Plasma Soluble Thrombomodulin Research Articles

28: BIOMARKER-BASED RISK STRATIFICATION IN PEDIATRIC SEPSIS FROM A LOW-MIDDLE-INCOME COUNTRY

Introduction: Most biomarker studies of sepsis and acute respiratory distress syndrome (ARDS) originate from high-income countries per World Bank classification, whereas mortality risk is higher in low- and middle-income countries. In children, the second iteration of the Pediatric Sepsis Biomarker Risk Model (PERSEVERE-II) has been validated in multiple North American pediatric intensive care units (PICUs) for prognosis, with areas under the receiver operating characteristic (AUROC) curve of > 0.80 for discriminating 28-day mortality. Given differences in epidemiology, we assessed the performance of PERSEVERE-II in septic children from Pakistan, a low-middle income country, as well as the utility of other select biomarkers. Methods: This was a prospective cohort study of children (age < 18 years) from Aga Khan University Hospital (AKUH) meeting modified pediatric Sepsis-3 criteria (pSOFA ≥ 2 and lactate > 2) between 11/2020 and 2/2022. Plasma was collected < 24 hours of PICU admission and biomarkers measured via single- and multiplex ELISAs. The AUROC for PERSEVERE-II to discriminate 28-day mortality was determined. Additional biomarkers were compared between PERSEVERE-II risk strata (low, medium, and high mortality risk), between survivors and non-survivors, and between those with and without ARDS. Projecting 15% mortality, 73 subjects were needed to detect AUROC > 0.80 at α = 0.05 and power = 0.90. Results: We enrolled 86 subjects with 20 (23%) 28-day non-survivors. PERSEVERE-II discriminated mortality with an AUROC of 0.83 (95% CI 0.72 to 0.94). Non-survivors had higher plasma angiopoietin-2 (ANG2), soluble thrombomodulin (sTM), and plasminogen activator inhibitor 1 (PAI1; all rank-sum p < 0.05), with increasing ANG2 across worsening PERSEVERE-II risk strata. Children who met ARDS criteria within 96 hours had elevated soluble receptor for advanced glycation end-products (sRAGE) and surfactant protein D (SPD; both rank-sum p < 0.05). Conclusions: PERSEVERE-II performs well in septic children from AKUH, representing the first validation of PERSEVERE-II in a low-middle income country. ANG2, sTM, and PAI1 were elevated in non-survivors, and sRAGE and SPD elevated in subjects with ARDS, suggesting a biomarker profile comparable to that of adult and pediatric sepsis and ARDS from high income countries.

Effects of convalescent plasma infusion on the ADAMTS13-von Willebrand factor axis and endothelial integrity in patients with severe and critical COVID-19

BackgroundConvalescent plasma infusion (CPI) was given to patients with COVID-19 during the early pandemic with mixed therapeutic efficacy. However, the impacts of CPI on the ADAMTS13-von Willebrand factor (VWF) axis and vascular endothelial functions are not known. ObjectivesTo determine the impacts of CPI on the ADAMTS13-VWF axis and vascular endothelial functions. MethodsSixty hospitalized patients with COVID-19 were enrolled in the study; 46 received CPI and 14 received no CPI. Plasma ADAMTS13 activity, VWF antigen, endothelial syndecan-1, and soluble thrombomodulin (sTM) were assessed before and 24 hours after treatment. ResultsPatients with severe and critical COVID-19 exhibited significantly lower plasma ADAMTS13 activity than the healthy controls. Conversely, these patients showed a significantly increased VWF antigen. This resulted in markedly reduced ratios of ADAMTS13 to VWF in these patients. The levels of plasma ADAMTS13 activity in each patient remained relatively constant throughout hospitalization. Twenty-four hours following CPI, plasma ADAMTS13 activity increased by ∼12% from the baseline in all patients and ∼21% in those who survived. In contrast, plasma levels of VWF antigen varied significantly over time. Patients who died exhibited a significant reduction of plasma VWF antigen from the baseline 24 hours following CPI, whereas those who survived did not. Furthermore, patients with severe and critical COVID-19 showed significantly elevated plasma levels of syndecan-1 and sTM, similar to those found in patients with immune thrombotic thrombocytopenic purpura. Both syndecan-1 and sTM levels were significantly reduced 24 hours following CPI. ConclusionOur results demonstrate the relative deficiency of plasma ADAMTS13 activity and endothelial damage in patients with severe and critical COVID-19, which could be modestly improved following CPI therapy.

