Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin. Jci human thrombomodulin is not an efficient inhibitor. Structure and expression of human thrombomodulin, a thrombin. One of these peptides, hirugen, is derived from the cterminal fragment of hirudin. Thrombin, thrombomodulin, and extracellular signalregulated. Gene structure of human thrombomodulin, a cofactor for. Pc activation by the thrombintm complex is further enhanced 20fold in vivo when pc is bound to the endo thelial cell protein c receptor epcr. The activation of protein c is greatly enhanced following the binding of thrombin to thrombomodulin, an integral membrane protein expressed by endothelial cells. Activated protein c apc and protein s form a complex on the plasma membrane of endothelial cells and possibly also on other cells. Exploring traditional and nontraditional roles for thrombomodulin. This tmthrombin binding increases the activation of protein c by 100fold and blocks the binding of thrombin to cellular receptors on platelets and inflammatory cells box 88. C by thrombin bound to the cofactor thrombomodulin on the surface of endothelial cells 1. The thrombin bound cyclic peptide assumes an overall conformation similar to those observed in the free but intact egf molecules.
The endothelial cell surface receptor thrombomodulin is a 450,000dalton protein whose only known ligand is thrombin. Surface bound thrombomodulin and heparin inhibit tissue factorinduced thrombin generation in a flow model. A critical step in this attenuation is the activation of protein c by the thrombinthrombomodulin tm complex. Localization of thrombomodulinbinding site within human. It has been demonstrated that the egflike domains of thrombomodulin essential for the activation of protein c. In contrast, the tmthrombin complex is a potent activator of pc that enhances. It is concluded that scupa bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on upamediated local fibrinolysis and extracellular. In response to thrombin, and especially thrombin bound to thrombomodulin, tafi is activated to tafia. Structure of thrombomodulin s egf domains in the thrombin bound and free states. Recombinant human soluble thrombomodulin art123, asahi. Aug 15, 2019 thrombomodulin plays a vital role in maintaining intravascular patency due to its anticoagulant, antiinflammatory, and cytoprotective properties. Thrombomodulin is a membrane glycoprotein expressed on endothelial cells1. Thrombomodulin is an endogenous anticoagulant protein that, in cooperation with protein c and thrombin activatable fibrinolysis inhibitor, serves to. However, human thrombin bound to pig tm was unable to ef.
Following thrombin cleavage apc hydrolyzes both factor va and viiia rendering them inactive. Thrombomodulin tm, cd141 or bdca3 is an integral membrane protein expressed on the surface of endothelial cells and serves as a cofactor for thrombin. Deregulated interplay between inflammation and coagulation plays a pivotal role in the pathogenesis of sepsis. The inhibited thrombin rapidly dissociates from thrombomodulin, regenerating the anticoagulant surface. Thrombomodulin binding selects the catalytically active form. Our servers in the cloud will handle the pdf creation for you once you have combined your files. Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein c and tafi. Laboratory issues in diagnosing abnormalities of protein c.
Plasma from patients undergoing liver transplantation is. Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Because the structure of thrombin is basically the same whether or not it is bound to the egf domains, the idea that thrombomodulin allosterically alters thrombin specificity 2 has been put to. Egflike domains are involved in the binding of thrombin and the cofactor activity 20. The ability to inhibit release of 3hserotonin from washed human platelets was at least 10 times less using human thrombomodulin compared with rabbit thrombomodulin. Once thrombin is bound to thrombomodulin, thrombins substrate specificity shifts from. Human thrombomodulin was also much less effective compared with rabbit thrombomodulin in inhibiting thrombin induced human coagulation factor v activation. The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein c zymogen, was isolated from a human genomic library by employing human thrombomodulin cdna as a probe. The nucleotide sequences of the gene and the adjacent 5 and 3 flanking regions were then determined.
In the structure of thrombin bound to natural heparin reported here arg 165 and his 230 are seen to make contacts with heparin in monomer ab. Tm is a glycosylated type i transmembrane molecule of 557 amino acids and does not possess intrinsic enzymatic activity. Pharmacokinetics of recombinant human soluble thrombomodulin. Pdf surface bound thrombomodulin and heparin inhibit tissue. However, under pathological conditions such as sepsis and systemic inflammation, endothelial thrombomodulin expression is downregulated and its function impaired.
