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Recombinant Human Thrombomodulin
Description
Thrombomodulin (TM) is the cell surface receptor for thrombin. When occupied, thrombomodulin converts thrombin from a procoagulant protein into the activator of Protein C. After activating Protein C, thrombomodulin acts as a major anticoagulant through its ability to inactivate various blood factors (Va, VIIIa, Xa and XIIIa). In competing for thrombin binding, thrombomodulin inhibits the proteolytic effect of thrombin in its clotting of fibrinogen, the inactivation of Protein S and the induction of platelet aggregation.
TM is similar in structure to the low-density lipoprotein (LDL) receptor. TM possesses several EGF repeats, of which numbers five and six are responsible for the high affinity binding of thrombin (Kd = 0.5 nM). In addition, the B chain of thrombin possesses a domain, distinct from the active catalytic site, termed anion-binding Exosite I, which is involved in the binding of thrombin to thrombomodulin. TM contains a chondroitin sulfate (glycosoaminoglycan) which accelerates the inactivation of thrombin by anti-thrombin III. On SDS-polyacrylamide gels, human thrombomodulin appears as a single band at Mr 75,000 D under non-reducing conditions and shows a band at approximately Mr 110,000 D following reduction of its disulfide bonds.
The thrombomodulin-thrombin complex enhances the catalytic activation of Protein C over 1,000 fold3. The binding of thrombin to thrombomodulin does not require calcium; however, interaction of the complex with Protein C is calcium dependent. Platelets, monocytes and neutrophils contain small amounts of TM in comparison to cultured endothelial cells. Immunohistochemical analysis has localized TM to the luminal surface of endothelium of blood vessels and lymphatics, the squamous epithelium, and the placental syncytiotrophoblast.
REF 2374 is a recombinant carboxy-terminal truncated form of thrombomodulin and lacks the putative transmembrane and cytoplasmic domains, approximately 38 amino acids.
Preparation
REF 2374 has been expressed by a HEK293 cell line and purified by reverse phase, ion exchange and size-exclusion chromatography. Purity is determined via SDS-PAGE analysis. Under reducing conditions, the protein migrates as a single band at 68,000 Da.
Presentation
Screw-capped clear glass vial containing 10 Dg of recombinant human thrombomodulin lyophilized from a 100 Dg/mL solution in 50 mM Tris, 100 mM NaCl, pH 7.4, with 100 mM Mannitol added as an excipient. Protein content was determined by spectrophotometric analysis at a wavelength of 280 nm using an extinction coefficient of 0.88 for a 0.1% solution, 1 cm pathlength.
Reconstitution
Add 100 DL of filtered deionized or sterile water to generate a 100 Dg/mL solution.
