High plasma soluble thrombomodulin levels indicated poor prognosis of decompensated liver cirrhosis: a prospective cohort study.

OBJECTIVE: Hepatic sinusoidal endothelial injury is a prominent characteristic of liver cirrhosis. We determined plasma soluble thrombomodulin (sTM) levels in cirrhosis patients to evaluate the relationship between vascular injury and long-term prognosis. METHODS: A prospective single-center study was performed. The participants were followed up for every 6 months or until death or transplantation. A chemiluminescent enzyme immunoassay was used to establish a baseline sTM. RESULTS: Among the 219 patients with decompensated liver cirrhosis, 53.42% were caused by hepatitis B and hepatitis C. Plasma sTM levels were much higher in cirrhosis than in healthy controls and increased parallel with Child-Pugh classification ( P < 0.01) and the amount of ascites ( P = 0.04). After adjusting for sex, age, international normalized ratio, bilirubin, and other potential factors, multivariate Cox regression revealed that per TU/ml elevation of plasma sTM causes an increase of 8% in mortality, and per-SD elevation of thrombomodulin causes a 53% increase in mortality. As the mortality rates in low (5.90-12.60 TU/ml) and medium (12.70-18.00 TU/ml) sTM levels were similar, so we chose the cutoff of 18.00 TU/ml to divide into two groups, and K-M analysis indicated that patients with sTM >18.0 TU/ml demonstrated an additional 2.01 times death risk (95% CI, 1.13-7.93; P = 0.01) than those with sTM ≤18.0 TU/ml. CONCLUSION: Plasma sTM in cirrhosis was significantly increased in parallel with the severity of liver dysfunction. sTM elevation than 18 TU/ml indicated a poor prognosis of decompensated liver cirrhosis.

Thrombomodulin is associated with increased mortality and organ failure in mechanically ventilated children with acute respiratory failure: biomarker analysis from a multicenter randomized controlled trial.

BACKGROUND: Acute respiratory failure (ARF) can progress to acute respiratory distress syndrome and death. Biomarkers may allow for risk stratification and prognostic enrichment in ARF. Thrombomodulin (TM) is a transmembrane antithrombotic mediator expressed in endothelial cells. It is cleaved into its soluble form (sTM) during inflammation and vascular injury. Levels of sTM correlate with inflammation and end organ dysfunction. METHODS: This was a prospective observational study of 432 patients aged 2 weeks-17 years requiring invasive mechanical ventilation. It was ancillary to the multicenter clinical trial, Randomized Evaluation of Sedation Titration for Respiratory Failure (RESTORE). After consent, patients had up to 3 plasma samples collected at 24-h intervals within 5 days after intubation. sTM was assayed by ELISA. The Hazard ratio (HR) for 90-day mortality was determined by Cox regression. Mixed effect models (MEM) were used to test for association with extrapulmonary multiorgan failure (MOF) and oxygenation index (OI). Age, race, sex and PRISM-III scores were used as confounding variables for multivariable analyses. RESULTS: sTM values ranged from 16.6 to 670.9 ng/ml within 5 days after intubation. Higher sTM was associated with increased 90-day mortality (n = 432, adjusted HR = 1.003, p = 0.02) and worse OI in the first 5 days after intubation (n = 252, Estimate = 0.02, p < 0.01). Both initial and slope of sTM were associated with increased extrapulmonary MOF in unadjusted and adjusted analyses (Intercept, Estimate = 0.003, p < 0.0001; and slope, Estimate = 0.01, p = 0.0009, n = 386). CONCLUSIONS: Plasma sTM is associated with mortality, severity of hypoxic respiratory failure and worsening extrapulmonary MOF in children with ARF. This suggests a role of vascular injury in the pathogenesis of ARF and provides potential applicability towards targeted therapies. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT00814099 . In healthy lung endothelium, thrombomodulin (TM) recruits thrombin to activate Protein-C (PC/APC), that inhibits plasminogen activator-1 (PAI-1) and thrombosis. In inflamed and damaged endothelium, TM is cleaved into its soluble form (sTM), precluding its usual regulation of thrombosis. In this study, we measured plasma sTM levels in pediatric patients with respiratory failure and found that sTM correlated with mortality and other clinical markers of poor outcomes.

Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex.

Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-ß-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced µs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased µs-ms dynamics in a ß-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two ß-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Endothelial Markers: Thrombomodulin and Von Willebrand Factor and Risk of Kidney Thrombosis After Transplantation.

OBJECTIVE: Endothelial disturbance is well known as one of the causes of thrombosis. This study measured von Willebrand factor (vWF) and soluble thrombomodulin (sTM) in renal vein blood to evaluate for the risk of thrombosis after kidney transplantation. METHODS: A cross-sectional study that included 61 consecutive recipients of kidney transplant. The sTM and activity of vWF were evaluated in blood of the renal vein at the time of reperfusion. RESULTS: The renal vein blood had higher values of vWF activity and sTM concentration than the peripheral blood. In the first minutes of reperfusion, the concentration of thrombomodulin was the highest but activity of vWF was the lowest. As the reperfusion continued, thrombomodulin gradually decreased, but vWF increased. The strong correlations of TMs and vWF with warm ischemia were observed (r = 0.5577 and r = 0.3429, respectively). Thrombosis was found in about 10% of all recipients. However, other complications, such as delayed graft function or ureter necrosis, were associated with high values of vWF and sTM. They were correlated with increased sTM concentration and activity of vWF (P < .006 and P < .05, respectively). This was confirmed by analysis of the receiver operator characteristic curve. The area under the curve values for TMs and vWF were 0.762 and 0.602, respectively (P < .0001 and P < .015, respectively). The cutoff points for sTM and vWF were 14.89 ng/mL and 129.89%, respectively. Positive prediction value sTM and vWF were 76% and 66% and negative prediction value 69% and 59%, respectively. CONCLUSIONS: The endothelium of a transplanted kidney could be involved in the pathogenesis of renal thrombosis. Endothelial protection during harvesting can greatly contribute to the improvement of transplantation outcomes. The renal pool of sTM and vWF could be a useful marker of the risk of renal thrombosis.

Thrombin generation in patients with COVID-19 with and without thromboprophylaxis.

OBJECTIVES: Thrombin generation (TG) with and without thrombomodulin (TM) was evaluated in COVID-19 patients with different disease severity and thromboprophylaxis regimen, in order to understand the prothrombotic profile. METHODS: We enrolled consecutive patients with confirmed diagnosis of COVID-19 admitted to Medical Departments (MD) or Intensive Care Units (ICU), and 54 healthy controls. RESULTS: Eighty-nine patients were included (mean age 60.4±16.1 years, 68.5% male); 33.7% admitted to ICU. Twenty-four patients (26.9%) were enrolled before thromboprophylaxis administration; 45 patients (50.6%) received standard and 20 (22.5%) intermediate sub-therapeutic dose thromboprophylaxis. Overall, patients with COVID-19 showed a TG profile comparable to that of healthy subjects (i.e. comparable peak height, endogenous thrombin potential [ETP] with and without TM). The only exception was lag time and time to peak, prolonged in COVID-19 patients vs. controls. MD patients showed a similar TG profile to healthy controls, and ICU patients showed significantly decrease ETP (p=0.030) compared to MD. As for thromboprophylaxis, TG profile was significantly increased in COVID-19 patients without thromboprophylaxis vs. controls and vs. those with thromboprophylaxis. In this latter group, ETP inhibition was significantly decreased (p=0.0003) and positively correlated with anti-Xa activity (r=0.49, p=0.0017). However, patients with thromboprophylaxis had similar TG profile vs. controls. Intermediate dose thromboprophylaxis more effectively inhibited TG in severe COVID-19 patients by increasing ETP inhibition via ETP with TM reduction vs. standard dose. CONCLUSIONS: COVID-19 patients showed increased TG at diagnosis. Standard thromboprophylaxis reduced TG to levels of healthy controls. Intermediate sub-therapeutic thromboprophylaxis more effectively inhibited TG by decreasing ETP with TM.

Intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation.

BACKGROUND: Intravenous fluids, an essential component of sepsis resuscitation, may paradoxically worsen outcomes by exacerbating endothelial injury. Preclinical models suggest that fluid resuscitation degrades the endothelial glycocalyx, a heparan sulfate-enriched structure necessary for vascular homeostasis. We hypothesized that endothelial glycocalyx degradation is associated with the volume of intravenous fluids administered during early sepsis resuscitation. METHODS: We used mass spectrometry to measure plasma heparan sulfate (a highly sensitive and specific index of systemic endothelial glycocalyx degradation) after 6 h of intravenous fluids in 56 septic shock patients, at presentation and after 24 h of intravenous fluids in 100 sepsis patients, and in two groups of non-infected patients. We compared plasma heparan sulfate concentrations between sepsis and non-sepsis patients, as well as between sepsis survivors and sepsis non-survivors. We used multivariable linear regression to model the association between volume of intravenous fluids and changes in plasma heparan sulfate. RESULTS: Consistent with previous studies, median plasma heparan sulfate was elevated in septic shock patients (118 [IQR, 113-341] ng/ml 6 h after presentation) compared to non-infected controls (61 [45-79] ng/ml), as well as in a second cohort of sepsis patients (283 [155-584] ng/ml) at emergency department presentation) compared to controls (177 [144-262] ng/ml). In the larger sepsis cohort, heparan sulfate predicted in-hospital mortality. In both cohorts, multivariable linear regression adjusting for age and severity of illness demonstrated a significant association between volume of intravenous fluids administered during resuscitation and plasma heparan sulfate. In the second cohort, independent of disease severity and age, each 1 l of intravenous fluids administered was associated with a 200 ng/ml increase in circulating heparan sulfate (p = 0.006) at 24 h after enrollment. CONCLUSIONS: Glycocalyx degradation occurs in sepsis and septic shock and is associated with in-hospital mortality. The volume of intravenous fluids administered during sepsis resuscitation is independently associated with the degree of glycocalyx degradation. These findings suggest a potential mechanism by which intravenous fluid resuscitation strategies may induce iatrogenic endothelial injury.

C1-esterase inhibitor enhances thrombin generation and spatial fibrin clot propagation in the presence of thrombomodulin.

Package inserts for C1-esterase inhibitor (C1INH) products include warnings for an elevated risk of possible thrombosis in certain individuals, referring to thromboembolic events (TEEs) that were reported to occur after C1INH infusions. However, the mechanism(s) that could explain possible development of TEEs due to C1INH remains unknown. In this work, we evaluated plausible impact of C1INH on the protein C (PC) anticoagulant system. We performed thrombin generation (TG) assays (TGA) and analyzed spatial fibrin clot propagation using thrombodynamics in plasma of individual donors after the addition of thrombomodulin (TM) and C1INH. The addition of C1INH was consistent with the plasma concentrations resulting from doses currently approved for the HAE treatment up to ones consistent with off-label use in patients with risk of inflammation. 16 IU/ml of C1INH significantly enhanced thrombin peak (TP) generation in the presence of 12 and 15 nM TM. TG enhancement was observed by the addition of C1INH to make concentrations equal to 2 and 4 IU/ml in some donor plasmas. C1INH addition in the presence of TM enhanced the stop time of spatial clot growth in Thrombodynamics assay. A chromogenic activity assay demonstrated that C1INH inhibited PC activation by thrombin in the presence of TM. Substitution of TM with APC in TGA attenuated the TP enhancing effect of C1INH. The collective results of the present study suggest a concentration dependent C1INH interaction with the PC system. This study introduces a plausible TM-dependent mechanism, that may explain reported TEEs via suppressed production of APC in the presence of C1INH.

Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

BACKGROUND: Microvascular free flap reconstruction has become a standard technique in head and neck reconstructive surgery. Pre-operative radiotherapy is associated with a higher incidence of free flap malperfusion and the need for operative revision. Irradiated vessels present characteristic histomorphological and structural changes. Alterations in endothelial cells of irradiated arteries remain incompletely investigated especially with regard to long-term changes in endothelial dysfunction supporting an intraluminal pro-thrombotic and pro-inflammatory milieu. METHODS: Endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E­ and P­selectin, endothelial NO-synthase (eNOS), thrombomodulin and plasminogen activator inhibitor-1 (PAI-1) in irradiated and non-irradiated arteries was analysed using immunohistochemistry and Remmele scale grading. The average radiation dose was 58.7 ± 7.0 Gy; the time interval between end of radiation and tissue sampling was 106.0 ± 86.8 months. RESULTS: Endothelial expression of ICAM-1, VCAM-1, E­ and P­selectin as well as PAI-1 was significantly increased in previously irradiated arteries compared with non-irradiated controls, whereas thrombomodulin and eNOS expression did not show any differences. However, when comparing non-irradiated free flap arteries with irradiated arteries from the head and neck area in respective individuals, eNOS expression was significantly lower in irradiated vessels whereas ICAM-1, VCAM-1, E­/p-Selectin and PAI-1 showed significantly higher expression levels. CONCLUSION: There is ongoing endothelial dysfunction in terms of increased expression of pro-thrombotic and pro-inflammatory markers in irradiated arteries even years after radiotherapy. Treating this endothelial dysfunction might reduce the complication rates associated with microvascular free flap reconstructions in irradiated patients.

Biomarkers of disseminated intravascular coagulation in pediatric intensive care unit in Thailand.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a systemic activation of hemostatic system caused by several causes. Biomarkers including antithrombin (AT), protein C (PC), and thrombomodulin (TM) were reported as the additional markers for DIC in adults. This study aimed to determine the association between biomarkers among patients with overt DIC (ODIC) and nonovert DIC (NDIC) in children in PICU. METHODS: We enrolled 103 subjects, aged 1 month-18 years, who were admitted to PICU at Chiang Mai University (CMU) Hospital >24 hours with underlying conditions predisposing to DIC were enrolled. Biomarkers were tested after 24 hours of admission. Subject who had NDIC on the 1st investigations would have other tests on days 3-5 of admission. RESULTS: The incidence of ODIC by the International Society on Thrombosis and Hemostasis (ISTH) DIC score was found 24%. The bleeding, thrombosis, and death were significantly higher in ODIC group (P < 0.05). Mean levels of AT and PC in ODIC group were significantly different from NDIC one (66.9% vs 79.9%, P < 0.001 and 46.1% vs 59.2%, P = 0.004, respectively) while mean level of TM was not different between two groups. Adding AT to DIC score was better than the original score for predict mortality [area under curve (AUC) = 0.662 vs AUC = 0.65] and bleeding (AUC = 0.751 vs AUC = 0.732). CONCLUSIONS: ODIC is prevalent among critically ill children. Adverse outcomes were more commonly found in children with ODIC. AT and PC levels after 24 hours of PICU admission seem to be the useful biomarkers for ODIC in PICU.

The combination of Caprini risk assessment scale and thrombotic biomarkers to evaluate the risk of venous thromboembolism in critically ill patients.