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.

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.

Unfractionated heparin attenuates histone-mediated cytotoxicity in vitro and prevents intestinal microcirculatory dysfunction in histone-infused rats.

Extracellular histones are major mediators of organ dysfunction and death in sepsis, and they may cause microcirculatory dysfunction. Heparins have beneficial effects in sepsis and have been reported to bind to histones and neutralize their cytotoxicity. The aim of this study was to investigate the impact of histones on intestinal microcirculation and the intestinal endothelium and to discuss the protective effect of unfractionated heparin (UFH) on the endothelial cytotoxicity and microcirculatory dysfunction induced by histones. Anesthetized rats were infused with 30 mg/kg calf thymus histones, and UFH was administered intravenously at a concentration of 100 IU/kg per hour. The intestinal microcirculation was visualized and measured with incident dark field microscope. Plasma von Willebrand factor (vWF) and soluble thrombomodulin were detected, and structural changes in the rat intestinal microvascular endothelium were examined. The effects of histones and UFH on cell survival rates, vWF release and calcium influx were investigated in human intestinal microvascular endothelial cells (HIMECs). Histone infusion caused severe intestinal microcirculatory dysfunction in the absence of obvious hemodynamic changes, and UFH protected intestinal microcirculation in histone-infused rats. Concentrations of the plasma endothelial injury markers vWF and soluble thrombomodulin were elevated, and structural abnormalities were found in the intestinal microvascular endothelium in the histone-infused rats. These events were attenuated by UFH. In vitro, UFH significantly reduced the histone-induced cytotoxicity of HIMECs, reduced the release of vWF from the cytoplasm into the culture medium, and inhibited calcium influx into HIMECs. Histones induce intestinal microcirculatory dysfunction followed by direct injury to the endothelial cells; UFH protects the intestinal microcirculation partly by antagonizing the endothelial toxicity of histones.

Pre-operative methylprednisolone decreased early vascular endothelial injury after total knee arthroplasty

Objective To investigate the effect of methylprednisolone on markers of early vascular endothelial activation and damage after total knee arthroplasty. Methods We randomly selected 88 patients undergoing unilateral total knee arthroplasty. The patients were divided into two groups by a completely random grouping method(n=44), they were given either pre-operative intravenous methylprednisolone 120 mg (number of patients for methylprednisolone group) or isotonic saline (number of patients for control group). All operations were performed under lumbar spinal and epidural anesthesia. Blood samples were collected at 2, 6, 12 h and 24 h pre- and post-operation, respectively. By enzyme-linked immunosorbent assay(ELISA), we analyzed concentrations of Syndecan-1, plasma soluble thrombomodulin (sTM), E-selectin, vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) at different time points. Results There was no significant difference in age, gender, body mass index (BMI), operation time and ropivacaine dose between patients in two groups (P>0.05). Syndecan-1、E-selectin and CRP were significantly decreased at 2, 6, 12 h and 24 h in methylprednisolone group, sTM and VEGF were significantly decreased at 6, 12 h and 24 h in methylprednisolone group (P<0.05) . Compared with preoperative, the level of Syndican-1 and E-selectin at 2, 6, 12 h and 24 h were significantly decreased, the level of CRP was significantly increased (P<0.05) , the level of sTM and VEGF were significantly decreased at 6, 12 h and 24 h in methylprednisolone group (P<0.05) . Conclusions Pre-operative administration of methylprednisolone 120 mg could reduce markers of endothelial activation, reduce the level of damage markers and reduce the systemic inflammatory response after total knee arthroplasty. Key words: Total knee arthroplasty; Syndecan-1; Soluble thrombomodulin; E-selectin; Vascular endothelial growth factor; C-reactive protein

Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice

AbstractCerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO) or Pdcd10 (Pdcd10ECKO), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

The relation of thrombomodulin G33A and C1418T gene polymorphisms to the risk of acute myocardial infarction in Egyptians

