Soluble Thrombomodulin Levels Research Articles

Iloprost and Organ Dysfunction in Adults With Septic Shock and Endotheliopathy

Soluble thrombomodulin is a marker of endotheliopathy, and iloprost may improve endothelial function. In patients with septic shock, high plasma levels of soluble thrombomodulin (>10 ng/mL) have been associated with worse organ dysfunction and mortality. To assess the effects of treatment with iloprost vs placebo on the severity of organ failure in patients with septic shock and plasma levels of soluble thrombomodulin higher than 10 ng/mL. This investigator-initiated, adaptive, parallel group, stratified, double-blind randomized clinical trial was conducted between November 1, 2019, and July 5, 2022, at 6 hospitals in Denmark. The trial had a maximum sample size of 380, with an interim analysis for futility only at 200 patients with 90 days of follow-up. In total, 279 adults in the intensive care unit (ICU) with septic shock and endotheliopathy were included. Patients were randomized 1:1 to masked intravenous infusion of iloprost, 1 ng/kg/min (n = 142), or placebo (n = 137) for 72 hours. The primary outcome was mean daily Sequential Organ Failure Assessment (SOFA) score in the ICU adjusted for trial site and baseline SOFA score for the per-protocol population. SOFA scores for each of the 5 organ systems ranged from 0 to 4, with higher scores indicating more severe dysfunction (maximum score, 20). The secondary outcomes included serious adverse reactions and serious adverse events at 7 days and mortality at 90 days. Of 279 randomized patients, data from 278 were analyzed (median [IQR] age, 69 [58-77] years; 171 (62%) male), 142 in the iloprost group and 136 in the placebo group. The trial was stopped for futility at the planned interim analysis. The mean [IQR] daily SOFA score was 10.6 (6.4-14.8) in the iloprost group and 10.5 (5.9-15.5) in the placebo group (adjusted mean difference, 0.2 [95% CI, -0.8 to 1.2]; P = .70). Mortality at 90 days in the iloprost group was 57% (81 of 142) vs 51% (70 of 136) in the placebo group (adjusted relative risk, 1.12 [95% CI, 0.91-1.40]; P = .33). Serious adverse events occurred in 26 of 142 patients (18%) for the iloprost group vs 20 of 136 patients (15%) for the placebo group (adjusted relative risk, 1.25 [95% CI, 0.73-2.15]; P = .52). Only 1 serious adverse reaction was observed. In this randomized clinical trial of adults in the ICU with septic shock and severe endotheliopathy, infusion of iloprost, 1 ng/kg/min, for 72 hours did not reduce mean daily SOFA scores compared with placebo. In a clinical context, administration of iloprost will be unlikely to improve outcome in these patients. ClinicalTrials.gov Identifier: NCT04123444.

COAGULATION AND ANTICOAGULATION PARAMETERS IN MULTIPLE SCLEROSIS PATIENTS WITH AND WITHOUT COVID-19

The aim. To investigate plasma levels of main coagulation and fibrinolytic factors in MS patients with and without COVID-19 history. Materials and methods. A total of 127 participants were enrolled in this study, including 97 MS patients and 30 healthy controls (HC). Patients with MS were divided into two groups: MS+Covid group (n=41) – patients with MS, who had a laboratory-verified diagnosis of COVID-19 in the past 3-6-month period and MS group (n=56) – patients with MS, who did not suffer from COVID-19 previously. Determination of plasma levels of prothrombin, plasminogen, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), protein C (PC), soluble thrombomodulin (TM) was performed by means of enzyme-linked immunosorbent assay. Spectrophotometric techniques were used to determine concentrations of fibrinogen, soluble fibrin monomeric complexes (SFMC) as well as plasminogen activity and inhibitory potential of α-2-antiplasmin. Results. The MS group was characterized by elevated levels of plasma prothrombin, fibrinogen, D-dimer, SFMC, soluble TM compared to HC, while PC concentration did not differ between MS and HC groups. Plasma plasminogen level as well as plasma level of the potential plasmin activity were significantly decreased in MS patients compared to HC group. The plasma tPA level was significantly reduced while plasma PAI-I level was significantly increased in MS patients compared to HC. Patients of MS group had an increased level of plasma α-2-antiplasmin activity compared with HC group. To note, most of studied parameters did not differ between two MS groups, except protein C, soluble thrombomodulin levels and plasma α-2-antiplasmin activity. Conclusions. The results of our study showed that MS patients have got altered hemostasis parameters; however, further study is necessary to find out the relationship between particular components of coagulation and fibrinolytic systems and pathophysiology of MS. Additionally, our findings demonstrated that a SARS-CoV-2 infection had a limited effect on hemostasis parameters in MS patients, causing changes in only a few parameters, including thrombomodulin and protein C levels as well as α-2-antiplasmin activity.

