Thus, while pig tm may be capable of binding thrombin generated in the vicinity. Expression of allosteric linkage between the sodium ion binding site. Protein c activation occurs when the serine protease thrombin binds to the cell surface proteoglycan thrombomodulin tm forming a complex that halts coagulation. In addition, the lectinlike domain is thought to bind to an unidentified receptor, thereby suppressing map kinase and nf b activation. Endothelium also produces protein s, a cofactor for protein c, and tissue factor pathway inhibitor tfpi, a cell surface protein that directly inhibits. The pathway is initiated when thrombin binds to the endothelial cell thrombin binding protein, thrombomodulin. Abstracton endothelial cells, thrombin binds to thrombomodulin tm, an integral membranebound glycoprotein, and to proteaseactivated receptors pars. Laboratory issues in diagnosing abnormalities of protein c. Jci inhibition by human thrombomodulin of factor xa.
Complex formation between thrombin and thrombomodulin inhibits. It is a cofactor for thrombin binding that mediates protein c activation and inhibits thrombin. Thrombomodulin bound thrombin can no longer generate blood clots. Thrombin is responsible for local fibrin formation, and upon binding to thrombomodulin for activating protein c which serves to limit clot propagation in the region of intact and healthy endothelium. Originally, thrombomodulin tm was identified as an antico agulant factor that. Thrombin, tm, and protein c bind simultaneously, thereby forming a complex on the surface of the endothelium that expresses endothelial cell. Ribbon and surface electrostatic representations of thrombin bound to heparin. Thrombomodulin allosterically modulates the activity of. Lectinlike domain of thrombomodulin binds to its specific ligand lewis y antigen and neutralizes lipopolysaccharideinduced inflammatory response running title. In addition to its role in activation of fibrin clot formation, thrombin plays an important regulatory role in coagulation. 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. Thrombomodulin binds to thrombin at an anionbinding exosite on the carboxylterminal side of the substrate binding cleft. Thrombomodulin allosterically modulates the activity of the.
Leftover thrombin \from a triggered clot formation event\ travels to healthy endothelium, binds to thrombomodulin and activates protein c \see next slide\ thrombin on an endothelial surface has anticoagulant activity. Thrombin and trypsin activate proteaseactivated receptors pars that modulate vascular tone. Thrombin bound to thrombomodulin becomes inactive and can no longer activate procoagulant factors or platelets. Smcs were cultured in media with thrombin at 1 uml alone solid bar, thrombin at 1 uml combined with rtm at 1. Thrombin binds to thrombomodulin with high affinity kd lo m and the thrombinthrom bomodulin complex specifically activates a vitamin kde pendent zymogen, protein c, by limited proteolysis 4, 5. The effect of thrombomodulin on the cleavage of fibrinogen. Tm is an endothelial transmembrane glycoprotein that plays a critical role in the control of coagulation. Thrombomodulin tm, a widely expressing glycoprotein and originally identified in vascular endothelium, is an important cofactor in the protein c anticoagulant system. Exosite 1 of thrombin consists of a cluster of basic residues arg35, lys36, arg67, lys70, arg73, arg75, and arg77 in chymotrypsinogen numbering that play key roles in the function of thrombin.
In contrast, in an intact vascular system, thrombin has anticoagulant function since it binds to thrombomodulin and activates protein c. Thrombomodulin tm, cd141 or bdca3 is an integral membrane protein expressed on the surface of endothelial cells and serves as a cofactor for thrombin. Moreover, thrombin binds thrombomodulin on the surface of endothelial cells and activates protein c, which then exerts its anticoagulant activity by digesting factor va and depriving the prothrombinase complex of its most important cofactor. Thrombomodulin prolongs thrombininduced extracellular signal. Thrombin binds to thrombomodulin with high affinity kd lo m and the thrombin throm bomodulin complex specifically activates a vitamin kde. Thrombin combines with thrombomodulin present on endothelial cell surfaces forming a complex that converts protein c to protein ca. We show that both ang1 and ang2 inhibit tm function in cultured endothelial cells, possibly by interfering with thrombintm binding. Thrombomodulin binds to thrombin, thereby blocking activation of proteaseactivated receptors. The exact molecular reason for the 20fold lower potency against thrombin. A minimal tm fragment consisting of the 4 th, 5 th, and most of the 6 th egflike domain tm456m has been prepared that has much improved solubility. Thrombomodulin tm is a cell surface glycoprotein which is widely expressed in a variety of cell types. 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. Binding studies using lz5ilabeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. Thrombomodulin, cd141 or bdca3 is an integral membrane protein expressed on the surface of endothelial cells and serves as a cofactor for thrombin.