BackgroundAcute myocardial infarction (AMI) is one of the major causes of morbidity and mortality worldwide. There is an increased interest in the genetic risk factors in the pathogenesis of ischemic heart disease. Thrombomodulin (TM), a natural anticoagulant, may play a critical role in the pathogenesis of AMI. Aim of the studyTo assess whether Thrombomodulin (TM) G33A and C1418T gene polymorphisms are related to the risk of AMI in Egyptians or not. Subjects and methods102 AMI patients were recruited vs 110 healthy controls. For every subject, measurement of plasma soluble Thrombomodulin level was done by enzyme-linked immunosorbent assay (ELISA). Further, DNA samples were genotyped by PCR-RFLP method for the TM G33A polymorphism and by PCR-ASO for the TM C1418T polymorphism. ResultsOur results revealed that the C1418T gene polymorphism was significantly associated with increased risk of AMI (CT: OR=2.34, 95% CI=1.28–4.29, P=0.006; TT: OR=8.03, 95% CI=0.97–66.47, P=0.026; CT+TT: OR=2.65, 95% CI=1.47–4.78, P=0.001; T allele: OR=2.51, 95% CI=1.51–4.18, P<0.001). On the other side, the TM G33A polymorphism was not detectable in any of patients or controls. Further, plasma soluble TM concentrations were higher in AMI patients, compared to the control group (P<0.001). ConclusionsTM 1418C>T gene polymorphism, but not TM 33-G>A, may be associated with an increased risk of AMI in the Egyptian population. These results may have clinical implication in the management of AMI in the future.

The effect of pre-operative methylprednisolone on early endothelial damage after total knee arthroplasty: a randomised, double-blind, placebo-controlled trial.

We wished to evaluate whether inhibition of the systemic inflammatory response by a single pre-operative dose of methylprednisolone reduced markers of early endothelial damage after fast-track total knee arthroplasty. We randomly allocated 70 patients undergoing elective unilateral total knee arthroplasty (1:1) to receive either pre-operative intravenous methylprednisolone 125 mg (methylprednisolone group) or isotonic saline (control group). All procedures were performed under spinal anaesthesia without a tourniquet, using a standardised multimodal analgesic regime. The outcomes included changes in Syndecan-1 concentrations, a marker of glycocalyx degradation, markers of endothelial cell damage and activation (plasma soluble thrombomodulin and sE-Selectin), and permeability by vascular endothelial growth factor, as well as C-reactive protein concentrations. Blood samples were collected at baseline and 2 h, 6 h and 24 h after surgery, with complete sampling from 63 patients for analyses. Methylprednisolone significantly reduced markers of endothelial damage at 24 h following surgery compared with saline (methylprednisolone group vs. control group, adjusted means (SEM)) expressed by circulating Syndecan-1: 11.6 (1.0) ng.ml-1 vs. 13.4 (1.1) ng.ml-1 p = 0.046; soluble thrombomodulin: 5.1 (0.1) ng.ml-1 vs. 5.7 (0.2) ng.ml-1 , p = 0.009; sE-Selectin: 64.8 (1.8) ng.ml-1 vs. 75.7 (1.9) ng.ml-1 , p = 0.001, and vascular endothelial growth factor: 35.3 (2.7) ng.ml-1 vs. 58.5 (2.8) ng.ml-1 , p < 0.001. The effect of the intervention increased with time for soluble thrombomodulin, sE-Selectin and vascular endothelial growth factor, and was more pronounced in patients with high baseline values. Finally, methylprednisolone reduced the C-reactive protein response 24 h postoperatively; 31.1 (1.1) mg.l-1 vs. 68.4 (1.1) mg.l-1 , p < 0.001. Pre-operative administration of methylprednisolone 125 mg reduced circulating markers of endothelial activation and damage, as well as the systemic inflammatory response (C-reactive protein) early after fast-track total knee arthroplasty. These findings may have a positive effect on surgical outcome, but require studies in major surgery.