Laboratory indicators of hemostasis, lipid metabolism and endothelial dysfunction in men aged 18–50 years with different subtypes of ischemic stroke

Objective: to evaluate laboratory parameters of hemostasis, lipid metabolism and endothelial dysfunction and their relationship in men aged 18–50 years with atherothrombotic (ATS), lacunar (LS) and cardioembolic (CES) stroke. Material and methods. The study included 89 men with ATS (n=36), LS (n=34) and CES (n=19). Neuroimaging, ultrasound and laboratory blood serum analyses were performed in all patients. Results. The mean age of the patients was 42.6±5.3 years. The main risk factors for ATS, LS and CES included: arterial hypertension (75; 97.8 and 73.7% of cases, respectively), dyslipidemia (60; 41.3 and 42.1%), tobacco smoking (71.7; 67.4 and 52.6%), regular alcohol consumption (35; 19.6 and 36.8%), obesity (23.3; 8.7 and 15.8 %), diabetes mellitus (8.3; 6.5 and 10.5 %). Lower tissue plasminogen activator levels were found in patients with CES (2.66±1.77 ng/ml) compared to patients with LS (3.38±3.0 ng/ml) and ATS (3.48±2.45 ng/ml). Plasminogen activator inhibitor-1 levels were significantly increased in all stroke subtypes. The mean level of soluble thrombomodulin was highest in patients with LS (100.86±58.22 pg/ml) compared to patients with ATS (96.37±85.71 pg/ml) and CES (75.28±39.36 pg/ml). The level of asymmetric dimethylarginine was higher in patients with ATS (1.46±0.42 μmol/l) and in patients with LS (0.79±0.37 μmol/l), and in patients with CES (0.4±0.13 μmol/l) it was within the reference values. Conclusion. We noted differences in laboratory parameters of the hemostasis, lipid metabolism and endothelial dysfunction in men aged 18–50 years with different stroke subtypes (ATS, LS and CES), as well as clinical and laboratory correlations.

Assessment of soluble thrombomodulin and soluble endoglin as endothelial dysfunction biomarkers in seriously ill surgical septic patients: correlation with organ dysfunction and disease severity.

Sepsis, a complex condition characterized by dysregulated immune response and organ dysfunction, is a leading cause of mortality in ICU patients. Current diagnostic and prognostic approaches primarily rely on non-specific biomarkers and illness severity scores, despite early endothelial activation being a key feature of sepsis. This study aimed to evaluate the levels of soluble thrombomodulin and soluble endoglin in seriously ill surgical septic patients and explore their association with organ dysfunction and disease severity. A case control study was conducted from March 2022 to November 2022, involving seriously ill septic surgical patients. Baseline clinical and laboratory data were collected within 24h of admission to the Surgical Intensive Care Unit. This included information such as age, sex, hemodynamic parameters, blood chemistry, SOFA score, qSOFA score, and APACHE-II score. A proforma was filled out to record these details. The outcome of each patient was noted at the time of discharge. The study found significantly elevated levels of soluble thrombomodulin and soluble endoglin in seriously ill surgical septic patients. The RTqPCR analysis revealed a positive correlation between soluble thrombomodulin and soluble endoglin levels with the qSOFA score, as well as, there was a positive association between RTqPCR soluble thrombomodulin and the SOFA score. These findings indicate a correlation between these biomarkers and organ dysfunction and disease severity. The study concludes that elevated levels of soluble thrombomodulin and soluble endoglin can serve as endothelial biomarkers for early diagnosis and prognostication in seriously ill surgical septic patients.

Endothelial dysfunction, thrombophilia, and nailfold capillaroscopic features in livedoid vasculopathy

BackgroundLivedoid vasculopathy (LV) is a rare, disabling disease characterized by painful ulcers, livedo reticularis and atrophy blanche. Hypercoagulation, endothelial, and microcirculatory dysfunction are believed to be responsible for the pathogenesis of this difficult-to-treat disease. ObjectivesThis study sought to investigate the frequency of endothelial dysfunction, hypercoagulability, and nailfold capillaroscopic features in LV patients to shed light on its etiology. MethodsThis case-control study included 16 patients with LV, 24 with systemic sclerosis (SSc), and 23 control subjects. Serum markers of endothelial dysfunction soluble endoglin, endocan, endothelin-1, lipoprotein a, plasminogen activator inhibitor-1 (PAI-1), soluble thrombomodulin, and von Willebrand factor were measured using enzyme-linked immunosorbent assays. Flow-mediated dilation and carotid intima-media thickness were examined as markers of endothelial dysfunction, and microcirculation was assessed with nailfold capillaroscopy. Thrombophilia-related parameters, including gene polymorphisms of factor V Leiden, prothrombin, PAI-1 genes, methylenetetrahydrofolate reductase (MTHFR) and factor XIII mutation and serum levels of protein C, protein S, antithrombin, homocysteine, D-dimer and antiphospholipid antibodies were investigated in LV patients. ResultsPlasminogen activator inhibitor-1 and soluble thrombomodulin levels were significantly higher in LV patients compared to control subjects (2.3 [2.05–2.79] ng/ml vs. 1.89 [1.43–2.33] ng/ml, p = 0.007; 1.15 [0.88–1.4] ng/ml vs. 0.76 [0.56–0.9] ng/ml, p = 0.004, respectively). Flow-mediated dilation was 25.4 % lower in the LV patients compared to the control group (14.77 % [11.26–18.26] vs. 19.80 % [16.47–24.88], p = 0.034). Capillaroscopic features, including ramifications (75 % vs. 8.7 %, p < 0.001), avascular areas (25 % vs. 0 %, p = 0.011) and dilatations (33.2 % vs. 0 %, p = 0.016), were significantly higher in LV patients than in controls. LV patients had multiple biochemical or genetic abnormalities related to thrombophilia, including heterozygous factor V Leiden mutations (6.3 %), MTHFR (C677T) mutations (heterozygous 43.8 %, homozygous 18.8 %), MTHFR (A1298C) mutations (heterozygous 37.5 %, homozygous 12.5 %), factor XIII heterozygous mutation (12.5 %), antithrombin deficiency (31.3 %), protein S deficiency (12.5 %), hyperhomocysteinemia (31.3 %), D-dimer elevation (25 %), anti-β2-glycoprotein I (12.5 %), lupus anticoagulant antibodies (6.3 %), and anticardiolipin antibodies (6.3 %). ConclusionsIn conclusion, LV patients were characterized by an increased presence of thrombophilia-related parameters, and also exhibited vascular endothelial and microcirculatory dysfunction, resembling SSc. These findings support the complex interaction of thrombophilia, endothelial dysfunction, and microcirculation dysregulation in the pathogenesis of LV. Thus, the treatment of LV patients should be individualized, based on the identification of the predominant pathological pathways.