To evaluate the correlation between the Caprini risk assessment scale and plasma thrombosis biomarkers and estimate the validity of this method in identifying critically ill patients at high risk of venous thromboembolism (VTE).Patients with VTE who were admitted to the intensive care unit (ICU) department of West China Hospital SiChuan University from October 2016 to October 2017 were enrolled in this case-control study. We retrieved relative clinical data and laboratory test results included in the Caprini risk assessment scale to calculate the Caprini score and compared thrombosis biomarkers between various risk stratifications (low, moderate, high, and highest).A total of 151 critically ill patients were enrolled in our research, including 47 VTE and 94 non-VTE patients. The differences in Caprini score and levels of thrombosis biomarkers between the VTE and control group were significant. Thrombomodulin (TM) was positively correlated with Caprini score (R-value was .451, P < .05). Based on the receiver operating characteristic analysis, TM, tissue plasminogen activator-inhibitor complexes, D-dimer, and fibrinogen degradation products had a certain diagnostic efficiency in distinguishing VTE from others (P < .05). Using the logistic regression model, we identified that 5 risk factors, namely drinking history, major surgery (>3 hours), swollen legs (current), TM, and D-dimer, were independent factors for the occurrence of VTE in critically ill patients admitted in the ICU.Thrombosis markers were positively correlated with Caprini risk stratification. The combination of plasma markers and Caprini risk assessment scale can further increase the predictive value in critically ill patients with VTE.

Thrombomodulin (TM) in tumor cell differentiation and periphery blood immune microenvironment in oral squamous cell carcinoma.

Thrombomodulin (TM, also known as CD141), which functions as an anticoagulant, is widely expressed on cell surface of a variety of cell types, including human blood cells as well as certain immune cells. To determine whether TM could be a potential marker for OSCC diagnosis as well as a molecular target for OSCC therapy, we examined the expression of TM in an oral cancer tissue microarray with 153 oral cancer tissues. Further, we also analyzed the expression of TM on DCs of 36 OSCC patients and 36 healthy donors. The expression of TM was determined using standard immunohistochemistry on a tissue microarray of 153 OSCC patients. Flow cytometric analyses were performed to determine the proportions of CD141+ DCs in the PBMC of 36 OSCC patients and 36 healthy donors. Clinicopathological correlations were performed based on the available clinical data. Our results showed that in the univariate analysis, high TM expression was significantly associated with well differentiation of tumor cells (P=.001), but not correlated with overall survival and disease-free survival (P>.05). In addition, CD141+ DCs were both present in OSCC patients and healthy donors with about 0.04%. There was no significant difference with the percentages of CD141+ DCs in the PBMC of OSCC patients and that of the normal control group (P>.05). This study indicates that TM expression might play the most critical role in the differentiation of OSCC tumors. Functional distinctions of CD141+ DCs in OSCC patients deserve further investigation to provide important therapeutic understandings for future immunotherapy.

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells.

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5­5%) and nicotine (10­3­10­9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription­quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6­h exposure, TM protein and mRNA expression levels decreased in a dose­dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking­associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences.

Markers of acute kidney injury in patients with sepsis: the role of soluble thrombomodulin.

