The interaction of thrombomodulin with Ca<sup>2+</sup>

Light, David R.; Glaser, Charles L.; Betts, Melissa; Blasko, Eric; Campbell, Elizabeth; Clarke, J H; McCaman, Michael T.; Mclean, Kirk; Nagashima, Mariko; Parkinson, John F.; Rumennik, Galina; Young, Tish; Morser, John

doi:10.1046/j.1432-1327.1999.00398.x

Cited by 30 publications

(23 citation statements)

References 39 publications

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“…The present studies indicate that the activation of both protein C and TAFI requires the fifth and sixth EGF domains of thrombomodulin, which contain structures needed for thrombin binding and high affinity Ca 2ϩ binding (25). Although these elements of structure are necessary for function, they are not sufficient to enhance activation of either protein C or TAFI.…”

Section: Discussionmentioning

confidence: 67%

“…Ca 2ϩ Dependence of Thrombomodulin-mediated Protein C and TAFI Activation-Others (25,26) have shown that protein C activation in the presence of thrombin and thrombomodulin without chondroitin sulfate exhibits a complex, biphasic dependence on the Ca 2ϩ concentration. As the Ca 2ϩ concentration is increased, the rate of protein C activation typically increases up to a maximum at about 0.25 mM Ca 2ϩ and then decreases toward a plateau at about 5.0 mM Ca 2ϩ .…”

Section: Fig 2 Effect Of Oxidation Of Met-388 Of Thrombomodulin On mentioning

confidence: 99%

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Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Wang¹,

Nagashima²,

Schneider³

et al. 2000

Journal of Biological Chemistry

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Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombinactivable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no loss of function in protein C activation for mutations within the c-loop of EGF3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reductions at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C. Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the rate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca 2؉ -dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mM Ca 2؉ . Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a 60-kDa plasma protein that circulates in plasma at concentration of about 75 nM (1). It is a zymogen that is activated to a carboxypeptidase B-like enzyme by a single thrombin-catalyzed cleavage at arginine 92 (2-4). The enzyme, designated TAFIa, catalyzes removal of carboxyl-terminal arginine and lysine residues in fibrin as it undergoes fibrinolysis (5). As a consequence, feedback up-regulation of plasminogen activation is eliminated, and the process of fibrinolysis is suppressed. The activation of TAFI by thrombin and subsequent actions of TAFIa define a molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses activity in the latter (6). Although thrombin at the high levels generated after the clotting of fibrin occurs can activate sufficient TAFI to suppress fibrinolysis, thrombin by itself is a relatively weak activator (2, 7). Thrombin bound to thrombomodulin, however, activates TAFI with a catalytic efficiency 1250-fold greater than that of thrombin alone (6). Thus, the thrombin-thrombomodulin complex is probably the physiologic ac...

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Section: Discussionmentioning

confidence: 67%

Section: Fig 2 Effect Of Oxidation Of Met-388 Of Thrombomodulin On mentioning

confidence: 99%

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Wang¹,

Nagashima²,

Schneider³

et al. 2000

Journal of Biological Chemistry

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“…Consisting of the extracellular domains of thrombomodulin, it is distinguished by several directed mutations, providing for lack of a chondroitin sulfate attachment site, resistance to exocarboxypeptidase/protease activity and to oxidation/irradiation, and, finally, abolishing the N-terminal heterogeneity arising in the wild-type sequence from 2 common signal cleavage sites. [15][16][17] Recently, we demonstrated that soluble thrombomodulin (Solulin) may be used to partially correct the premature lysis defect in fVIIIdeficient plasma through an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-dependent mechanism, 18 which supports the hypothesis that bleeding in hemophilia may be due to unregulated fibrinolysis 19 in addition to the well-documented clotting defect. 20 This hypothesis is also supported by a preliminary clinical study showing that ⑀-amino caproic acid, an antifibrinolytic lysine analog, may be used adjunctively with fVIII inhibitor bypass activity or activated prothrombin complex to control bleeding.…”

Section: Introductionmentioning

confidence: 80%

Solulin increases clot stability in whole blood from humans and dogs with hemophilia

Foley

Petersen

Rea

et al. 2012

Blood

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Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)-deficient plasma by an activated thrombinactivatable fibrinolysis inhibitor (TAFIa)-dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor-initiated fibrin formation and tissueplasminogen activator-induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombinSolulin complex were determined to be 1.53 and 0.02/M/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses. (Blood. 2012;119(15): 3622-3628) IntroductionPatients with hemophilia A have a bleeding diathesis that is usually predicted by their factor VIII (fVIII) activity level (fVIII:C). 1,2 The primary form of treatment for severe hemophilia A is replacement therapy, which involves administration of recombinant or plasmaderived fVIII. FVIII can be given either on demand or by prophylaxis, 3 and the amount needed can vary drastically depending on the treatment schedule and the type and severity of the bleed in the case of on-demand treatment. 4 The treatment developments to date have greatly improved both mortality and morbidity for individuals with hemophilia 5,6 ; however, current treatments are not 100% effective, are expensive, and are often considered inconvenient. Because single bleeding events can have devastating consequences, it is important to continue to strive for maximally effective treatments. The recent improvements in mortality and morbidity have only been observed in developed countries with the resources to fund treatment. It is currently estimated that 80% of the world's hemophilia population has little or no access to therapy 7 ; therefore, the development of costeffective alternate treatment strategies or effective factor-sparing regimes to treat bleeding is clearly necessary.Many new and adjunctive therapeutic options have been explored, including platelet infusion, 8 tranexamic acid, 9 ⑀-amino caproic acid, 10 molecules that block tissue factor pathway inhibitor, 11,12 and a combination of phospholipid and fXa 13 and fXIII. 14 Solulin is a recom...

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“…Indeed, mouse models of TM gene mutation (TMpro/pro mouse) and endothelial cell-specific gene deletion (TMLox Ϫ mouse) both exhibit greatly reduced ability to generate activated protein C (APC) within the circulation, leading to thrombosis and hypercoagulable state (66,127). Thrombin binds to the TM EGF5-6 repeat domain and also, with lower affinity, to the TM CS moiety within the Ser/Thr-rich domain via the thrombin anion binding exosite I and II regions, respectively (86,189). This has the effect of blocking the interaction of thrombin with circulating procoagulant substrates (e.g., fibrinogen) and enhancing its specificity for PC, leading to a substantially elevated rate of PC activation (over 1,000-fold relative to unbound thrombin) (3).…”

Section: Thrombomodulin Functionsmentioning

confidence: 99%

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

Martin

Murphy

Cummins

2013

American Journal of Physiology-Heart and Circulatory Physiology

180

163

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The interaction of thrombomodulin with Ca²⁺

Cited by 30 publications

References 39 publications

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Solulin increases clot stability in whole blood from humans and dogs with hemophilia

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

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The interaction of thrombomodulin with Ca2+

Cited by 30 publications

References 39 publications

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Elements of the Primary Structure of Thrombomodulin Required for Efficient Thrombin-activable Fibrinolysis Inhibitor Activation

Solulin increases clot stability in whole blood from humans and dogs with hemophilia

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

Contact Info

Product

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About

The interaction of thrombomodulin with Ca²⁺