Initial Tumor Necrosis Factor-Alpha and Endothelial Activation Are Associated with Hemorrhagic Complications during Extracorporeal Membrane Oxygenation.

Studies on inflammatory markers, endothelial activation, and bleeding during extracorporeal membrane oxygenation (ECMO) are lacking. Blood samples were prospectively collected after ECMO initiation from 150 adult patients who underwent ECMO for respiratory failure between 2018 and 2021. After excluding patients who died early (within 48 h), 132 patients were finally included. Their tumor necrosis factor-alpha (TNF-α), tissue factor (TF), soluble thrombomodulin (sTM), and E-selectin levels were measured. A Cox proportional hazards regression model was used to estimate the hazard ratio for hemorrhagic complications during ECMO. The 132 patients were divided into hemorrhagic (n = 23, H group) and non-complication (n = 109, N group) groups. The sequential organ failure assessment score, hemoglobin level, and ECMO type were included as covariates in all Cox models to exclude the effects of clinical factors. After adjusting for these factors, initial TNF-α, TF, sTM, E-selectin, and activated protein C levels were significantly associated with hemorrhagic complications (all p < 0.001). TNF-α, TF, and E-selectin better predicted hemorrhagic complications than the model that included only the aforementioned clinical factors (clinical factors only (area under the curve [AUC]: 0.804), reference; TNF-α (AUC: 0.914); TF (AUC: 0.915); E-selectin (AUC: 0.869)). Conclusions: TNF-α levels were significantly predictive of hemorrhagic complications during ECMO.

Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19

BackgroundSevere COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. ObjectivesWe investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis. MethodsThirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor [VWF], angiopoietin-2 [Angpt-2], and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. ResultsMarkedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis. ConclusionWe propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.

Elevated endothelial dysfunction-related biomarker levels indicate the severity and predict sepsis incidence

This study was conducted to investigate the relationship between serum endothelial dysfunction-related biomarker levels and organ dysfunction severity in septic patients and the predictive value of these levels during sepsis. In total, 105 patients admitted to the Department of Critical Care Medicine were enrolled between September 2020 and November 2021. Serum syndecan-1 and soluble thrombomodulin(sTM) levels were measured by enzyme-linked immunosorbent assay, and clinical and laboratory data were recorded. Enroll patients were divided into the infection (n = 28), septic nonshock (n = 31), and septic shock (n = 46) groups . Serum syndecan-1 (102.84 ± 16.53 vs. 55.38 ± 12.34 ng/ml), and sTM(6.60 ± 1.44 ng/ml vs. 5.23 ± 1.23 ng/ml, P < 0.01) levels were increased in the septic group compared with those in the infection group. Serum syndecan-1 levels were closely positively correlated with serum sTM (rs = 0.712, r2 = 0.507, P < 0.001). Additionally, serum syndecan-1(rs = 0.687, r2 = 0.472, P < 0.001) and sTM levels (rs = 0.6, r2 = 0.36, P < 0.01) levels were significantly positively correlated with the sequential organ failure assessment scores respectively. Syndecan-1 (AUC 0.95 ± 0.02, P < 0.0001) was more valuable for prediction sepsis than was sTM (AUC 0.87 ± 0.04, P < 0.0001). Compared with sTM (AUC 0.88 ± 0.03, P < 0.001), syndecan-1 (AUC 0.95 ± 0.02, P < 0.001) and SOFA score (AUC 0.95 ± 0.02, P < 0.001) were better predictors of septic shock. Serum syndecan-1 and sTM levels were associated with organ dysfunction severity in septic patients, and both were good predictors for early identification of sepsis, particularly in patients undergoing septic shock.