BACKGROUND: Endothelial activation and damage occur early during sepsis, with activated coagulopathy and playing a major role in the pathophysiology of sepsis-induced acute kidney injury (AKI). The aim of this study was to compare the various biomarkers of endothelial injury with the biomarkers of coagulation and inflammation and to determine a significant predictor of AKI in patients with sepsis. METHODS: We conducted a single-center, retrospective, observational study on patients with sepsis fulfilling the Third International Consensus Definitions for Sepsis and Septic Shock criteria admitted to an adult intensive care unit (ICU) at a university hospital from June 2011 to December 2016. Levels of 13 biomarkers were measured on ICU admission, including markers of endothelial injury (soluble thrombomodulin [sTM], E-selectin, protein C, and plasminogen activator inhibitor-1 [PAI-1]) and markers of coagulation derangement (platelet count, fibrin degradation product [FDP], prothrombin time [PT], fibrinogen, α2-plasminogen inhibitor [α2-PI], antithrombin III [AT III], plasminogen, thrombin-antithrombin complex, and plasmin-α2-plasmin inhibitor complex). All patients with sepsis were reviewed, and the development of AKI was evaluated. Multivariate logistic regression analysis was performed to identify significant independent predictive factors for AKI. RESULTS: Of the 514 patients admitted with sepsis, 351 (68.3%) developed AKI. Compared with the non-AKI group, all the endothelial biomarkers were significantly different in the AKI group (sTM [23.6 vs. 15.6 U/ml, P < 0.0001], E-selectin [65.5 vs. 46.2 ng/ml, P = 0.0497], PAI-1 [180.4 vs. 75.3 ng/ml, P = 0.018], and protein C [45.9 vs. 58.7 ng/ml, P < 0.0001]). Biomarkers of coagulopathy and inflammation, platelet counts, FDP, PT, α2-PI, AT III, plasminogen, and C-reactive protein were significantly different between the two groups. Multivariable logistic regression analysis showed that sTM was an independent predictive factor of AKI, with an AUROC of 0.758 (P < 0.0001). CONCLUSIONS: Endothelial biomarkers were significantly changed in the sepsis patients with AKI. Particularly, sTM was an independent predictive biomarker for the development of AKI that outperformed other coagulation and inflammation biomarkers as well as organ function in patients with sepsis.

Highly sensitive turn-on fluorescence detection of thrombomodulin based on fluorescence resonance energy transfer.

As an integral glycoprotein on the surface of endothelial cells, thrombomodulin (TM) has very high affinity for thrombin. TM has been regarded to be a marker of endothelial damage since it can be released during endothelial cell injury. In this work, a highly sensitive fluorescence method for the quantitative detection of TM was developed. TM antibody (Ab) and bovine serum albumin (BSA) were bound on gold nanoparticles (AuNPs) to construct BSA-AuNPs-Ab nanocomposites and they were characterized by transmission electron microscope and UV-vis spectrophotometry. The fluorescence of acridine orange (AO) was quenched by the prepared gold nanocomposites based on fluorescence resonance energy transfer (FRET). In the presence of TM, the fluorescence was turned on due to the effective separation of AO from the surface of gold nanocomposites. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the logarithm of the TM concentration from 0.1pgmL-1 to 5ngmL-1 with a low detection limit of 12fgmL-1. The release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells in the presence of H2O2 was investigated using the proposed method. The released sTM content in the growth medium was found to be increased with the enhancement of contact time of the cells with H2O2.

The involvement of endothelial mediators in leprosy.

Leprosy is a chronic infectious disease that requires better understanding since it continues to be a significant health problem in many parts of the world. Leprosy reactions are acute inflammatory episodes regarded as the central etiology of nerve damage in the disease. The activation of endothelium is a relevant phenomenon to be investigated in leprosy reactions. The present study evaluated the expression of endothelial factors in skin lesions and serum samples of leprosy patients. Immunohistochemical analysis of skin samples and serum measurements of VCAM-1, VEGF, tissue factor and thrombomodulin were performed in 77 leprosy patients and 12 controls. We observed significant increase of VCAM-1 circulating levels in non-reactional leprosy (p = 0.0009). The immunostaining of VEGF and tissue factor was higher in endothelium of non-reactional leprosy (p = 0.02 for both) than healthy controls. Patients with type 1 reaction presented increased thrombomodulin serum levels, compared with non-reactional leprosy (p = 0.02). In type 2 reaction, no significant modifications were observed for the endothelial factors investigated. The anti-inflammatory and antimicrobial activities of the endotfhelial factors may play key-roles in the pathogenesis of leprosy and should be enrolled in studies focusing on alternative targets to improve the management of leprosy and its reactions.