This tm thrombin binding increases the activation of protein c by 100fold and blocks the binding of thrombin to cellular receptors on platelets and inflammatory cells box 88. Furthermore, thrombin bound to thrombomodulin is inactivated by plasma protease inhibitors 20 times faster than free thrombin, resulting in increased clearance of thrombin from the circulation. Activation rates oftheprotein cderivative lackingtheycarboxyglutamicaciddomain,whichis required for binding to epcr, are not altered by the antiepcr antibodies. Thrombomodulin blocks the ability of thrombin to activate platelets. The binding of thrombin to cellsurface thrombomodulin accelerates protein c activation. The ability of thrombin to specifically recognize a wide range of substrates derives from interactions that occur outside of the active site of thrombin. Recombinant human soluble thrombomodulin in patients with. We developed a recombinant thrombomodulin analog and compared the effects of recombinant thrombomodulin 100 pgea, saline controls. Thus, thrombomodulin plays a role as an anticoagulant on the endothelium of the vascular wall by converting thrombin from a procoagulant into an anticoagulant. Thrombin also initiates an anticoagulant pathway by binding to thrombomodulin and catalyzing protein c activation. The contact region on exosite i is depicted in red. Activated protein c, together with its cofactor, protein s ps, forms a complex on cell membrane surfaces and proteolytically degrades activated coagulation factors v va and viii viiia, thus inactivating them. The method comprises combining a blood plasma test sample and a coagulationactivating substance in a calciumfree solution, then adding soluble thrombomodulin. The structure of thrombin bound to hirugen was solved with the active site free vijayalakshmi et al.
Pdf surface bound thrombomodulin and heparin inhibit. When bound to intact tm, thrombin effectively invokes the onset of the protein c pathway, involving activation of the circulating proenzyme protein c. Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells david m. Tissue factor and thrombomodulin are cell membrane proteins that extend a 20 to 35aminoacid residue tails into the cell cytoplasm. The present study aimed at clarifying the role of thrombin in tm. Pdf effect of bovine thrombomodulin on thrombin specificity. It reduces blood coagulation by converting thrombin to an anticoagulant enzyme from a procoagulant enzyme. Furthermore, the activation of protein c by the thrombinthrombomodulin complex is enhanced 20fold when protein c is bound to the endothelial cell protein c receptor 8. The surface topology of the thrombin molecule indicates positions pi p3, differ from those of the canonical serineproteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to proteinc activation on thrombomodulin complex formation.
And when you want to do more, subscribe to acrobat pro dc. The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin jan hofsteenge and stuart r. This is a pdf file of an unedited manuscript that has. Thrombomodulin prolongs thrombininduced extracellular. To merge pdfs or just to add a page to a pdf you usually have to buy expensive software. Thrombomodulin is a receptor for thrombin that participates in feedback inhibition of thrombin generation.
Thrombomodulin binding selects the catalytically active. Thrombin bound to a fibrin clot confers angiogenic and. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smcs by prostaglandins, endogenously formed via the cox2 pathway. Thrombinactivatable fibrinolysis inhibitor tafi is cleaved to its activated form by the thrombinthrombomodulin complex.
Like membrane bound tm, art123 binds to thrombin to inactivate coagulation via activation 7of protein c. The turnover of thrombinthrombomodulin complex in cultured. Thrombin complexed with thrombomodulin cannot cleave fibrinogen or activate factor v or factor xiii but rather accelerates the conversion from protein c to apc, a potent anticoagulant by virtue of its capacity to inactivate clotting factors va and viiia. Us5525478a soluble thrombomodulinbased onestage assay. This subject is discussed in detail in the article by dr. Molecular basis of thrombomodulin activation of slow thrombin ncbi. Thrombin binding to tm modulates endothelial cell and smooth muscle cell proliferation mediated through par1. Structural basis for the anticoagulant activity of the. Thrombin bound to thrombomodulin loses all procoagulant activity and instead activates the protein c anticoagulant pathway.