Complement Activation Is Associated with Endothelial Damage in Hematopoietic Stem Cell Transplant Associated-Thrombotic Microangiopathy

Introduction: Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) is a life-threatening complication of HSCT. Identification of at-risk patients is challenging, leading to delays in diagnosis and poor outcomes. Degradation of the glycocalyx and dysregulated complement activation are integral to the pathophysiology. The glycocalyx components thrombomodulin (TM) and syndecan-1 (SYND1) are shed during cell damage, and detection in patient plasma may serve as biomarkers of endothelial cell (EC) dysfunction. Therapeutic intervention to preserve glycocalyx integrity may protect the endothelium and also improve the clinical outcome during HSCT-TMA. Additionally, plasma Ba, an alternative complement pathway (AP) activation product, has been identified as a prognostic biomarker of HSCT-TMA. Our objective was to explore the link between excessive AP activation and endothelial damage in the context of HSCT-TMA and to potentially identify biomarkers to aid in its diagnosis. Methods: Biomarker levels of soluble TM, SYND1, and Ba were measured in plasma of pediatric HSCT patients with and without (controls) TMA obtained from Texas Children's Hospital. Sample acquisition was approved by Baylor College of Medicine Institutional Review Board. Biomarkers were also measured in a pre-clinical in vivo model of inflammation-mediated complement activation similar to HSCT-TMA, in which C57BL/6J mice were injected with lipopolysaccharide (LPS) to induce complement activation, followed by treatment with AP (anti-fB IgG) and terminal complement pathway (anti-C5 IgG) inhibitors or an IgG isotype control. Finally, as the immunosuppressant cyclosporin A (CsA) is a known trigger of HSCT-TMA, the role of complement in CsA-mediated damage in human ECs (HMEC-1 cells and HUVECs) was assessed in vitro. ECs were incubated with CsA followed by normal human serum (NHS) in the presence or absence of the C5 complement inhibitor eculizumab. TM was then measured by fluorescent staining on the cell surface and quantified using mean fluorescence intensity (MFI). Results: HSCT-TMA patients (n=11), compared to HSCT control patients (n=7), had significantly elevated levels of biomarkers of glycocalyx damage (TM: 18.3 ± 10.9 ng/mL and 6.6 ± 2.9 ng/mL, respectively; P<0.01; SYND1: 175.3 ± 177.0 ng/mL and 35.8 ± 18.9, respectively; P<0.05). A strong positive correlation between TM and SYND1 was observed (Pearson r=0.89). Plasma Ba was also significantly elevated in patients with TMA compared to those without (1790.0 ± 1262.0 ng/mL and 652.1 ± 233.5 ng/mL, respectively; P<0.05), and correlated positively with both TM and SYND1 levels (Pearson r=0.72 and 0.50, respectively). We have previously shown in an in vivo model of inflammation-mediated complement activation that LPS-injected mice, compared to saline-injected control mice, had significantly increased plasma Ba levels. Here we demonstrated LPS-injected mice (which also received IgG isotype control) had higher circulating levels of TM than untreated mice (35.1 ± 5.8 ng/mL [n=6] and 10.9 ± 1.6 ng/mL [n=6], respectively; P<0.0001). Inhibition of alternative (anti-fB IgG) and terminal (anti-C5 IgG) pathways of complement significantly attenuated TM levels (compared to isotype control [35.1 ± 5.8 ng/mL]; anti-FB: 25.2 ± 5.3 ng/mL, P<0.01; anti-C5: 26.2 ± 4.9 ng/mL, P<0.05, Fig. 1). TM was positively correlated with Ba (Pearson r=0.68). In vitro treatment of HMEC-1 cells with CsA led to deposition of iC3b and C5b-9, and C5 inhibition with eculizumab significantly reduced C5b-9 deposition (P< 0.0001) but had no effect on iC3b deposition. Treatment of HUVECs with CsA also reduced surface expression of TM (P<0.0001), and TM loss was partially restored by eculizumab (P<0.05, Fig 2). Conclusion: Plasma biomarkers of endothelial damage were elevated in patients with HSCT-TMA and in mice with inflammatory-mediated complement activation. Furthermore, AP activation positively correlated with biomarkers of endothelial damage. Blockade of complement activation significantly reduced markers of endothelial damage in both in vivo and in vitro models. These results provide further evidence linking complement activation and endothelial damage in HSCT-TMA and suggest that the use of these biomarkers may aid in the diagnosis of HSCT-TMA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasma thrombomodulin levels are associated with acute kidney injury in patients with acute heart failure