The involvement of endothelial mediators in leprosy

Leprosy is a chronic infectious disease that requires better understanding since it continues to be a significant health problem in many parts of the world. Leprosy reactions are acute inflammatory episodes regarded as the central etiology of nerve damage in the disease. The activation of endothelium is a relevant phenomenon to be investigated in leprosy reactions. The present study evaluated the expression of endothelial factors in skin lesions and serum samples of leprosy patients. Immunohistochemical analysis of skin samples and serum measurements of VCAM-1, VEGF, tissue factor and thrombomodulin were performed in 77 leprosy patients and 12 controls. We observed significant increase of VCAM-1 circulating levels in non-reactional leprosy (p = 0.0009). The immunostaining of VEGF and tissue factor was higher in endothelium of non-reactional leprosy (p = 0.02 for both) than healthy controls. Patients with type 1 reaction presented increased thrombomodulin serum levels, compared with non-reactional leprosy (p = 0.02). In type 2 reaction, no significant modifications were observed for the endothelial factors investigated. The anti-inflammatory and antimicrobial activities of the endotfhelial factors may play key-roles in the pathogenesis of leprosy and should be enrolled in studies focusing on alternative targets to improve the management of leprosy and its reactions.

Endothelial activation/injury and associations with severity of post-cardiac arrest syndrome and mortality after out-of-hospital cardiac arrest.

BACKGROUND: Post-cardiac arrest syndrome (PCAS) is characterized by whole-body ischemia triggering systemic inflammation and damage of the endothelium. This study investigated the relationship between systemic inflammation, endothelial damage and severity of PCAS and the association between endothelial damage and outcome after out-of-hospital cardiac arrest (OHCA). METHODS: In this post hoc study, we analyzed 163 comatose patients included at a single center in the target temperature management (TTM) trial, randomly assigned to TTM at 33°C or 36°C for 24h. Endothelial biomarkers (syndecan-1, thrombomodulin, sE-selectin, sVE-cadherin) and the inflammatory biomarker interleukin-6 (IL-6) were measured at admission (baseline) and 24, 48 and 72h after OHCA. Severity of PCAS was assessed by Sequential Organ Failure Assessment score. Mortality at 30-days was evaluated by Cox regression analysis. RESULTS: By linear regression, baseline IL-6 levels (two-fold) was independently associated with glycocalyx damage (syndecan-1 (10.3ng/ml (p=0.01))), endothelial activation (sE-selectin (2.0ng/ml (p=0.03))) and endothelial damage (thrombomodulin 0.7ng/ml (p=0.0005)) at 24h after OHCA. Adjusted for baseline IL-6, a two-fold increase in thrombomodulin from baseline to 48h (1.7 (0.9-2.4), p<0.0001) and 72h (1.5 (0.6-2.3), p<0.0007) was more closely associated with severity of PCAS than IL-6. Levels of syndecan-1, thrombomodulin and sVE-cadherin was not influenced by level of target temperature but levels of sE-selectin was significantly lower in the 36°C group (-55ng/ml (95%CI: -53 to -58ng/ml), p=0.005) compared to the 33°C group. High levels of thrombomodulin at 24h (HR=2.1 (1.3-3.3), p=0.001) and 48h (HR=1.75 (1.0-2.8), p=0.02) were associated with increased 30-day mortality in univariate analysis, but not in multivariable analyses. CONCLUSION: In comatose survivors after OHCA treated with TTM, systemic inflammation was associated with endothelial activation and endothelial damage. Sustained endothelial damage was independently associated with severity of PCAS, adjusted for level of systemic inflammation. TTM at 36°C compared to 33°C after OHCA was associated with lower endothelial activation, but not endothelial damage. CLINICAL TRIAL REGISTRATION: URL: clinicaltrials.gov/ct2/show/NCT01020916. Unique identifier: NCT01020916.

