Thrombotic Hazard Markers: Thrombomodulin

A 750 bp bovine Thrombomodulin (TM) cDNA fragment was used as an hybridization probe to screen an oligo-dT primed Lambda gtll. cDNA library prepared from human umbilical vein endothelial cell mRNA. A 2.4 kb positive human clone was isolated which showed an 80% nucleotide sequence homology with bovine TM cDNA. This clone and a 550 bp fragment from its 5' end were used to further screen the oligo-dT primed library as well as randomly primed library prepared from the same mRNA. The cDNA clones obtained allow us to describe the overall structure of human TM and reveal that it is extremely similar to the structure of bovine TM, especially as the bovine TM is organized like the receptor for low density lipoprotein (LDL R). Both TM and LDL R exhibit short cytoplasmic C-terminal tails which are either neutral or negatively charged. Other coated pit receptors such as the insulin receptor or the epidermal growth factor (EGF) receptor have very large cytoplasmic regions with a complex tyrosine kinase segment as well as multiple sites for phosphorylation. Both TM and LDL R possess a transmembrane region and an immediately adjacent extracellular serine/threonine rich region which in LDL R has been shown to bear 0-1inked sugars. Both TM and LDL R contain a more distal area of cysteine rich repeats, first noted in the EGF precursor and termed EGF type B. However, the TM EGF type B repeats appear to have been duplicated in TM resulting in their being 6 of them rather than the 3 found in LDL R. The N-terminal half of LDL R is thought to contain the ligand binding region of the receptor and is constructed from multiple cysteine rich repeats similar to those of Complement factor C9. The structure of this region of TM is quite different from that of LDL R, possessing few cysteines. We suspect that protein C and/or thrombin may bind to this unique domain of TM.

Novel p.C252G and p.C280X mutations in the epidermal growth factor-1 domain of thrombomodulin lead to a thrombosis-bleeding syndrome.

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Background:Venous thromboembolism (VTE) is an important complication in patients with malignant tumors. Its exact diagnosis and treatment are still lacking. We used a high-sensitive chemiluminescence method to detect thrombin–antithrombin III complex (TAT), plasmin-α2-plasmininhibitor complex (PIC), thrombomodulin (TM), and tissue plasminogen activator–inhibitor complex(t-PAIC) in combination with D-dimer and fibrin degradation product (FDP) to analyze their diagnostic and prognostic value in patients with malignant tumors.Methods:In total, 870 patients with confirmed malignant tumors were included, 82 of whom had diagnosed VTE; 200 healthy individuals were classified as the control group. The TAT, PIC, TM, and t-PAIC were detected using Sysmex HISCL5000 automated analyzers, whereas FDP and D-dimer were detected using Sysmex CS5100 coagulation analyzer. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficiency. Survival probabilities were determined using Kaplan–Meier analysis, and multivariate analyses were performed using a Cox regression model.Results:Compared with healthy controls, patients with malignant tumors showed significantly elevated TAT, PIC, TM, t-PAIC, D-dimer, and FDP. Similarly, compared with patients in the non-thrombosis group, those in the thrombosis group showed significantly elevated levels of the above mentioned markers. Logistic regression analysis showed that TAT, PIC, TM, t-PAIC, D-Dimer, and FDP were all associated with VTE. ROC analysis showed that “TAT+PIC+TM+t-PAIC+D-dimer+FDP”showed the highest sensitivity and specificity. Patients with elevated TAT, PIC, TM, and t-PAIC had a significantly shorter survival. Multivariate Cox survival analysis showed that TM and t-PAIC were significantly associated with poor prognosis. In addition, the incidence of VTE was significantly lower in patients with malignant tumors who were treated with low-molecular-weight heparin (LMWH), and their survival period was significantly longer than that of patients with malignant tumors who were not treated with LMWH.Conclusion:TAT, PIC, TM, and t-PAIC combined with D-dimer and FDP were better than the application of a single marker in the diagnosis of VTE in patients with malignant tumors. TAT and PIC can be used as sensitive markers in the diagnosis of VTE but not as prognostic markers. TM and t-PAIC might be independent prognostic indicators in patients with malignant tumors, regardless of the state of thrombus.