Cardiorenal syndrome type I (CRS I) is defined as the development of acute kidney injury (AKI) following acute decompensated heart failure (ADHF). The clinical significance of endothelial markers in ADHF-associated AKI has yet to be clarified. This study therefore investigated the biological processes linking ADHF and AKI with the aim of determining whether the plasma markers of endothelial injury and activation are associated with AKI in patients with ADHF. The study prospectively recruited 125 consecutive patients admitted to a coronary critical unit due to ADHF. Patients with and without AKI were compared in terms of soluble thrombomodulin (sTM), angiopoietin (Ang)-1 and −2 plasma levels as well as baseline characteristics. Among the study population, 14 (11.2%) patients developed CRS within 7 days after admission. The hemoglobin levels (median [IQR]11.3[10.8–12.6] vs. 13.5 [12.2–15.0] g/dL, p = 0.003) and baseline eGFR (66.5[35.7–87.9] vs. 78.5 [64.9–107.5] mL/minute/1.73m2, p = 0.044) of patients with CRS were lower than those of patients without CRS. Patients with CRS also presented elevated plasma levels of BNP (1317.5 [222.6–3375.5] vs. 258.2 [63.2–925.8] pg/mL, p = 0.008), Ang-2 (3993.0 [1561.3–15722.7] vs. 1805.9 [1196.9–3302.3] pg/mL, p = 0.006), and sTM (6665.7 [4707.1–11947.3] vs. 4132.2 [3338.0–5531.8] ng/mL, p < 0.001), compared to patients without CRS. Multivariate logistic regression analysis based on forward stepwise method identified that log sTM was the only independent risk factor for AKI (OR, 13.83; 3.02–63.28, p = 0.001). Furthermore, higher sTM levels were associated with AKI in patients with ADHF. These findings suggest a novel approach to dealing with kidney injury in the context of ADHF, involving the use of baseline biomarker profiles to identify individuals at risk of developing AKI. KEY MESSAGES The clinical significance of endothelial markers in acute decompensated heart failure (ADHF)-associated acute kidney injury (AKI) has not previously been clarified. This study revealed that markers of endothelial injury (i.e. plasma soluble thrombomodulin (sTM) levels) were higher in ADHF patients with AKI than in those without AKI. Multivariate analysis identified sTM level > cutoff value of 4,855.2 pg/mL as an independent factor associated with the development of AKI. sTM could potentially be used as a biomarker to predict the development of AKI in patients with heart failure. These findings suggest a novel approach to dealing with kidney injury in the context of ADHF, involving the use of baseline biomarker profiles to identify individuals at risk of developing AKI.

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

ObjectiveHepatic 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.MethodsA 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.ResultsAmong 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.ConclusionPlasma 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.

A missense mutation in lectin domain of thrombomodulin causing functional deficiency

Thrombomodulin (TM) functions in coagulation, fibrinolysis and inflammation by its cofactor activity for protein C, thrombin-activatable fibrinolysis inhibitor (TAFI) activation and high mobility group box 1 (HMGB1) degradation induced by thrombin. It has been widely reported that mutations in TM are related to thromboembolic diseases but hardly in lectin domain. Here we report our findings about the functional deficiencies in TM caused by substitution of aspartate with tyrosine at residue 126. Three patients suffering from recurrent thromboembolic diseases were identified with this mutation and their plasma soluble TM levels were decreased. Transfected cells expressing wild-type TM or the variant and corresponding proteins were used to examine TM functions in vitro. The cofactor activity of the mutant for protein C, TAFI activation was reduced to approximately 50% and 60% respectively. Loss in anti-inflammation due to weakened HMGB1 degradation was also observed. And the study with thrombosis models of mice suggested the decreased inhibition of thrombus development of the mutant. Together the results showed deleterious changes on TM function caused by this mutation, which may explain the thrombophilia tendency of the patients. This work provided supportive evidence that mutation in lectin domain of TM might be related to thrombotic diseases and may help us better understand the physiological roles of TM.

A prospective study to assess serum level of soluble thrombomodulin as early marker of acute traumatic coagulopathy in polytrauma patients

Background: Coagulopathy following major trauma is conventionally attributed to activation of coagulation factors. We hypothesized that early coagulopathy is due to tissue hypoperfusion and investigated thrombomodulin (TM) as early marker of endothelial injury in poly trauma patient.Methods: This was a prospective cohort study of major trauma patients admitted to a single trauma center. Blood was drawn within 10 minutes of arrival for analysis of TM. We assess its association with blood transfusion, length of hospital stays and mortality.Results: A total of 90 patients were enrolled. An increasing lactate was associated with high soluble TM. High TM was significantly associated with increased mortality, blood transfusion requirements, hospital stay.Conclusions: Acute traumatic coagulopathy (ATC) occurs only in the presence of tissue hypoperfusion which we have measured in form of lactate and coagulopathy measured using international normalized ratio (INR) as standard. Admission serum TM can be predictive of clinical outcomes following major trauma.