Localization of thrombomodulinbinding site within human. Our current concepts of the mechanisms of the protein c anticoagulant pathwayare that thepathwayis triggeredwhen thrombin binds to the endothelial cell receptor, thrombo. Egf4 protrudes away from egf5 and the thrombin surface 3 and is essential for the binding and activation of protein c by the tmthrombin complex. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombinmediated activation of protein c, an. Activated protein c proteolytically inactivates coagulation cofactor proteins factors va and viiia 6, 7. Thrombomodulin tm is central to the normal function of all organs, as it is expressed by endothelial cells that line all vessels arteries, veins, capillaries, and lymphatics. Thrombomodulin prolongs thrombininduced extracellular. Smc proliferation was quantified with mtt mitochondrial function assay on. Recombinant thrombomodulin inhibits arterial smooth muscle. Thrombomodulin binds to thrombin and converts it from a procoagulant into an anticoagulant via its ability to activate protein c, which inhibits clotting by inactivating factors vaand viiia. Structural basis for the anticoagulant activity of the. The substitution gln387pro in thrombomodulin inhibits protein c activation and interferes with this feedback inhibition.
Jan 11, 2018 thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin mediated activation of protein c, an. Used with permission the dynamics of hemostasis, haematologic technology, k. Thrombin on an endothelial surface has anticoagulant activity \1\ procoagulant activity generates thrombin \coagulant\ \2\ thombin binds to thrombomodulin on a healthy cell \anticoagulant\ and activates protein c \apc\ \3\ apc binds to protein s. Nov 03, 2015 although many thrombin substrates do not require binding at an allosteric site, it is fascinating to surmise that fibrinogen, which binds to both abe1 and the active site, can conformationally select the active form of thrombin since its interaction with abe1 would stabilize the nterminusinconformation and its interaction with the active. There are several studies that have shown that thrombomodulin indicates endothelial injury and correlates with dic, multiple organ failure, and mortality 1, 24, 28. Thrombomodulinactivated protein c system in septic. Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by factor xa in the presence of phospholipid or platelets, calcium, and factor va. The yshaped thrombomodulin fragment binds to thrombins anionbinding exositei, preventing binding of procoagulant substrates. Schreiber md, in consultative hemostasis and thrombosis third edition, 20. Pdf angiopoietins bind thrombomodulin and inhibit its. 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. The lectinlike domain binds to, and enhances degradation of, proinflammatory molecule hmgb1. Thrombin bound to thrombomodulin activates protein c, an inhibitor of the coagulation cascade.
Thrombin also initiates an anticoagulant pathway by binding to thrombomodulin and catalyzing protein c activation. Pig thrombomodulin binds human thrombin but is a poor cofactor for. Thrombomodulin, plasminogen activator inhibitor1 and. Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein c and tafi j. The endothelial cell surface membrane protein thrombomodulin binds thrombin with high affinity and acts as both a cofactor for protein c activation and an inhibitor of fibrinogen hydrolysis. Review open access the role of thrombomodulin lectinlike. Thrombomodulin is an endothelial cell surface protein and an essential negative regulator of coagulation through its function as a cofactor in the thrombinmediated generation of activated protein c apc 47 51. Thrombin has also been implicated as a mitogen for smooth muscle cell smc proliferation that contributes to arterial restenosis. In addition to its anticoagulant activity, recent evidence has revealed that tm, especially its lectinlike domain, has potent antiinflammatory function through a variety of molecular.