The thrombin thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin activatable fibrinolysis inhibitor. Disseminated intravascular coagulation fibrinolysis obstetrics placenta pregnancy thrombin summary as activation of the coagulation pathway is a physiological response to injury, the development of disseminated intravascular coagulation dic is a warning signal to the clinician that the primary pathological disease state is decompensating. Thrombin inhibition by cyclic peptides from thrombomodulin. Thrombomodulin serves as a receptor for thrombin and protein c allowing an interaction between the two proteins. Copinion targeting activated protein c to treat hemophilia. However, human thrombin bound to pig tm was unable to. After generation from prothrombin, thrombin plays multiple roles in the blood coagulation cascade that are mediated by interaction with a number of physiologic substrates, effectors, and inhibitors.
Thesedata indicate that the protein cactivation complexinvolves protein c, thrombin, thrombomodulin,and epcr. Cascading into the thrombincascading into the thrombin. Thrombomodulin is present in the body in two forms2. When bound to thrombomodulin, thrombins procoagulant activities e. Thrombomodulin, a cell membrane bound glycoprotein lining the vascular endothelium, specifically binds thrombin so as to convert it to a form with decreased ability to catalyze clot formation but with a 1,000fold increased capacity to activate protein c.
Thrombin generated in the vicinity of intact endothelial cells binds to thrombomodulin and efficiently activates protein c. The uptake and degradation of thrombin bound to thrombomodulin is very similar to the receptormediated endocytosis of epidermal growth factor egf irl press limited, oxford, england carpenter and cohen, 1976, low density lipoprotein ldl goldstein et al. During severe inflammation, protein c levels are decreased from impaired synthesis and degradation by neutrophil elastase, and the system is defective due to downregulation of tm at the endothelial. Thrombin mediated activation of human tafi is significantly enhanced by the addition of thrombomodulin. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin tm, it acts as an anticoagulant, cleaving protein c. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release c serotonin. As a result, administering thrombomodulin represents a potential therapeutic modality. On the other hand, thrombin behaves as anticoagulant upon binding to thrombomodulin to favor activation of protein c. Clinical importance of thrombomodulin serum levels european. Thrombomodulin weiler 2003 journal of thrombosis and. Thrombomodulin is an endogenous anticoagulant protein that, in cooperation with protein c and thrombin activatable. Thrombomodulin in disseminated intravascular coagulation and. In addition, thrombin, particularly when it is bound to thrombomodulin, catalyzes the activation of the zymogen thrombinactivatable fibrinolysis inhibitor tafi to the antifibrinolytic, carboxypeptidase.
Functional domains of membrane bound human thrombomodulin the fifth and sixth growth factorlike domains of thrombomodulin bind to the anionbinding exo 1993. Under physiologic conditions, protein c is activated by thrombin bound to the ec membraneassociated thrombomodulin tm. Thrombomodulin plays a vital role in maintaining intravascular patency due to its anticoagulant, antiinflammatory, and cytoprotective properties. Thrombin feedback amplifies the system by activating not only factor v, but also factors viii and xi. Structure and expression of human thrombomodulin, a. Recombinant soluble htm shtm which has been recently approved for treatment of disseminated intravascular coagulation might be potentially available. Thrombomodulin tm is an integral component of a multimolecular system, localized primarily to the vascular endothelium, that integrates crucial biological processes and biochemical pathways, including those related to coagulation, innate immunity, inflammation, and cell proliferation. Used with permission the dynamics of hemostasis, haematologic technology, k. Then you can edit, export, and send pdfs for signatures. Thrombomodulin binds to thrombin to form a complex that inactivates the coagulant activity of thrombin and activates protein c. Therapeutic approaches that simultaneously target both inflammation and coagulation hold great promise for the treatment of sepsis.
Regulation of thrombomodulin expression in human vascular. We demonstrate here that thrombomodulin and thrombin, in the presence of serum, activate progelatinase a and that activated protein c alone can convert the. The efficient inactivation of cell bound scupa suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. The effect of thrombomodulin on the cleavage of fibrinogen. Thrombin iia procoagulant anticoagulant fibrinogen fv, fviii, fxi fxiii tafi fibrinolysis inhibitor platelet activation protein cs bound to thrombomodulin thrombin has both pro and anticoagulant functions. The discovery of thrombomodulin wiley online library. Thrombomodulin is required for the antithrombotic activity of. Human thrombomodulin is a 557 amino acid type i transmembrane protein, consisting of six epidermal growth factor egflike domains and is highly glycosylated. Abstracton endothelial cells, thrombin binds to thrombomodulin tm, an integral membrane bound glycoprotein, and to proteaseactivated receptors pars. Thrombomodulin tm is a monomeric, type1 transmembrane protein that serves as a cell surface receptor for thrombin. Thrombinthrombomodulin connects coagulation and fibrinolysis. Only with adobe acrobat reader you can view, sign, collect and track feedback, and share pdfs for free. Thrombomodulin tm, an endothelial membrane protein, and soluble tm, known as tm. Thrombomodulin is an important component in the anticoagulation system by functioning as a cell surface receptor for thrombin, and has also been shown to play a role in anti.
