Thrombomodulin (TM; CD141 or BDCA-3) is a 557 amino acid transmembrane glycoprotein predominantly expressed on the luminal surface of endothelial cells of most blood vessels, but also present in other gestational tissues like placenta and myometrium, polymorphonuclear neutrophils and monocytes. TM plays a central role in protection against excessive coagulation via protein C pathway, with subsequent inactivation of coagulation V and VIII. After processing and removal of the N-terminal site a 70-100KDa protein is obtained. TM consist of 5 domains: D1. CTLD, EGF-like D2, extracellular serine/threonine D3, transmembrane D4 and cytoplasmic D5. TM forms a complex with thrombin. This complex is needed in the conversion of protein C into its activated form (APC). APC leads to degradation of coagulation factor Va and VIIIa leading to continuation of its downstream pathway and consumption of thrombin. The binding to thrombin is 1:1 and the complex accelerated the activation of protein C significantly. When the coagulation cascade is activated, prothrombin is converted to thrombin by coagulation factors Va and VIIIa, ultimately leading to fibrin clot formation.
Although its primary function is to mediate endothelial thromboresistance, soluble variants comprising the extracellular domains are found in eg. serum and urine. Soluble TM (sTM) is considered as a marker of endothelial damage and retains its anticoagulant characteristics. Increased levels of sTM have been associated with inflammatory diseases related to damage endothelium like atherosclerosis and lung injury. STM has also been linked to inflammatory conditions. TM/thrombin complex can suppress cytokine production via APC. TM is also effective in the complement system by accelerating factor I inactivation of C3b. Mutations in TM can lead to overactive complement possibly leading to atypical hemolytic uremic syndrome.
Thrombomodulin assay HK383 is a 4.5h ELISA- assay making use of two monoclonal antibodies and uses a recombinant protein as standard. Parallelism between this standard and native human thrombomodulin has been shown, validating the use of this assay to reliably quantify human TM is samples.
The Human soluble Thrombomodulin (sThrombomodulin) ELISA kit is to be used for the in vitro quantitative determination of sThrombomodulin in serum and plasma samples.
The Human sThrombomodulin ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours. The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay. Samples and standards are incubated in microtiter wells coated with antibodies recognizing Human sThrombomodulin. Biotinylated tracer antibody will bind to the captured Human sThrombomodulin. Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody. Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB). The enzyme reaction is stopped by the addition of oxalic acid. The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the Human sThrombomodulin standards (log). The Human sThrombomodulin concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Normal human blood samples (plasma) containing baseline levels of human sThrombomodulin, were mixed with human blood samples (plasma) with high values of sThrombomodulin. Samples were incubated for 30 minutes at room temperature and measured using the ELISA. Values for human sThrombomodulin, ranged between 89% and 100%.