In culture, rtm blocks thrombin s activation of proteaseactivated receptors pars, that mediate thrombin killing of neurons and glial reactivity. Activated protein c inactivates factors va and viiia. It is a cofactor for thrombin binding that mediates protein c activation and inhibits thrombin activity. It reduces blood coagulation by converting thrombin to an anticoagulant enzyme from a procoagulant enzyme. A, the asymmetric unit consists of four thrombin monomers organized into two nearly equivalent dimers.
These observations open new questions about the control ofcoagulation reactions onvascularendothelium. Once thrombin binds with endothelial thrombomodulin, protein c is activated and stm is released into the bloodstream. Instead, it cleaves and activates protein c which shuts down further thrombin generation. Once thrombin binds to thrombomodulin on the cell surface, the resulting complex is considered to be transferred into the cell where thrombin is then degraded, and the internalized thrombomodulin recycles onto the surface of the membrane maruyama and majerus, 1985. 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. Thrombin binds to the membrane receptor thrombomodulin and activates protein c to activated protein c apc. Because of this negative feedback pathway, prothrombinthrombin behaves as indirect anticoagulant. Thrombomodulin is also expressed on human mesothelial cell, monocyte and a dendritic cell subset. Mar 30, 2000 the yshaped thrombomodulin fragment binds to thrombins anionbinding exositei, preventing binding of procoagulant substrates. In culture, rtm blocks thrombins activation of proteaseactivated receptors pars, that mediate thrombin killing of neurons and glial reactivity. In addition to the pars, thrombin also binds to thrombomodulin via exosite 1, a domain also involved in the interaction of thrombin with par1 but not par2. Exploring traditional and nontraditional roles for. Results aid in the classification of mesothelioma 1.
Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein c and tafi article in american journal of transplantation 86. The yshaped thrombomodulin fragment binds to thrombins anionbinding exositei. Tm is also synthesized by several cell types, in mice and humans, including. May 30, 2008 pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein c and tafi j. In addition, the thrombintm complex activates protein c to produce apc, which. The complex exhibits dramatically altered macromolecular specificity. Thrombin takes part in many other reactions, including the establishment of inflammation, neoplastic transformation, angiogenesis, arteriosclerosis, and tissue repair. Tmmediated binding of thrombin is known to enhance the specificity of the latter serine protease towards both protein c and thrombin activatable fibrinolysis. Oct 14, 2003 exosite 1 of thrombin consists of a cluster of basic residues arg35, lys36, arg67, lys70, arg73, arg75, and arg77 in chymotrypsinogen numbering that play key roles in the function of thrombin. In addition, the thrombin tm complex activates protein c to produce apc, which inactivates factors. Thrombin, thrombomodulin, and extracellular signalregulated. Cascading into the thrombincascading into the thrombin. Activated protein c apc can cleave the phospholipidmembranebound cofactors factors va and viiia, which results in.
Thrombomodulin is an endothelial cell thrombin receptor that converts. Thrombomodulin binds to thrombin at an anionbinding exosite on the carboxyl terminal side of the substrate binding cleft. Thrombomodulin as a model of molecular mechanisms that. On the other hand, thrombin behaves as anticoagulant upon binding to thrombomodulin to favor activation of protein c. The role of thrombomodulin lectinlike domain in inflammation yiheng li1,2, chenghsiang kuo2,3, gueyyueh shi2,3 and hualin wu2,3,4 abstract thrombomodulin tm is a cell surface glycoprotein which is widely expressed in a variety of cell types. Thrombin generated in high concentrations at the sites of vascular injury plays a central role in thrombosis and hemostasis. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombinmediated activation of protein c, an anticoagulant protein, as well as thrombinactivatable fibrinolysis inhibitor tafi. It has been suggested that molecular incompatibilities in the coagulation cascade, such as failure of pig thrombomodulin tm to bind primate thrombin, may contribute to this process. Structural data suggest that the side chain of arg35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin tm. Structurefunction relationships of the thrombin thrombomodulin. Angiopoietins bind thrombomodulin and inhibit its function as. The yshaped thrombomodulin fragment binds to thrombin s anionbinding exositei, preventing binding of procoagulant substrates.