Persistent endotheliopathy in the pathogenesis of long COVID syndrome

BackgroundPersistent symptoms including breathlessness, fatigue, and decreased exercise tolerance have been reported in patients after acute SARS‐CoV‐2 infection. The biological mechanisms underlying this “long COVID” syndrome remain unknown. However, autopsy studies have highlighted the key roles played by pulmonary endotheliopathy and microvascular immunothrombosis in acute COVID‐19. ObjectivesTo assess whether endothelial cell activation may be sustained in convalescent COVID‐19 patients and contribute to long COVID pathogenesis. Patients and MethodsFifty patients were reviewed at a median of 68 days following SARS‐CoV‐2 infection. In addition to clinical workup, acute phase markers, endothelial cell (EC) activation and NETosis parameters and thrombin generation were assessed. ResultsThrombin generation assays revealed significantly shorter lag times (p < .0001, 95% CI −2.57 to −1.02 min), increased endogenous thrombin potential (p = .04, 95% CI 15–416 nM/min), and peak thrombin (p < .0001, 95% CI 39–93 nM) in convalescent COVID‐19 patients. These prothrombotic changes were independent of ongoing acute phase response or active NETosis. Importantly, EC biomarkers including von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), and factor VIII were significantly elevated in convalescent COVID‐19 compared with controls (p = .004, 95% CI 0.09–0.57 IU/ml; p = .009, 95% CI 0.06–0.5 IU/ml; p = .04, 95% CI 0.03–0.44 IU/ml, respectively). In addition, plasma soluble thrombomodulin levels were significantly elevated in convalescent COVID‐19 (p = .02, 95% CI 0.01–2.7 ng/ml). Sustained endotheliopathy was more frequent in older, comorbid patients, and those requiring hospitalization. Finally, both plasma VWF:Ag and VWFpp levels correlated inversely with 6‐min walk tests. ConclusionsCollectively, our findings demonstrate that sustained endotheliopathy is common in convalescent COVID‐19 and raise the intriguing possibility that this may contribute to long COVID pathogenesis.

Platelet and Red Blood Cell Transfusions and Risk of Acute Graft-versus-Host Disease after Myeloablative Allogeneic Hematopoietic Cell Transplantation

Transfusion therapy is a critical part of supportive care early after allogeneic hematopoietic cell transplantation (allo-HCT). Platelet and RBC transfusions elicit immunomodulatory effects in the recipient, but if this impacts the risk of acute graft-versus-host disease (aGVHD)has only been scarcely investigated. We investigated if platelet and RBC transfusions were associated with the development of aGVHD following myeloablative allo-HCT in a cohort of 664 patients who underwent transplantation between 2000 and 2019. Data were further analyzed for the impact of blood donor age and sex and blood product storage time. Exploratory analyses were conducted to assess correlations between transfusion burden and plasma biomarkers of inflammation and endothelial activation and damage. Between day 0 and day +13, each patient received a median of 7 (IQR, 5 to 10) platelet transfusions and 3 (IQR, 2 to 6) RBC transfusions (Spearman's ρ=0.49). The cumulative sums of platelet and RBC transfusions, respectively, received from day 0 to day +13 were associated with subsequent grade II-IV aGVHD in multivariable landmark Cox models (platelets: adjusted hazard ratio [HR], 1.27; 95% confidence interval [CI], 1.06 to 1.51; RBCs: adjusted HR, 1.41; 95% CI, 1.09 to 1.82; both per 5 units; 184 events). For both platelet and RBC transfusions, we did not find support for a difference in the risk of aGVHD according to age or sex of the blood donor. Transfusion of RBCs with a storage time longer than the median of 8 days was inversely associated with aGVHD (HR per 5 units, 0.54; 95% CI, 0.30 to 0.96); however, when using an RBC storage time of ≥14 days as a cutoff, there was no longer evidence for an association with aGVHD (HR, 1.03 per 5 units; 95% CI, 0.53 to 2.00). For platelets, there was no clear association between storage time and the risk of aGVHD. The transfusion burdens of platelets and RBCs were positively correlated with plasma levels of TNF-α, IL-6, and soluble thrombomodulin at day +14. In conclusion, platelet and RBC transfusions in the first 2 weeks after myeloablative allo-HCT were associated with subsequent development of grade II-IV aGVHD. We did not find evidence of an impact of blood donor age or sex or blood product storage time on the risk of aGVHD. Our findings support restrictive transfusion strategies in allo-HCT recipients.

Vascular and Hemostatic Alterations Associated with Pulmonary Hypertension in β-Thalassemia Hemoglobin E Patients Receiving Regular Transfusion and Iron Chelation