The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was comparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing conditions, respectively, in the two-dimensional electrophoresis. Each subspecies under non-reducing conditions, except the 200 and 52 kDa molecules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies of reducing conditions and a dimer of the 33 kDa subspecies, respectively. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspecies maintained Ala1-Pro2-Ala3- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu137-Gln138-, Gln214-Gly215-, Ser228-Val229- and Ala240-Ile241-, respectively. All subspecies obtained under non-reducing conditions exhibited cofactor activity for thrombin-dependent protein C activation ranging from 58 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of the subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa subspecies were suggested to lack both chondroitin sulfate glycosaminoglycan (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic properties and the interaction with phospholipid vesicles composed from phosphatidylcholine and phosphatidylethanolamine.

Structure-Function Analyses of Thrombomodulin by Gene-Targeting in Mice: The Cytoplasmic Domain Is Not Required for Normal Fetal Development

In the past decade, it became apparent that immune mediated cell death in a number of autoimmune endocrine diseases was due to the induction of apoptosis in target organ cells. This was conclusively demonstrated for thyroid follicular cells in Hashimoto’s (destructive autoimmune) thyroiditis, but the mechanisms underlying this cell death were not clear. Several hypotheses were put forth involving the role of deathsignalling molecules expressed on thyroid cells. While many of these hypotheses did not hold up under close scrutiny, this stimulated work on the molecular mechanisms of thyroid destruction. Several apoptosis signalling pathways, initiated by molecules such as Fas ligand (FASL) and tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. In this review we will attempt to sort out the inconsistencies in published data on the mechanisms of death-receptor mediated thyroid destruction. We will also review recently proposed models of these mechanisms, and outline directions for research that we feel might lead to discoveries of benefit to the clinician in the treatment and prevention of destructive autoimmune thyroiditis.

Evaluation of an individual's thrombin-generating capacity enables a global assessment of the coagulation cascade and is therefore thought to better reflect the clotting function of blood. However, the lack of standardization still hampers the use in routine clinical practice. Nineteen healthy subjects were sampled once a week for 5 consecutive weeks. Thrombin generation assay (TGA) was performed in duplicate by calibrated automated thrombogram (CAT) on platelet poor plasma with and without thrombomodulin. After exclusion of outliers, a nested analysis of variance (ANOVA) was performed to evaluate the biological variability (BV) results. Analytical variation (CVA ), within-individual variation (CVI ), between-individual variation (CVG ), index of individuality (II), and reference change value (RCV) were calculated. All parameters taken together, the CVA, CVI , and CVG without TM, ranged from 2.8% to 6.5%, from 4.1% to 13.3% and from 10.4% to 28.4%, respectively. For TG with TM, CVI and CVG were higher and ranged from 5.0% to 18.1% and from 14.9% to 35.3%, respectively. For endogenous thrombin potential (ETP), a CVI of 4.1% and CVG of 10.4% were obtained without addition of thrombomodulin (TM). With addition of TM, both CVI and CVG were higher: 14.0% and 34.8%, respectively. The II was low and the RCV ranged from 17.2% to 50.4%. CAT parameters are highly individualized and population-based reference values could be called into question. The assessment of BV and RCV for thrombin generation assays could optimize interpretation of serial patient results and guide setting of analytical specification goals.