Thrombomodulin lectin domain neutralizes lps chungsheng shi,1,2 gueyyueh shi, 1,2 shiming hsiao,1,2 yuanchung kao,1,2 kuanlin. Thrombin binds to thrombomodulin with high affinity kd lo m and the thrombinthrom bomodulin complex specifically activates a vitamin kde. 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. In addition, the lectinlike domain is thought to bind to an unidentified receptor. Tissue factor and thrombomodulin are cell membrane proteins that extend a 20 to 35aminoacid residue tails into the cell cytoplasm. Thrombomodulinbound thrombin can no longer generate blood clots.
Structure and expression of human thrombomodulin, a. The activation of protein c is greatly enhanced following the binding of thrombin to thrombomodulin, an integral membrane protein expressed by endothelial cells. Tm binds to thrombin and changes the enzymes conformation, allowing thrombin to activate protein c figure. Neuroprotective effects of recombinant thrombomodulin in. Restenosis after angioplasty or bypass grafting to restore circulation to ischemic organs is still an unsolved problem. Request pdf pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein c and tafi incompatibility between pig thrombomodulin tm and primate thrombin is. Thrombomodulin definition of thrombomodulin by medical. Tm expressed on vascular endothelium binds to thrombin, forming a 1.
Lectinlike domain of thrombomodulin binds to its specific. Pig thrombomodulin binds human thrombin but is a poor cofactor. Thrombin binding to tm modulates endothelial cell and smooth muscle cell proliferation mediated through par1. Exploring traditional and nontraditional roles for thrombomodulin. The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin jan hofsteenge and stuart r. Thrombomodulin weiler 2003 journal of thrombosis and. Response by blocking the functions of cd14 lipopolysaccharide. Heparin was modeled into positive difference density contoured at 2. Pig thrombomodulin binds human thrombin but is a poor. The normal distribution of tm includes the lining of blood and lymphatic vessels, mesothelial cells and some macrophages of the lung, meningeal lining cells, synovial cells, syncytiotrophoblasts, megakaryocytes and platelets. Structure and expression of human thrombomodulin, a thrombin. Its nterminal extracellular region comprises several.
We studied the phosphorylation and nuclear translocation of extracellular signalregulated kinases erks 1 and 2 in human. Thrombin is a naturally occurring enzyme that converts fibrinogen into fibrin, which is an integral step in clot formation. Pdf macromolecular specificity determinants on thrombin for. Angiopoietins bind thrombomodulin and inhibit its function. Aug 25, 2004 protein c activation occurs when the serine protease thrombin binds to the cell surface proteoglycan thrombomodulin tm forming a complex that halts coagulation. This process inactivates the procoagulant function of thrombin. Thrombomodulin can stimulate endothe lial cell growth. The purpose of this study was to determine whether thrombomodulin would alter thrombininduced vasoconstriction, thought to be. Vascular contraction and relaxation to thrombin and trypsin. The protein c anticoagulant system generates an on demand physiologic anticoagulant response. Inhibition by human thrombomodulin of factor xamediated cleavage. Thrombomodulin tm is an endothelial cell transmembrane glycoprotein. Vascular contraction and relaxation to thrombin and.
Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin mediated activation of protein c, an anticoagulant protein, as well as thrombin activatable fibrinolysis inhibitor tafi. The glycoprotein thrombomodulin binds to exosite 1 of thrombin via its peptide chain while simultaneously binding to exosite 2 using its chondroitin sulfate chain. Thrombomodulin blocks the ability of thrombin to activate platelets. The thrombomodulin tmactivated protein c apc system plays an important role in maintaining the homeostasis of thrombosis and hemostasis and maintaining vascular integrity in vivo. Thrombin from bovine plasma t75 product description. We presently have demonstrated that recombinant nterminal lectin. Tm is a glycosylated type i transmembrane molecule of 557 amino acids and does not possess intrinsic enzymatic activity. Pdf macromolecular specificity determinants on thrombin. Heparin modelingone heparin molecule was built for each of the two thrombin dimers of the asymmetric unit. Tm appears to exhibit antiinflammatory ability through both protein c dependent and independent pathways.
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