Background: β-thalassemia hemoglobin E (β-thal/HbE) is currently the commonest β-thalassemia disease worldwide. Pulmonary hypertension (PH) is the most common cardiovascular complication in this genotype, especially in splenectomized patients. Yet the pathogenesis of this major complication remains poorly characterized. Most of the previous reports investigated pathophysiology of PH in β-thal/HbE with inadequate transfusion. In this study we aimed to define the prevalence and comprehensively explore mechanisms of PH in β-thal/HbE patients receiving regular transfusion and iron chelation which were reported to alleviate PH. Our findings will provide deeper insights into pathogenesis and identify targets for management of persistent PH in thalassemia patients after adequate transfusion and chelation.Methods: β-thal/HbE patients who received regular transfusion ≥ 1 unit per month and any forms of iron chelation over 1 year were eligible for the study. Normal healthy subjects were used as controls. Blood sample collection and echocardiographic assessment were performed on the same days. Patients at risk for PH were defined by the tricuspid regurgitant jet velocity (TRV) ≥ 2.5 m/s.Results: There were 68 β-thal/HbE and 38 healthy controls enrolled between May and September 2015. The ages (mean ± SD) were 34.1 ± 12.6, ranging from 19-67 years, and 37.2 ± 8.6 years, ranging from 18-70 years, respectively. Among 68 β-thal/HbE, the average transfusion duration was 8.1 ± 2.2, ranging from 2.2-10.6 years, and the quantity of transfusion was 22.3 ± 9.6 units per year. The pre-transfusion hemoglobin (Hb) level was 8.1 ± 1.3 g/dL. There were 31 (45.6%) splenectomized patients. β-thal/HbE showed markedly increased erythropoiesis (growth differentiation factor, GDF15, and soluble transferrin receptor, sTfR, levels) and iron load (serum ferritin) compared to controls, all p < 0.001. Thrombin antithrombin (TAT) complex and microparticle's procoagulant activity (MPA) were significantly elevated (both p < 0.001), but total tissue factor antigen (TFAg) and microparticle-associated tissue factor activity (MPTFA) were not different from those of controls, suggesting TF-independent hypercoagulability. Serum nitric oxide metabolites (NOx) and prostaglandin E2 (PGE2) levels were significantly lower than those of controls (both p < 0.001), while soluble thrombomodulin (sTM) levels were significantly elevated (p= 0.045). Serum endothelin-1 (ET-1) and soluble p-selectin levels were not different between patients and controls. Echocardiographic profiles demonstrated preserved biventricular systolic functions in β-thal/HbE with a significant increase in TRV (p < 0.001).Among β-thal/HbE, there were 29 (42.6%; 16 non-splenectomized and 13 splenectomized) cases at risk for PH. Duration and amount of transfusion, pre-transfusion Hb, GDF15 and sTfR levels were not statistically different between PH and non-PH subgroups indicating the similar degree of erythropoiesis. Notably, β-thal/HbE with PH had a significant reduction in NOx levels (p < 0.001) but elevations in TAT, ET-1 and sTM levels compared to those without PH (p= 0.046, 0.005 and 0.026, respectively). Additionally, TRV was significantly correlated with NOx, TAT, sTM and ET-1 levels (r = -0.514, p < 0.001; r = 0.281, p= 0.004; r = 0.313, p= 0.001; and r = 0.245, p= 0.012, respectively). Serum ferritin, MPA, MPTFA, TFAg and PGE2 levels were not statistically different between PH and non-PH subgroups. Surprisingly, there were no differences in any biomarkers and echocardiographic profiles between splenectomized and non-splenectomized patients as well as no significant associations between splenectomy statuses and PH (Χ2 = 0.012, p= 0.914).We reviewed another β-thal/HbE cohort with available flow mediated dilatation (FMD) data. 9 β-thal/HbE with PH had significantly lower FMD (3.43%) compared to 18 β-thal/HbE without PH (6.37%) and 27 age-sex matched controls (9.87%; p= 0.025 and p < 0.001, respectively) indicating a greater degree of endothelial dysfunction in β-thal/HbE having PH. In addition, TRV was significantly correlated with FMD (r = -0.474, p < 0.001).Conclusions: PH is still prevalent in β-thal/HbE patients receiving long-term regular transfusion and iron chelation. In this group, PH is not associated with splenectomy statuses but correlated with NO depletion, TF-independent hypercoagulation and endothelial dysfunction. [Display omitted] DisclosuresSutcharitchan:Chulalongkorn University: Consultancy, Employment; Celgene: Research Funding.

Remote Ischemic Preconditioning Protects Against Endothelial Dysfunction in a Human Model of Systemic Inflammation: A Randomized Clinical Trial.

OBJECTIVE: Inflammation, oxidative stress, and endothelial dysfunction are known to contribute to ischemia-reperfusion injury. Remote ischemic preconditioning (RIPC) protects from endothelial dysfunction and the damage induced by ischemiareperfusion. Using intensive periodontal treatment (IPT), an established human model of acute systemic inflammation, we investigated whether RIPC prevents endothelial dysfunction and modulates systemic levels of inflammation and oxidative stress. APPROACH AND RESULTS: Forty-nine participants with periodontitis were randomly allocated to receive either 3 cycles of ischemia-reperfusion on the upper limb (N=24, RIPC) or a sham procedure (N=25, control) before IPT. Endothelial function assessed by flow-mediated dilatation of the brachial artery, inflammatory cytokines, markers of vascular injury, and oxidative stress were evaluated at baseline, day 1, and day 7 after IPT. Twenty-four hours post-IPT, the RIPC group had lower levels of IL-10 (interleukin-10) and IL-12 (interleukin-12) compared with the control group (P<0.05). RIPC attenuated the IPTinduced increase in IL-1β (interleukin-1β), E-selectin, sICAM-3 (soluble intercellular adhesion molecule 3), and sTM (soluble thrombomodulin) levels between the baseline and day 1 (P for interaction <0.1). Conversely, oxidative stress was differentially increased at day1 in the RIPC group compared with the control group (P for interaction <0.1). This was accompanied by a better flow-mediated dilatation (mean difference 1.75% [95% CI, 0.428–3.07], P=0.011). After 7 days from IPT, most of the inflammatory markers, endothelial-dependent and -independent vasodilation, were similar between groups. CONCLUSIONS: RIPC prevented acute endothelial dysfunction by modulation of inflammation and oxidation processes in patients with periodontitis following exposure to an acute inflammatory stimulus. REGISTRATION: URL: https://clinicaltrials.gov/ct2/show/NCT03072342; Unique identifier: NCT03072342.

