Recognition Sites for Thrombomodulin, Procoagulant and Anticoagulant Proteins around the Active Center of Thrombin
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Thrombomodulin
Summary Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.
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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.
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Vascular endothelial cells have several mechanisms which play an active role in preventing blood clot formation in vivo. One of the mechanisms by which prevention is achieved involves a cell surface thrombin receptor, thrombomodulin, which converts thrombin from a procoagulant into an anticoagulant due to accelerating thrombin-catalyzed activation of an anticoagulant protease zymogen, protein C. Activated protein C then proteolytically inactivates coagulation cofactors, Factors Va and VIIIa, in concert with another anticoagulant protein S. Activated protein C is finally neutralized by protein C inhibitor. The physiological relevance of the anticoagulant protein C-thrombomodulin pathway is demonstrated by the identification of hereditary deficiency of protein C or protein S with severe thrombotic complications. The recombinant protein C or thrombomodulin would be useful for treatment of the thrombotic diseases.
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Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.
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Background : The concept of immunothrombosis has established as a central pathogenic factor leading to thrombosis complications, respiratory failure, and a multiple organ failure in patents with COVID-19. Studying of the hypercoagulability in patients with COVID-19 could be useful for improving of disease's outcomes. Estimation of anticoagulants efficiency can be informative for thrombotic risk assessment. The highest interest presents protein C system, because, as we know, infection and inflammation lead to disfunction of this anticoagulant system. Aims : To estimate the efficiency of protein C anticoagulant system in COVID-19 patients. Methods : The study included 60 patients with COVID-19 and 21 healthy controls . Thrombin generation was assessed by CAT according to Hemker et al. Measure was conducted in platelet poor plasma with or without presence of thrombomodulin. The following parameters were determined: endogenous thrombin potential (ETP, nM∗min), peak thrombin (Peak, nM). Sensitivity ETP and Peak for thrombomodulin were calculated as percent of decreasing of these parameters after adding thrombomodulin to the assay. Reduction of sensitivity for thrombomodulin indicates dysfunction of anticoagulant protein C system. Protein C activity and free protein S level were measured by 'ACL ELIT PRO', Instrumentation Laboratory, USA. STATISTICA 12.0 package was used. Results are presented as median with 95% confidence intervals, P < 0.05 was considered statistically significant (∗). Results : Sensitivity ETP and Peak for thrombomodulin, protein C activity and free protein S level are presented in the table. Conclusions : The efficiency of protein C anticoagulant system is depressed in COVID-19 patients. It may be associate with protein S level decreasing. The disability of anticoagulant protein C system can lead to hypercoagulability and it may be cause of thrombotic complication.
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Thrombomodulin and tissue-factor activities were measured on the surface of confluent human saphenous-vein endothelial cells (HSVEC) cultivated in 96-multiwell plates. Thrombomodulin activity was measured in the presence of purified human thrombin (2.2 nM) and protein C (65 nM). Tissue-factor activity was measured with purified human Factor VII (5 nM) and Factor X (400 nM). Generated activated protein C and Factor Xa released in the supernatant were assayed with chromogenic substrates. Resting cells exhibited significant thrombomodulin activity, but no detectable tissue-factor activity. After 4 h of preincubation with tumour necrosis factor (TNF, 22-2200 pM), interleukin-1 (IL-1, 5.7-570 nM) or phorbol myristate acetate (PMA, 1.61-161 nM) there was an increase in tissue-factor activity and a concomitant decrease in thrombomodulin activity. However, the extent of both responses varied according to the nature of the stimulus. Thrombin (0.44-44 nM) also induced an increase in tissue-factor activity, but had no effect on thrombomodulin activity. Kinetic studies showed that for all stimuli the increase in tissue factor was transient, reaching a maximum after 4-8 h of preincubation with the stimulating agent and returning to normal values after 24 h. IL-1 and TNF induced a time-dependent decrease in thrombomodulin, by respectively 47% and 67% of control values after 24 h. However, PMA induced only a transient down-regulation of thrombomodulin, full activity being recovered after 18 h. Hence this simultaneous assay system, using intact HSVEC and purified human coagulation factors, enabled us to observe that the regulation of thrombin generation could be diversely affected by various substances known to stimulate the endothelium. This suggests that the simultaneous and opposite modulation of these proteins does not represent an unified response of the endothelial cells to procoagulant stimuli. These results also confirm the absence of effect of thrombin on the expression of thrombomodulin on the cell surface.
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The protein C anticoagulant system is a well established pathway regulating thrombin generation and therefore clot formation [reviewed in]. When thrombin binds to the endothelial cell transmembrane protein, thrombomodulin (TM), its potent procoagulant functions are reversed and its substrate specificity is redirected towards protein C. Protein C (the zymogen of APC) is then activated on the surface of endothelial cells by the thrombin/TM complex. Regulation is achieved via the degradation of procoagulant activated factors V (Va) and VIII (VIIIa) by activated protein C (APC), a serine proteinase, in conjunction with its non-enzymatic cofactor, protein S. Factor V also participates in factor VIIIa degradation in synergy with protein S. Protein C and protein S deficiencies are well recognized risk factors for venous thrombosis.
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