Research on the relationship between microalbuminuria and plasma soluble thrombomodulin in elderly patients with asymptomatic coronary heart disease

Objective To investigate the relationship between microalbuminuria and plasma soluble thrombomodulin in elderly patients with asymptomatic coronary heart disease. Methods 106 elderly patients with asymptomatic coronary heart disease were divided into non-microalbuminuria group and microalbuminuria group, and another 50 patients without coronary heart disease were set as control group.The levels of plasma soluble thrombomodulin and microalbuminuria were determined, the differences between two groups and the correlation between plasma soluble thrombomodulin and microalbuminuria were assessed. Results The level of plasma soluble thrombomodulin was(3.36±1.27)μg/L in the control group, (13.96±1.33)μg/L in non-microalbuminuria group, and(17.36±7.48)μg/L in microalbuminuria group, with obvious difference among groups.There were positive correlations of microalbuminuria with plasma soluble thrombomodulin, course of CHD, history of diabetes, hypertension, and serum creatinine levels in elderly patients with coronary heart disease(all P 0.05). The multiple linear regression analysis showed that after adjusting for gender, blood lipid and UA, plasma soluble thrombomodulin, history of diabetes, hypertension and age were independent risk factors for microalbuminuria in elderly patients with coronary heart disease. Conclusions Microalbuminuria and plasma soluble thrombomodulin can appear in asymptomatic stage in elderly patients with coronary heart disease, and there is a positive correlation between microalbuminuria and plasma soluble thrombomodulin, which suggests that early monitoring of microalbuminuria and plasma soluble thrombomodulin could probably contribute to the delayed coronary heart disease progression. Key words: Thrombomodulin; Albuminuria; Coronary disease

Plasma Von Willebrand Factor Antigen and Soluble Thrombomodulin Levels in Male Patients with Acute Coronary Syndrome at Diagnosis

Plasma Von Willebrand Factor Antigen and Soluble Thrombomodulin Levels in Male Patients with Acute Coronary Syndrome at Diagnosis

Radix Puerariae lobatae (Gegen) suppresses the anticoagulation effect of warfarin: a pharmacokinetic and pharmacodynamics study.

BackgroundRadix Salvia miltiorrhiza (Danshen) and Radix Puerariae lobatae (Gegen) are used in Traditional Chinese Medicine to treat cardiovascular diseases. However, adverse herb-drug interactions were observed between warfarin and herbal remedies containing Danshen and Gegen. This study aims to investigate the pharmacokinetic and pharmacodynamic interactions between warfarin and the different components found in Danshen and Gegen.MethodsSixty Sprague–Dawley rats were used to investigate the effects of warfarin (0.2 mg/kg), Danshen (240 or 480 mg/kg) and Gegen (240 or 480 mg/kg) both in isolation and combination. The rats in the warfarin and Danshen/Gegen combination groups were given an oral dose of Danshen or Gegen 2 h after being given an oral dose of warfarin. After five consecutive days of treatment, the pharmacokinetic interactions between Danshen/Gegen and warfarin were investigated by simultaneously monitoring and comparing the cytochrome P450 (CYP) activities, mRNA and protein expression levels in the livers of the rats from the different treatment groups. The pharmacodynamic interactions were evaluated by monitoring and comparing the vitamin K epoxide reductase (VKOR) activities, mRNA and protein expression levels in the livers of rats from the different groups, as well as the thrombomodulin (TM) activities, mRNA and protein in the lungs of these animals. The rat plasma soluble thrombomodulin concentrations of the different treatment groups were also evaluated. Microsomes incubation, Real Time-Polymerase Chain Reaction and Western blot was applied respectively to study the activity, mRNA expression and protein expression of CYP, VKOR and TM.ResultsThe activities and expression levels of the CYP and VKOR enzymes in the warfarin-Gegen combination groups increased by nearly 30 % (P = 0.02) compared with the warfarin-alone group, whereas those of TM decreased by almost 25 % (P = 0.02). The administration of Danshen did not lead to any changes in the activities or the expression levels of the CYP, VKOR or TM enzymes compared with those of the control group. Gegen induced several warfarin-metabolizing CYP enzymes and neutralized the effects of warfarin towards VKOR and TM.ConclusionGegen, rather than Danshen at the same tested dosage, offsets the anticoagulant effects of warfarin by accelerating the phase I liver metabolism of warfarin, as well as increasing the activity, mRNA and protein expression of VKOR while decreasing those of TM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0078-9) contains supplementary material, which is available to authorized users.