Thrombomodulin is an endothelial cell thrombin receptor that converts. Pig thrombomodulin binds human thrombin but is a poor. Thrombin bound to thrombomodulin activates protein c, an inhibitor of the coagulation cascade. Activated protein c inactivates factors va and viiia. Activated protein c degrades factors v and viii reducing the production of thrombin. The thrombin bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear overhauser effects transferred noes and by distance geometry and monte carlo calculations.
Thrombin is the final protease in the blood coagulation cascade and serves both pro and anticoagulant functions through the cleavage of several targets. Functional difference between membranebound and soluble. Interestingly, apc exhibits important cytoprotective functions, including. Thrombomodulin tm, a membranebound glycoprotein expressed on endothelial cells, has a high affinity of binding to thrombin and converts thrombin from a procoagulant to an anticoagulant. Thus, thrombin has the capacity to express both procoagulant and anticoagulant functions depending on the context under which it is generated. Thrombin cleaves protein c resulting in its activation apc. Directing thrombin blood american society of hematology. At sites of vascular disruption, the procoagulant effects of thrombin are fully expressed. Thrombin binds to thrombomodulin with high affinity kd lo m and the thrombinthrom bomodulin complex specifically activates a vitamin kde. Disseminated intravascular coagulation in obstetric. Tm binds to thrombin and changes the enzymes conformation, allowing thrombin to activate protein c figure. A minimal tm fragment consisting of the fourth, fifth, and most of the sixth egflike domain tm456m that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity.
Stone friedrichmiescherinstitut, basel received march 3may 18, 1987 ejb 87 0316 thrombomodulin acts as a linear competitive inhibitor of thrombin with respect to the substrate fibrinogen. The essential components of the pathway involve thrombin, thrombomodulin, the endothelial cell protein c receptor epcr, protein c, and protein s. Thrombin activatable fibrinolysis inhibitor tafi, plasma procarboxypeptidase b, carboxypeptidase u is a single chain glycoprotein zymogen mr60,000 synthesized in the liver and circulating at a plasma concentration of 50 nm 14. The present invention provides a novel onestage assay which uses soluble thrombomodulin for directly determining the functional status in plasma of the vitamin kdependent coagulationinhibiting proteins, protein c and protein s. Thrombomodulin binds thrombin, directly inhibiting its clotting and cell activation potential while at the same time augmenting protein c and thrombin activatable fibrinolysis inhibitor tafi activation. The concentration of tm bound to the cell surface is. Request pdf functional difference between membranebound and soluble human thrombomodulin. This event is accelerated 20fold by the binding of protein c to the same cell surface via an interaction between its gcarboxyglutamic acid gla domain adepartment of haematology, university of cambridge, cambridge. When bound to thrombomodulin on the surface of vascular.
Thrombomodulin prolongs thrombininduced extracellular signal. Abstracton endothelial cells, thrombin binds to thrombomodulin tm, an integral membranebound glycoprotein, and to proteaseactivated receptors pars. We studied the phosphorylation and nuclear translocation of extracellular signalregulated. When thrombin is bound to thrombomodulin it cannot activate platelets or cleave fibrinogen to make blood clots. It is then activated by thrombin bound to an adjacent membrane protein, thrombomodulin tm. Competition for thrombin exosites is nicely illustrated by recent crystal structures of thrombin complexes with fibrin, thrombomodulin, gpib. Structural and mutagenesis studies have helped unravel the molecular basis of thrombin interactions in the context of both wellestablished and emerging new roles of the enzyme. Thrombin takes part in many other reactions, including the establishment of inflammation, neoplastic transformation, angiogenesis, arteriosclerosis, and tissue repair.
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