Researchers should investigate the coagulopathy that accompanies Coronavirus Disease 2019 (COVID-19), as the incidence, as well as the mortality associated with coagulopathy, is higher than expected in COVID-19 patients. We read with interest the article published by Tang N et al. suggesting that elevated plasma levels of the fibrin degradation product D-dimer might be a predictive marker for mortality of patients with COVID-19 [1]. The same authors also reported that the use of heparin improved the prognosis of COVID-19 patients with severe coagulopathy and D-dimer levels >6-fold of the normal upper limit [2]. The high plasma levels of D-dimer suggest that the fibrinolytic system is not inhibited; nonetheless, thrombotic events are frequently noted in COVID-19 patients transferred to intensive care units [3]. Curiously, severe cases of COVID-19 are complicated by cytokine release syndrome in conjunction with Hemophagocytic Lymphohistiocytosis (HLH) [4], suggesting that extremely exaggerated hypercoagulability is induced in COVID-19. The pathophysiology of COVID-19-induced coagulopathy has not been fully elucidated. However, recently, postmortem examination of patients with COVID-19 who died of respiratory failure found thrombi in parallel with the deposition of the C5b-9 complex in the microvasculature, indicating terminal complement activation [5]. These pathological findings are reminiscent of the pathogenesis of Transplant-Associated Thrombotic Microangiopathy (TA-TMA) and atypical Hemolytic Uremic Syndrome (aHUS), in which activation of the complement system plays a role. Other possible factors involved in the pathogenesis of COVID-19-induced coagulopathy may be related to the tropism of Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, to vascular endothelial cells [6]. SARS-CoV-2 infects vascular endothelial cells via the Angiotensin-Converting Enzyme 2 (ACE2) receptor, leading to vascular derangement, so-called endotheliitis, which may result in coagulopathy and TMA. Thrombomodulin (TM) is a cell-surface expressed glycoprotein comprising six distinct domains, including an NH2-terminal lectinlike region, six tandem Epidermal Growth Factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain [7]. TM binds to thrombin via its EGF-like region and Converts Protein C (PC) to Activated PC (APC), which inhibits coagulant factors FVIIIa and FVa in the presence of protein S, thereby inhibiting further thrombin generation. Interestingly, TM has an anti-inflammatory effect; the lectin-like domain of TM binds and degrades High Mobility Group Box-1 (HMGB1) and attenuates inflammation. Additionally, after interacting with thrombin, TM converts carboxypeptidase Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to activated TAFI (TAFIa), which inactivates C3 and C5. Furthermore, the fifth domain of EGF-like region of TM (TME5) interacts with the cell-surface chemokine receptor G protein-coupled receptor protein 15 (GPR15) and then exerts cytoprotective effects on vascular endothelial cells in association with upregulation of antiapoptotic MCl-1 proteins, which is mediated by the activation of extracellular signal-regulated kinase [7]. Recombinant human soluble TM (rhTM) comprises extracellular domains of TM. A clinical trial comparing the efficacy and safety of rhTM with Unfractionated Heparin (UFH) in patients with Intravascular Coagulation (DIC) caused by hematological malignancies or infection found that a greater number of patients treated with rhTM recovered from DIC with acceptable toxicity profiles than those who received UFH. Based on these results, the use of rhTM has been approved for the treatment of DIC in Japan since 2008. The use of rhTM effectively treated many patients with DIC with various types of underlying diseases, including transplant-associated complications such as TA-TMA, hepatic sinusoidal obstruction syndrome/veno-occlusive disease and engraftment syndrome [8]. Importantly, endothliitis plays a role in the development of all of these conditions, suggesting the possible cytoprotective roles of rhTM in vascular endothelial cells. Moreover, prompt improvement of DIC caused by HLH developed in association with immune reconstitution syndrome after initiation of highly active antiretroviral therapy for acquired immunodeficiency syndrome was noted in the individual who received rhTM [9]. Thus, the use of anticoagulant rhTM with additional activities for immune systems and vascular endothelial cells could counteract severe COVID-19 with coagulopathy, although a systematic review and meta-analysis of rhTM therapy for sepsis-induced coagulopathy failed to prove the significant improvement of clinical outcomes of patients with sepsis-induced coagulopathy [10].

Thrombomodulin (TM) is a membrane protein with significant roles in coagulation hemostasis and immune response. Its soluble form (sTM) has recently emerged as a key biomarker for severe invasive bacterial infections, including Necrotizing Soft Tissue Infections (NSTI). While various mechanical, chemical, and enzymatic mechanisms have been linked to TM shedding, this study investigates the direct impact of bacterial stimuli on soft tissue cells as primary sources of TM release. We stimulated organotypic models, composed of fibroblast and endothelial cells, with NSTI clinical isolates and found that while Group A Streptococcus and Escherichia coli had minimal effect on TM release, Staphylococcus aureus infection triggered a significant increase of sTM levels. We further assessed whether the secreted proteins of S. aureus led to higher TM levels by increased expression, increased cell toxicity, or direct cleavage of TM from the endothelial cell membrane. To investigate these mechanisms, we performed in vitro stimulations of endothelial monolayers with secreted proteins of two S. aureus isolates differing in their agr-system functionality. Our results indicate that S. aureus agr-regulated proteins induce TM shedding by direct cleavage from the cell membrane, an effect that was inhibited by metalloproteinase inhibitors. Stimulation with the pore-forming protein α-toxin showed similar results, suggesting a potential involvement of ADAM10 in TM cleavage. Additionally, we observed that other agr-regulated proteins can cleave TM directly. Altogether, this study reveals a pathogen-specific mechanism for TM release during S. aureus invasive infection, contributing to its elevated plasma levels and providing deeper insights into the pathophysiology of NSTI.

