Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
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PMID:Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin. 131 33

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.
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PMID:[Endothelial cells and vascular hemostasis]. 131 12

Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.
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PMID:Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli. 131 19

Thrombomodulin is an endothelial surface thrombin receptor. Thrombin bound to thrombomodulin loses all procoagulant activity and instead activates the protein C anticoagulant pathway. We developed a recombinant thrombomodulin analog and compared the effects of recombinant thrombomodulin (100 micrograms/ea), saline (controls), recombinant hirudin (1.0 mg/kg), and heparin (100 units/kg) on thrombus formation, activated partial thromboplastin time, and tail transection bleeding time in a rat model of stasis-induced venous thrombosis. Results showed that thrombus was detected in the vena cava in six of the six rats treated with saline solution, in zero of the six rats treated with recombinant thrombomodulin (p less than 0.05), in one of six rats treated with recombinant hirudin (p less than 0.05), and in zero of six rats treated with heparin (p less than 0.05). The activated partial thromboplastin time in rats receiving recombinant thrombomodulin was slightly longer than controls (22 +/- 8 vs 37 +/- 6, p less than 0.05). The bleeding times in rats receiving recombinant thrombomodulin were approximately twice as long as controls (215 +/- 68 vs 545 +/- 173, p = 0.037). In all rats treated with recombinant hirudin or heparin, activated partial thromboplastin times were greater than 120 seconds and bleeding times were greater than 1200 seconds. We conclude that recombinant thrombomodulin inhibits venous thrombosis in a rat model with less prolongation of activated partial thromboplastin time and bleeding time than heparin or hirudin.
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PMID:Intravenous recombinant soluble human thrombomodulin prevents venous thrombosis in a rat model. 165 3

Thrombomodulin is a thrombin endothelial cell membrane receptor. The thrombomodulin-thrombin complex rapidly activates protein C resulting in anticoagulant activity. We investigated the anticoagulant effects and pharmacokinetic behavior of detergent-solubilized purified rabbit thrombomodulin labeled with iodine 125 when intravenously injected into rabbits. Thrombomodulin half-life (t1/2) was determined by tracking the 125I-radiolabeled protein and the biologic activity as determined by the prolongation of the activated partial thromboplastin time (APTT) and thrombin clotting time (TCT). When 200 micrograms/kg 125I-thrombomodulin was injected into rabbits, the APTT and TCT were immediately prolonged, whereas no effect on the prothrombin time was seen. In vitro calibration curves enabled us to convert the prolongations of the clotting times into micrograms per milliliter thrombomodulin equivalents. The best fit (r greater than 0.99) for the disappearance curves was provided by a two-compartment model with mean t1/2 alpha (distribution phase) of 18 minutes for 125I, 12 minutes for APTT, and 20 minutes for TCT, and mean t1/2 beta (elimination phase) of 385 minutes for 125I, 460 for APTT, and 179 for TCT. The administration of two doses of endotoxin (50 micrograms/kg) 24 hours apart did not accelerate the turnover rate of 125I-thrombomodulin as measured by the disappearance of 125I from the circulation. Thus, detergent-solubilized purified thrombomodulin administered intravenously circulates in a biologically active form for appreciable time periods.
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PMID:In vivo behavior of detergent-solubilized purified rabbit thrombomodulin on intravenous injection into rabbits. 215 45

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.
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PMID:The activation of bovine protein C by factor Xa. 255 Apr 35

Thirty consecutive children scheduled for pediatric cardiac operation with cardiopulmonary bypass were included in the study. Before the operation, the patients were randomly divided into two groups: with aprotinin (n = 15, 30,000 U/kg after induction of anesthesia, 30,000 U/kg added to the prime of the cardiopulmonary bypass or without aprotinin (n = 15). Thrombomodulin, (free) protein S, protein C, and thrombin/antithrombin III complex were measured from arterial blood samples taken after induction of anesthesia (at baseline, before aprotinin) and before, during, and after cardiopulmonary bypass until the first postoperative day. Standard coagulation parameters (antithrombin III, fibrinogen, platelet count, and partial thromboplastin time) were without differences between the groups. Thrombomodulin plasma concentrations were within normal range ( < 40 micrograms/L) and were similar in both groups at baseline. During cardiopulmonary bypass and until 5 hours after cardiopulmonary bypass, however, thrombomodulin plasma levels were significantly lower in the children treated with aprotinin. No further differences were observed on the first postoperative day. Protein C and protein S plasma levels did not differ between the two groups. Thrombin/antithrombin III-complex plasma concentrations increased significantly during cardiopulmonary bypass, however, without showing differences between children with (225 +/- 49 micrograms/L) and without (149 +/- 31 micrograms/L) aprotinin treatment. Blood loss and the need for homologous blood and blood products did not differ significantly between the two groups. We concluded that administration of aprotinin resulted in reduced thrombomodulin plasma levels in pediatric patients undergoing cardiac operation without altering protein C/protein S plasma concentration. The exact role of aprotinin in endothelium-derived coagulation should be further studied.
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PMID:Influence of aprotinin on the thrombomodulin/protein C system in pediatric cardiac operations. 751 76

