The effect of bovine thrombomodulin on the specificity of bovine thrombin.
106
Citation
27
Reference
10
Related Paper
Citation Trend
Abstract:
Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.Keywords:
Thrombomodulin
Hirudin
Thrombomodulin (TM) is a cell surface anticoagulant glycoprotein that plays a key role in the protein C pathway. TM expression in endothelial cells may be modulated by a variety of extracellular signals. Most notably, TM has been shown to be downregulated by inflammatory mediators, such as tumor necrosis factor-α and lipopolysaccharide. The objective of this study was to determine the effect of thrombin on TM expression and activity. Thrombin resulted in reduced TM in primary cultures of human endothelial cells by approximately 40% at the level of mRNA, protein, and activity. These effects were blocked by the thrombin inhibitor hirudin. These results suggest that activation of the coagulation cascade may result in a positive-feedback loop consisting of thrombin-mediated repression of TM-dependent protein C activation.
Thrombomodulin
Thrombin Generation
Primary (astronomy)
Cite
Citations (10)
Thrombomodulin
Hirudin
Dissociation constant
Prothrombinase
Cite
Citations (33)
In the present in vivo study the stabilities of the thrombin-hirudin and thrombin-antithrombin III complexes were investigated. After incubation of the complexes with free inhibitors the thrombin-antithrombin III levels were determined by ELISA. The thrombin-hirudin complex proved to be stable in the presence of antithrombin III or heparin. However, in the presence of heparin and plasma equivalent concentrations of antithrombin III, the thrombin-hirudin comples dissociated and was displaced. In contrast, both thrombin-antithrombin III and thrombin-antithrombin-III/heparin complexes are very stable even in the presence of a large excess of hirudin.
Hirudin
Cite
Citations (8)
Thrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.
Thrombomodulin
Thrombin Generation
Cite
Citations (21)
Conversion of human α‐thrombin to γ‐thrombin by limited proteolysis resulted in a decrease in the inactivation rate of the enzyme by antithrombin III. The second‐order rate constants were similar but significantly different: 11 ± 1.7 × 10 3 and 7 ± 0.5 × 10 3 M −1 s −1 for α‐ and γ‐thrombin respectively. This difference is probably related to a slight change in reactivity of the catalytic site, rather than to a structural alteration of the recognition site for antithrombin III. The rate of protein C activation, measured in the absence of thrombomodulin, was greatly reduced by conversion of α‐thrombin to γ‐thrombin. In addition, γ‐thrombin failed to displace α‐thrombin from its complex with thrombomodulin, as demonstrated by measuring either the rate of protein C activation by thrombin‐thrombomodulin, or the fibrinogen clotting activity of thrombin‐thrombomodulin, in the presence of competing diisopropylphospho‐thrombin. It is concluded that the recognition sites involved in protein‐C–thrombin and thrombomodulin–thrombin interactions are both dramatically affected by the loss of peptide material occurring during the conversion of α‐thrombin to γ‐thrombin and/or by the resulting conformational changes.
Thrombomodulin
Cite
Citations (85)
Thrombomodulin
Zymogen
Cleavage (geology)
Cite
Citations (49)
Summary Thrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.
Thrombomodulin
Thrombin Generation
Cite
Citations (16)
Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.
Thrombomodulin
Thrombin Generation
Cite
Citations (57)
Thrombomodulin
Cite
Citations (0)