This study suggests that protein S is a gatekeeper for the function of the protein C pathway. Clinically, in addition to measuring protein S levels and/or function, the degree of the inflammatory state should be considered as the level of C4b binding protein would be altered affecting the functionality of protein S. Our study has shown that abnormal levels of protein S and CRP are components that establish a hypercoagulable environment in LVAD implanted patients where pump thrombus develops weeks later. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies on the Anticoagulant Actions of Recombinant Thrombomodulin and Heparin and Their Neutralization By FEIBA As Measured By Thromboelastography

INTRODUCTIONThromboelastographic (TEG) analysis represents a global approach to monitor the clotability of the native whole blood and is sensitive to the cellular and plasma components of the blood which affect the rate or structure of a thrombus or its breakdown. This method can be used for the evaluation of the point of care hemostatic status of whole blood. rTM (recombinant Thrombomodulin) is currently developed for various clinical indications and exerts multiple actions on blood and its components to reduce thrombogenesis without increasing a bleeding risk. This is in contrast to Heparins which mediate their actions accompanied by some bleeding risk. FEIBA (Factor VII Inhibitor Bypass Activity) contains non activated Factors II, IX and X and activated factor VII. It has been approved by the FDA for use in Hemophilia A and B patients with inhibitors for routine prophylaxis and to prevent or reduce the frequency of bleeding episodes. The purpose of this study is to compare the anticoagulant effects related to bleeding risk of rTM and Unfractionated Heparin (UH) at various levels and their neutralization by FEIBA. MATERIALS AND METHODSCitrated Whole Blood samples were supplemented with rTM or UH in a concentration range of 0-10ug/ml. (n=10). TEG analysis was performed on a TEG 5000 system in which the clotting was initiated by recalcification of the whole blood and set parameters as R time, K Time, MA and Angle were measured. The relative neutralization profiles of the UH and rTM by FEIBA at 1 and 0.1 U/ml were also investigated. All results were analyzed in terms of Mean Values ± Standard Deviation. RESULTSIn comparison to UH, rTM exhibited much weaker anticoagulant effects which were evident in all parametric evaluations.At 1ug/ml rTM did not produce any anticoagulant effects as evident by TEG profile. At higher levels greater than 2.5ug/ml, rTM produced a concentration dependent anticoagulant effect which altered the TEG profile of the whole blood. In contrast, UH produced relatively stronger anticoagulant effects and at a concentration greater than 2.5ug/ml, totally inhibited the clot formation in the TEG analysis. The anticoagulant effects of a 1ug/ml rTM as compared to 1ug/ml UH are tabulated in the following table.At higher concentrations, UH produced strong anticoagulation of whole blood whereas rTM at a concentration of 10ug/ml produced weaker effects. FEIBA at a concentration of 1U/ml completely neutralized the anticoagulant effect of rTM at 10ug/ml and UH at 0.5ug/ml. Furthermore, FEIBA at a concentration of 0.1U/ml neutralized the anticoagulant effects of rTM completely at a concentration of 10ug/ml. At this level, it only partially neutralized the anticoagulation effects of Heparin at 0.5ug/ml. The neutralization of anticoagulation effects of rTM at 10ug/ml and UH at 0.5ug/ml by FEIBA at 0.1U/ml are tabulated in the following table. CONCLUSIONThese studies suggest that rTM is a relatively weaker anticoagulant in comparison to UH. Furthermore, in the given indications, the expected concentration of rTM is in the range of 0.5-1.5ug/ml. At these concentrations, this agent is not expected to produce any anticoagulant effects. In contrast UH, at therapeutic concentrations of 1-2ug/ml produces, strong anticoagulant effect. While these TEG studies indicate that rTM is an antithrombotic agent mediating its effects via multiple mechanisms whereas heparins primarily produce anticoagulant effects. Moreover, the supratheraputic levels of thrombomodulin and heparin are neutralizable by FEIBA. [Display omitted] [Display omitted] DisclosuresTsuruta:Asahi Kasei Pharma America: Employment.

The Relationship between Endothelial Dysfunction and Endothelial Cell Markers in Peripheral Arterial Disease.

IntroductionThe endothelial function plays key roles in both promoting and protecting against atherosclerotic disease. Several methods, including peripheral arterial tonometry (PAT), have been reported to be useful for investigating endothelial dysfunction. Furthermore, the level of thrombomodulin (TM) is assumed to reflect the endothelial dysfunction. In the present study, we investigated endothelial dysfunction in patients with peripheral arterial disease (PAD) by measuring their TM levels, and evaluated the correlation between TM and various parameters.Materials and MethodsWe measured the TM levels and performed PAT in 17 patients with PAD. We also recorded the patients’ demographic information, including comorbidities, and investigated their hemodynamic status by measuring the ankle brachial pressure index (ABI). The PAT results were used to calculate the reactive hyperemia index (RHI), which reflected the patients’ level of endothelial dysfunction.ResultsThe RHI and ABI values and the serum level of creatinine were found to be significantly correlated with the TM level (P = 0.040, 0.041 and 0.005, respectively). After setting an RHI value of 1.67 as the cut-off, the patients with RHI values of <1.67 were found to have significantly higher TM levels (median, 20.3 U/mL) than the patients with RHI values of ≥1.67 (median, 13.7 U/mL) (P = 0.023).ConclusionsThe degree of endothelial dysfunction, as calculated by the TM level, might influence the prognostic value of the RHI, which is determined by PAT. The measurement of the TM level and PAT might both be useful methods of measuring endothelial dysfunction in patients with PAD.