Plasma soluble thrombomodulin levels are associated with mortality in the acute respiratory distress syndrome.

Thombomodulin (TM) is an activator of protein C and a biomarker for endothelial injury. We hypothesized that (1) elevated plasma levels would be associated with clinical outcomes and (2) polymorphisms in the TM gene would be associated with plasma levels. We studied 449 patients enrolled in the Fluid and Catheter Treatment Trial (FACTT) for whom both plasma and DNA were available. We used logistic regression and receiver operator curves (ROC) to test for associations between soluble TM (sTM) and mortality at 60days. Plasma sTM levels were higher in non-survivors than survivors at baseline [median 147 (IQR, 95-218) vs. 89 (56-129) ng/mL, p<0.0001] and on day 3 after study enrollment [205 (146-302) vs. 127 (85-189), p<0.0001]. The odds of death increased by 2.4 (95% CI 1.5-3.8, p<0.001), and by 2.8 (1.7-4.7, P<0.001) for every log increase in baseline and day 3 sTM levels, respectively, after adjustment for age, race, gender, severity of illness, fluid management strategy, baseline creatinine, and non-pulmonary sepsis as the primary cause of ARDS. By ROC analysis, plasma sTM levels discriminated between non-survivors and survivors [AUC=72% (66-78%) vs. AUC=54% for severity based on Berlin criteria). Addition of sTM improved discrimination based on APACHE III from 77 to 80% (P<0.03). sTM levels at baseline were not statistically different among subjects stratified by genotypes of tag SNPs in the TM gene. Higher plasma sTM levels are associated with increased mortality in ARDS. The lack of association between the sTM levels and genetic variants suggests that the increased levels of sTM may reflect severity of endothelial damage rather than genetic heterogeneity. These findings underscore the importance of endothelial injury in ARDS pathogenesis and suggest that, in combination with clinical markers, sTM could contribute to risk stratification.

Association of the Thrombomodulin Gene c.1418C&gt;T Polymorphism With Thrombomodulin Levels and With Venous Thrombosis Risk

To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane-bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

Common Genetic Risk Factors for Venous Thrombosis in the Chinese Population

Venous thrombosis is a major medical disorder caused by both genetic and environmental factors. Little is known about the genetic background of venous thrombosis in the Chinese population. A total of 1,304 individuals diagnosed with a first venous thrombosis and 1,334 age- and sex-matched healthy participants were enrolled in this study. Resequencing of THBD (encoding thrombomodulin) in 60 individuals with venous thrombosis and 60 controls and a functional assay showed that a common variant, c.-151G>T (rs16984852), in the 5' UTR significantly reduced the gene expression and could cause a predisposition to venous thrombosis. Therefore, this variant was genotyped in a case-control study, and results indicated that heterozygotes had a 2.80-fold (95% confidence interval = 1.88-4.29) increased risk of venous thrombosis. The THBD c.-151G>T variant was further investigated in a family analysis involving 176 first-degree relatives from 38 index families. First-degree relatives with this variant had a 3.42-fold increased risk of venous thrombosis, and their probability of remaining thrombosis-free was significantly lower than that of relatives without the variant. In addition, five rare mutations that might be deleterious were also identified in thrombophilic individuals by sequencing. This study is the largest genetic investigation of venous thrombosis in the Chinese population. Further study on genetics of thrombosis should focus on resequencing of THBD and other hemostasis genes in different populations.

Plasma soluble thrombomodulin and soluble endothelial protein C receptor levels in colorectal cancer patients

Thrombomodulin (TM) and endothelial protein C receptor (EPCR) are 2 transmembrane proteins that are thought to play an important role in cancer. We aimed to discover whether these 2 proteins are prognostic indicators in colorectal cancer. Materials and methods: Plasma TM and EPCR levels were measured using the ELISA method in 50 patients in different tumor stages that had been recently diagnosed with colorectal cancer and in a healthy control group of 50 people. Results: In colorectal cancer patients, higher plasma TM (21.3 ± 22.8 ng/mL, 13.2 ± 16.2 ng/mL, P = 0.010) and plasma EPCR levels (149.9 ± 79.6, 113.3 ± 49.3, P = 0.007) were determined compared to the control group. No statistically significant relationship was present between plasma TM, EPCR levels and tumor stage, tumor localization, tumor differentiation, lymphovascular invasion state, microvascular thrombus existence, and thrombosis progression (P > 0.05). Conclusion: We think that these 2 proteins are released into plasma as an indicator of endothelial dysfunction and can play a role in pathogenesis and biology of colorectal cancer.