Biomarkers of coagulation, endothelial function, and fibrinolysis in critically ill patients with COVID‐19: A single‐center prospective longitudinal study

BackgroundImmunothrombosis and coagulopathy in the lung microvasculature may lead to lung injury and disease progression in coronavirus disease 2019 (COVID‐19). We aim to identify biomarkers of coagulation, endothelial function, and fibrinolysis that are associated with disease severity and may have prognostic potential. MethodsWe performed a single‐center prospective study of 14 adult COVID‐19(+) intensive care unit patients who were age‐ and sex‐matched to 14 COVID‐19(−) intensive care unit patients, and healthy controls. Daily blood draws, clinical data, and patient characteristics were collected. Baseline values for 10 biomarkers of interest were compared between the three groups, and visualized using Fisher's linear discriminant function. Linear repeated‐measures mixed models were used to screen biomarkers for associations with mortality. Selected biomarkers were further explored and entered into an unsupervised longitudinal clustering machine learning algorithm to identify trends and targets that may be used for future predictive modelling efforts. ResultsElevated D‐dimer was the strongest contributor in distinguishing COVID‐19 status; however, D‐dimer was not associated with survival. Variable selection identified clot lysis time, and antigen levels of soluble thrombomodulin (sTM), plasminogen activator inhibitor‐1 (PAI‐1), and plasminogen as biomarkers associated with death. Longitudinal multivariate k‐means clustering on these biomarkers alone identified two clusters of COVID‐19(+) patients: low (30%) and high (100%) mortality groups. Biomarker trajectories that characterized the high mortality cluster were higher clot lysis times (inhibited fibrinolysis), higher sTM and PAI‐1 levels, and lower plasminogen levels. ConclusionsLongitudinal trajectories of clot lysis time, sTM, PAI‐1, and plasminogen may have predictive ability for mortality in COVID‐19.

Soluble thrombomodulin and cardiovascular disease risk factors in Japanese children.

This study aimed to establish standard reference values for soluble thrombomodulin in healthy prepubertal school-aged children and elucidate the relationship between soluble thrombomodulin levels and obesity, metabolic syndrome-associated indices, and other markers of vascular endothelial damage. The participants in this study were healthy Japanese children aged 9-10 years (315 boys and 267 girls). Blood tests for soluble thrombomodulin, leptin, fibrinogen, and general biochemical markers were performed, and the mean and 10th, 50th, and 90th percentiles for each marker were determined. Participants were divided into two groups based on their waist circumference (≥75 vs. <75 cm), and each parameter was compared between the two groups. Analyses were performed to compare subgroups with different numbers of risk factors for cardiovascular disease (CVD). We found that as CVD risk factors accumulated, the levels of total cholesterol, alanine aminotransferase, uric acid, soluble thrombomodulin, fibrinogen, and leptin were significantly elevated, whereas the level of high-density lipoprotein cholesterol significantly decreased. We determined reference values for soluble thrombomodulin in prepubertal children, and our results suggest that soluble thrombomodulin levels contribute to the latent progress of arteriosclerosis from childhood.

Thrombomodulin is upregulated in the kidneys of women with pre-eclampsia

The endothelial glycoprotein thrombomodulin regulates coagulation, vascular inflammation and apoptosis. In the kidney, thrombomodulin protects the glomerular filtration barrier by eliciting crosstalk between the glomerular endothelium and podocytes. Several glomerular pathologies are characterized by a loss of glomerular thrombomodulin. In women with pre-eclampsia, serum levels of soluble thrombomodulin are increased, possibly reflecting a loss from the glomerular endothelium. We set out to investigate whether thrombomodulin expression is decreased in the kidneys of women with pre-eclampsia and rats exposed to an angiogenesis inhibitor. Thrombomodulin expression was examined using immunohistochemistry and qPCR in renal autopsy tissues collected from 11 pre-eclamptic women, 22 pregnant controls and 11 hypertensive non-pregnant women. Further, kidneys from rats treated with increasing doses of sunitinib or sunitinib in combination with endothelin receptor antagonists were studied. Glomerular thrombomodulin protein levels were increased in the kidneys of women with pre-eclampsia. In parallel, in rats exposed to sunitinib, glomerular thrombomodulin was upregulated in a dose-dependent manner, and the upregulation of glomerular thrombomodulin preceded the onset of histopathological changes. Selective ETAR blockade, but not dual ETA/BR blockade, normalised the sunitinib-induced increase in thrombomodulin expression and albuminuria. We propose that glomerular thrombomodulin expression increases at an early stage of renal damage induced by antiangiogenic conditions. The upregulation of this nephroprotective protein in glomerular endothelial cells might serve as a mechanism to protect the glomerular filtration barrier in pre-eclampsia.