Circulating Biomarkers in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral microangiopathy presenting with variable features, including migraine, psychiatric disorders, stroke, and cognitive decline and variable disability. On neuroimaging, CADASIL is characterized by leukoencephalopathy, multiple lacunar infarcts, and microbleeds. Previous studies suggest a possible role of endothelial impairment in the pathogenesis of the disease. We assessed plasma levels of von Willebrand factor (vWF) and thrombomodulin (TM) and the blood levels of endothelial progenitor cells (EPCs) and circulating progenitor cells (CPCs) in 49 CADASIL patients and 49 age-matched controls and their association with clinical/functional and neuroimaging features. In multivariate analysis, CADASIL patients had significantly higher vWF and lower EPC levels. TM levels were similar in the 2 groups. CADASIL patients with a more severe clinical phenotype (history of stroke or dementia) presented lower CPC levels in comparison with patients with a milder phenotype. On correlation analysis, lower CPC levels were associated with worse performances on neuropsychological, motor and functional tests, and with higher lesion load on brain magnetic resonance imaging (degree of leukoencephalopathy and number of lacunar infarcts). This is the first CADASIL series in which multiple circulating biomarkers have been studied. Our findings support previous studies on the presence and the possible modulating effect of endothelial impairment in the disease. Furthermore, our research data suggest that blood CPCs may be markers of disease severity.

Abstract 13974: Loss of Endothelial Primary Cilia Promotes Thrombus Formation in Mice

Introduction: Recent evidence suggest that endothelial cilia may suppress anti-inflammatory signaling and protect against atherosclerosis. Given that atherosclerosis predisposes to thrombosis, we hypothesized that endothelial cilia may also play a fundamental role in thrombogenesis. Hypothesis: We hypothesize that endothelial cilia is important for protection against thrombosis, and that loss of endothelial cilia promotes thrombus formation in mice. Methods: We focused on the intraflagellar transport 88 (IFT88) gene since endothelial deletion of IFT88 has been associated with decreased atherosclerotic lesions in a murine model of atherosclerosis. Key thrombosis-associated molecules were evaluated in IFT88-silenced human umbilical vein endothelial cells (HUVECs). Thrombi formation were monitored in the laser-injured cremasteric arteries of 8 week old male endothelial cell (EC) specific IFT88 knockout (EC-IFT88 -/- ) mice. Results: Compared to scrambled siRNA-treated HUVECs, those exposed to siIFT88 exhibited lower mRNA and protein levels of tissue factor pathway inhibitor (TFPI) and thrombomodulin, two key regulators of blood coagulation (-27% and -20%, respectively; N=3; p &lt; 0.05). We next examined the effect of IFT88 loss on thrombosis in mice. Upon laser-induced injury, we determined, via mean fluorescence intensity, that although the thrombi formed in the cremasteric arteries of EC-IFT88 -/- mice were similar in size to those produced in the wild type (WT) mice (2.2 ± 0.6 and 2.0 ± 0.3 AU, respectively; x10 6 ; N=32 thrombi; p=0.8), the average rate at which thrombi developed was significantly faster in the EC-IFT88 -/- mice relative to that in WT mice (97 ± 11 and 57 ± 3 AU/s, respectively; N=32 thrombi; p&lt;0.001). Conclusions: Loss of endothelial IFT88 results in decreased TFPI mRNA and protein expression in HUVECs. EC-IFT88 -/- mice exhibited earlier thrombi formation in our laser injury model of thrombosis. These results collectively suggest that endothelial cilia intricately regulates thrombus formation in a TFPI-dependent manner and represents a previously unrecognized target to modulate thrombosis susceptibility.

Angiogenic Factors Correlate with T Cell Immune Reconstitution and Clinical Outcomes after Double-Unit Umbilical Cord Blood Transplantation in Adults

Umbilical cord blood (UCB) is a valuable graft source for allogeneic hematopoietic stem cell transplantation (HSCT) in patients who lack adult donors. UCB transplantation (UCBT) in adults results in delayed immune reconstitution, leading to high infection-related morbidity and mortality. Angiogenic factors and markers of endothelial dysfunction have biologic and prognostic significance in conventional HSCT, but their role in UCBT has not been investigated. Furthermore, the interplay between angiogenesis and immune reconstitution has not been studied. Here we examined whether angiogenic cytokines, angiopoietin-1 (ANG-1) and vascular endothelial growth factor (VEGF), or markers of endothelial injury, thrombomodulin (TM) and angiopoietin-2 (ANG-2), associate with thymic regeneration as determined by T cell receptor excision circle (TREC) values and recovery of T cell subsets, as well as clinical outcomes in adult recipients of UCBT. We found that plasma levels of ANG-1 significantly correlated with the reconstitution of naive CD4+CD45RA+ and CD8+CD45RA+ T cell subsets, whereas plasma levels of VEGF displayed a positive correlation with CD4+CD45RO+ T cells and regulatory T cells and a weak correlation with TRECs. Assessment of TM and ANG-2 revealed a strong inverse correlation of both factors with naive T cells and TRECs. The angiogenic capacity of each patient's plasma, as determined by an in vitro angiogenesis assay, positively correlated with VEGF levels and with reconstitution of CD4+ T cell subsets. Higher VEGF levels were associated with worse progression-free survival and higher risk of relapse, whereas higher levels of TM were associated with chronic graft-versus-host disease and nonrelapse mortality. Thus, angiogenic factors may serve as valuable markers associated with T cell reconstitution and clinical outcomes after UCBT.