Increased Plasma Soluble Thrombomodulin Levels in Cardioembolic Stroke

Soluble thrombomodulin (sTM) has been proposed as a potential marker of ischemic stroke. Results from previous studies remain controversial among different populations. We performed an analysis of plasma levels of sTM in Thai patients with acute ischemic stroke and determined whether sTM levels correlate with stroke subtypes, severity, and risk factors. Ninety-three patients and 76 controls were enrolled. Blood samples were obtained within 24 hours after stroke onset. Plasma sTM levels, measured using quantitative enzyme-linked immunosorbent assay, were significantly higher in patients than controls (P < .005), with the mean ± standard deviation (SD) levels of 3.08 ± 1.05 and 2.57 ± 1.15 ng/mL, respectively. Plasma levels of sTM in patients with cardioembolic subtype were significantly higher than in patients with other stroke subtypes, with the mean ± SD levels of 3.79 ± 1.26, 2.38 ± 0.68 (P < .009), and 2.38 ± 0.44 (P < .05) ng/mL for cardioembolism, large artery atherosclerosis, and small artery occlusion, respectively. Plasma sTM levels were not associated with stroke severity and risk factors of stroke; however, there was a slight relationship between high sTM levels and the presence of atrial fibrillation in the patient group. In conclusion, plasma sTM levels were increased in Thai patients with cardioembolic stroke and may be a potential marker during the acute phase.

SRAGE in diabetic and non-diabetic critically ill patients: effects of intensive insulin therapy

IntroductionHyperglycemia represents an independent prognostic factor in critically ill non-diabetic patients but not in those with diabetes. In this context, there is an ongoing debate on the benefit of an intensive insulin therapy, particularly in diabetic patients. We tested the hypothesis that expression of the receptor for advanced glycation end-products (RAGE), an important signal transduction receptor that elicits long-lasting nuclear factor kappa B (NF-κB) activation, may underlie this difference. RAGE expression is regulated by multiple ligands, including high mobility group box-1 (HMGB-1), and is reflected by its released soluble form (sRAGE).MethodsA predesigned analysis was conducted of prospectively collected samples from 76 hyperglycemic critically ill patients (33 type-2 diabetes, 43 non-diabetes) aged ≥18 years with blood glucose of > 6.1 mmol/L enrolled in a randomized controlled trial comparing intensive insulin therapy with conventional insulin therapy. sRAGE and its ligand HMGB-1 together with IL-6, and soluble thrombomodulin (as markers of inflammation and endothelial cell injury, respectively) were evaluated in ICU, at Days 1, 3, 5 and 7. Plasma samples from 18 healthy subjects were used as controls.ResultsBoth diabetic and non-diabetic hyperglycemic patients showed increased plasma sRAGE, HMGB-1 and soluble thrombomodulin levels at the time of admission to ICU. Plasma IL-6 concentration was only increased in non-diabetic patients. Plasma levels of sRAGE were higher in diabetic compared with non-diabetic patients. Intensive insulin therapy resulted in a significant decrease of sRAGE and thrombomodulin at Day 7, in diabetic but not in non-diabetic patients. Circulating sRAGE levels correlated positively with IL-6 and soluble thrombomodulin levels and inversely with HMGB-1. Multivariate regression analysis demonstrated that sRAGE remains independently correlated with HMGB-1 only in diabetic patients. Neither sRAGE nor any inflammatory markers are associated with mortality.ConclusionsThese findings support the hypothesis that sRAGE release, time-course and response to intensive insulin therapy differ between hyperglycemic diabetic and non-diabetic critically ill patients. Whether this difference underlies the dissimilarity in clinical outcome of hyperglycemia in these two conditions warrants further studies.