Thrombomodulin (TM) is a glycoprotein originally isolated from rabbit lung vasculature and characterized as a natural endothelial anticoagulant. Thrombin binds to TM noncovalently with high affinity. Thrombin-TM complexes can activate protein C efficiently. Activated protein C inactivates factors Va and VIIIa and regulates the blood coagulation cascade. Thus TM converts thrombin from a procoagulant protease to an anticoagulant. TM is found on endothelial cells in veins, arteries and capillaries. Our previous study has shown that TM is also expressed on the cell surface of squamous epithelium. In the present study, we aimed to disclose differences in TM expression among normal, dysplastic, and malignant squamous epithelium in human oral mucosa by counting TM-positive cells in each lesion. TM was uniformly expressed in the spinous layer of normal human oral squamous epithelium. The number of TM-positive cells was not significantly different between normal epithelium, lichen planus and mild dysplasia. In contrast, in moderate and severe dysplasia and well-differentiated squamous cell carcinoma (SCC), there were significantly fewer positive cells compared with normal epithelium. In SCCs, the periphery and the central keratinized area of tumor islands were often negative. The proportion of TM-positive cells in poorly differentiated SCC was significantly lower than in well-differentiated SCC. These results indicate that TM may have diagnostic value in the histological examination of oral premalignant and malignant lesions.

Frailty is a multidimensional syndrome. It necessitates individualized therapies, particularly in oncological surgery, due to the increased risk of perioperative complications in frail patients. Recent literature suggests that biomarkers in saliva and blood may serve as valuable tools in clinical practice. To identify effective methods for screening frail patients with solid tumors using appropriate biomarkers, we conducted a systematic review of the literature. A comprehensive literature search was conducted in Pubmed, Cochrane Library and Web of Science from the inception until 5th February 2024. Our inclusion criteria mandated a clear definition of frailty and a significant association with specific biomarkers in saliva or blood in patients with solid tumors. From 2604 screened abstracts 17 publications were included in the analysis. The results regarding biomarkers that were significantly associated with frailty, varied across the studies. Regarding frailty instruments, the G-8 geriatric screening tool and the Fried scale were the most frequently used tools. Both, elevated levels of CRP and IL-6 correlated with frailty in some research, while other studies identified links with the neutrophil-lymphocyte ratio, amino acids, or immune cells. Notably, no salivary biomarkers for frailty were found in patients with solid tumors. There are currently no established blood or salivary biomarkers for frailty. However, an elevated inflammatory status and related markers are often significantly correlated with frailty in solid tumor patients. These findings highlight the potential to develop suitable biomarkers for frail oncology patients, emphasizing the need for further research in this area.

Accelerated Molecular Dynamics Simulations of Thrombin-Thrombomodulin Reveal Potential for Entropic Allostery

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

Objective To explore the changes of thrombomodulin (TM) expression of different degree of smoke inhalation induced acute lung injury (SI-ALI). Methods Animals were divided into 4 groups, control group (C), 10 min smoke inhalation group(10 min), 20 min smoke inhalation group (20 min), 30 min smoke inhalation group (30 min). Samples (6 rats) were collected randomly from each group in 0, 6, 24 h after smoke inhalation.Immunohistochemistry (IHC) and Western blot (WB) were carried to show the protein level of TM in lung tissue and reverse transcription-polymerase chain reaction (rt-PCR) was used to show the mRNA level of TM.Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the soluble TM in bronchoalveolar lavage fluid (BALF). Results WB and IHC showed that TM was gradually increased in 10 min group in 0 h and came to normal in 24 h. There showed no significant difference in 20 min group compared with control group in all time point.TM expression was gradually decreased in 30 min group.Rt-PCR showed that TM mRNA increased in 10 min group after smoke inhalation and gradually decreased to normal.In 20 min group, mRNA increased in 0 h and decreased afterwards, while 30 min group showed continuous decrease.There showed no significant difference of soluble TM in BALF between control, 10 min and 20 min groups, while 30 min group dropped gradually.When analyzed by lung injury score (LIS), sTM overall showed a decrease trend when LIS increased.There showed significant difference of sTM when LIS>5 (P<0.05). Conclusions The differential expression of TM is related to the severity of SI-ALI.Mild injury could increase the expression and severe injury decrease the expression of TM in lung.TM in BALF shows diagnostic value in SI-ALI. Key words: Smoke inhalation injury; Acute lung injury; Thrombomodulin