Aprotinin has been reported to reduce bleeding in cardiac surgery patients. Its mechanisms of action on coagulation have not been fully elucidated. In a prospectively randomized study of 40 patients undergoing elective aortocoronary bypass grafting, the influence of high-dose aprotinin (2 million IU of aprotinin before CPB, 500,000 IU/h until the end of operation, 2 million IU added to the prime) (N = 20) on endothelial-related coagulation was compared to a nontreated control group (N = 20). Thrombomodulin (TM), protein C and (free) protein S as well as thrombin/antithrombin-III (TAT) plasma concentrations were measured by enzyme-linked immunosorbent assays (ELISA) before the aprotinin infusion, before cardiopulmonary bypass (CPB), during CPB and after CPB, at the end of surgery, 5 hours after CPB, and on the first postoperative day. All standard coagulation parameters (AT-III and fibrinogen plasma levels, platelet count, partial thromboplastin time) did not differ between the two groups. At baseline, TM plasma levels were within the normal range (< 40 ng/mL) and similar in both groups. During CPB, TM plasma concentrations decreased similarly in both groups (aprotinin: 18 +/- 6 ng/mL, control: 17 +/- 7 ng/mL) followed by a comparable increase in the postbypass period until the first postoperative day (aprotinin: 60 +/- 10 ng/mL, control: 53 +/- 11 ng/mL). Protein C and (free) protein S plasma levels also showed no differences between the two groups. On the first postoperative day, baseline values for protein C and protein S had not yet been reached.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Does aprotinin influence endothelial-associated coagulation in cardiac surgery? 752 60

Recent advances in determining anti-thrombogenic functions of vascular endothelial cells are reviewed. The following anticoagulant and fibrinolytic systems of endothelial cells are physiologically important; (1) Endothelial cell-derived metabolites including prostacyclin and nitric oxide (NO) support platelet inactivity. (2) Antithrombin III and tissue factor pathway inhibitor (TFPI) bound to heparin-like proteoglycans on endothelial cell membrane inhibit activated serine protease coagulation factors such as thrombin, factor Xa and factor VIIa-tissue factor complex. (3) Thrombomodulin converts thrombin from procoagulant into anticoagulant. Thrombin associated to thrombomodulin on endothelial cells activates protein C. Activated protein C in concert with protein S bound to endothelial cell membrane inactivates factors Va and VIIIa. (4) A receptor for both tissue plasminogen activator and plasminogen on endothelial cells provides an efficient plasmin generating system. Perturbation of these anti-thrombogenic systems of endothelial cells is caused by endotoxin (LPS), cytokines such as interleukin-1 and tumor necrosis factor (TNF), and risk factors for atherogenesis including lipoprotein(a) and homocysteine may result in arterial or venous thrombosis with subsequent development of atherosclerosis.
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PMID:[Anticoagulant and fibrinolytic systems of the injured vascular endothelial cells]. 817 40

Thrombomodulin (TM) is an endothelial cell membrane glycoprotein which neutralizes thrombin procoagulant activity and accelerates the thrombin-catalyzed activation of protein C. We expressed recombinant human soluble TM (rhs-TM) in Chinese hamster ovary cells and compared the effects of rhs-TM and heparin on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats. Experimental DIC was induced by a continuous intravenous infusion of endotoxin for four hours. rhs-TM or heparin was infused simultaneously with endotoxin. Treatment with rhs-TM significantly reversed the endotoxin-induced changes in significantly reversed the endotoxin-induced changes in following parameters: platelet count, fibrinogen level and fibrinogen and fibrin degradation products. Furthermore, glomerular fibrin deposits elevated by endotoxin treatment were reduced by the rhs-TM administration. Heparin showed the similar effects to rhs-TM. Activated partial thromboplastin time (APTT) in rats receiving rhs-TM were slightly longer than APTT in endotoxin-treated rats, but rats receiving heparin had much more prolonged APTT. From these results, we concluded that rhs-TM may be useful for the clinical treatment of DIC while having only minor adverse effects on APTT.
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PMID:Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats. 823 61


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