The involvement of endotelial mediators in leprosy

Leprosy is a chronic infectious disease that requires better understanding since it continues to be a significant health problem in many parts of the world. Leprosy reactions are acute inflammatory episodes regarded as the central etiology of nerve damage in the disease. The activation of endothelium is a relevant phenomenon to be investigated in leprosy reactions. The present study evaluated the expression of endothelial factors in skin lesions and serum samples of leprosy patients. Immunohistochemical analysis of skin samples and serum measurements of VCAM-1, VEGF, tissue factor and thrombomodulin were performed in 77 leprosy patients and 12 controls. We observed significant increase of VCAM-1 circulating levels in non-reactional leprosy (p = 0.0009). The immunostaining of VEGF and tissue factor was higher in endothelium of non-reactional leprosy (p = 0.02 for both) than healthy controls. Patients with type 1 reaction presented increased thrombomodulin serum levels, compared with non-reactional leprosy (p = 0.02). In type 2 reaction, no significant modifications were observed for the endothelial factors investigated. The anti-inflammatory and antimicrobial activities of the endotfhelial factors may play key-roles in the pathogenesis of leprosy and should be enrolled in studies focusing on alternative targets to improve the management of leprosy and its reactions.

Elevated soluble thrombomodulin is associated with organ failure and mortality in children with acute respiratory distress syndrome (ARDS): a prospective observational cohort study.

INTRODUCTION: The significance of endothelial injury in children with the acute respiratory distress syndrome (ARDS) has not been well studied. Plasma levels of soluble thrombomodulin (sTM), an endothelial surface protein involved in coagulation, have been associated with endothelial injury. We hypothesized that elevated plasma sTM would correlate with mortality and organ failure in children with ARDS. METHODS: We conducted a multicenter prospective observational study of pediatric patients with ARDS between 2008 and 2014. sTM was measured in plasma collected less than 24 hours from ARDS diagnosis. Outcomes were intensive care unit mortality and organ dysfunction by pediatric logistic organ dysfunction scores. Logistic regression was used to adjust for clinically relevant covariates. RESULTS: Plasma sTM was higher in patients with indirect lung injury compared to direct lung injury (100 ng/mL vs. 86 ng/mL, p = 0.02). Increased sTM levels were correlated with more organ dysfunction in the entire study population (Spearman's rho = 0.37, p < 0.01). Overall mortality was 16%. sTM levels were associated with increased mortality in patients with indirect lung injury (OR 2.7 per log(sTM), p = 0.02). These relationships were independent of age, oxygenation defect, or presence of acute kidney injury. CONCLUSION: Elevated plasma sTM levels are associated with organ dysfunction in children with ARDS and with higher mortality in children with indirect lung injury. These findings highlight the importance of endothelial injury in children with ARDS and may guide the development of future therapies targeted toward endothelial stabilization, repair, or functional replacement in this population.

Label-Free and Sensitive Detection of Thrombomodulin, a Marker of Endothelial Cell Injury, Using Quartz Crystal Microbalance.

Thrombomodulin (TM), an integral glycoprotein on the surface of endothelial cells, can be released during endothelial cell injury and the levels of serum TM are regarded as an important parameter of activity in vasculitides in vivo. Quantitative detection of TM and investigation on the release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells using quartz crystal microbalance (QCM) were achieved in this work. Anti-antibody (AAb) and bovine serum albumin (BSA) were bound on gold nanoparticles (GNPs) to construct BSA-GNPs-AAb nanocomposites and they were characterized by transmission electron microscope, UV-vis, and infrared spectrophotometry, respectively. The capture of the nanocomposites on the TM antibody modified electrode, which was tested by scanning electron microscope, could result in a great decrease of the resonant frequency (f0). This binding was effectively inhibited by the beforehand immobilized TM proteins on the electrode surface due to the strong steric hindrance effect. It led to the decrease of the frequency changing extent. The relative frequency-shift was found to be proportional to the logarithm of the TM concentration from 10 to 5000 ng mL(-1) with a detection limit of 2 ng mL(-1). By analyzing the growth medium used for cell incubation, the release of sTM by the injured HUVEC-C cells in the presence of H2O2 was confirmed. The sTM amount in the growth medium was increased with the enhancement of contact time of the cells with H2O2, proving that sTM may serve as a specific marker of endothelial cell injury.