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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms underlying the superinduction of procoagulant activity by cycloheximide (CHX) on LPS-activated human monocytes have been investigated. Tissue factor (TF) activity of intact, viable cells was quantitated with a plasma recalcification assay and assays using chromogenic substrates specific for thrombin and factor Xa (FXa). TF antigen was measured simultaneously by immunocytochemical staining and immunoblotting with an anti-TF monoclonal antibody (MAb). Peripheral blood mononuclear cells (PBMC) activated with LPS in the presence of low dose CHX expressed more TF activity (approx. 100% increase) than cells activated with LPS alone. However, TF antigen levels were decreased approximately 70% by CHX. This discordant relationship was due primarily to differences in rates of activation of factor X (FX); LPS/CHX-treated PBMC activated nearly twice as much FX as LPS-treated cells (2.19 +/- 0.37 versus 1.10 +/- 0.21 ng FXa/10(6) PBMC/min, respectively). These studies indicate that TF cofactor activity on LPS/CHX-treated monocytes was approximately 7 times greater than that present on LPS-treated cells. Increased TF functional activity may be due to CHX-induced alterations in the type and content of phospholipids (PL) in the cell membrane. Results showed that exogenous mixed PL markedly increased TF activity on LPS-activated monocytes, but not on LPS/CHX-activated cells, without increasing TF antigen levels or altering cell viability. Membrane alterations may occur on monocytes in certain pathological or iatrogenic conditions resulting in a highly active form of TF in vivo.
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PMID:Discordant expression of tissue factor antigen and procoagulant activity on human monocytes activated with LPS and low dose cycloheximide. 180 19

An experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.
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PMID:The factor VIII bypassing activity of prothrombin complex concentrates: the roles of factor VIIa and of endothelial cell tissue factor. 180 20

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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PMID:Heterogeneous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester. 184 20

Although it is well established that calcium is an essential cofactor in blood coagulation, recent experimental evidence suggests that zinc may also play an important role in hemostasis. In the present study, we have examined the effect of zinc ions on the amidolytic and proteolytic activity of recombinant factor VIIa in the presence of physiological levels of calcium ions. The amidolytic activity of factor VIIa was inhibited half-maximally by 20 microM zinc. The amidolytic activity of a derivative of factor VIIa lacking the gamma-carboxyglutamic acid domain was also inhibited half-maximally by 20 microM zinc, suggesting that the mechanism of zinc inhibition of factor VIIa amidolytic activity did not involve its gamma-carboxyglutamic acid residues. The amidolytic activity of a complex of recombinant tissue factor and factor VIIa was inhibited half-maximally by 70 microM zinc. In contrast to the results obtained with factor VIIa, the amidolytic activities of other human vitamin K-dependent coagulation proteases including factor Xa, thrombin and activated protein C were not appreciably affected by 50-100 microM zinc. The proteolytic activation of factor X by a complex of factor VIIa and relipidated tissue factor apoprotein was inhibited half-maximally by 40 microM zinc, whereas activation of factor IX in this system was inhibited half-maximally by 70 microM zinc ions. Considerably higher levels of zinc (approximately 100 microM) were required to inhibit half-maximally the rate of factor X activation by a complex of factor VIIa and functional tissue factor on the surface of either a human bladder carcinoma cell line, J82, or stimulated human umbilical vein endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of recombinant human blood coagulation factor VIIa amidolytic and proteolytic activity by zinc ions. 187 14

Mechanisms of coagulation activation in situ were studied by means of immunohistochemical techniques applied to surgically resected primary adenocarcinomas and squamous cell carcinomas of the lung. Findings in these two histologic types were similar. Double-labeling techniques using macrophage-specific antibody together with antibody to either tissue factor, factor VII, factor X, or factor V revealed coincident staining for each of these coagulation factors on tumor-associated macrophages. Staining of tumor cells for these factors was rare and inconsistent. Both macrophages and fibroblasts in the tumor connective tissue stained for the a subunit of factor XIII. Fibrinogen was abundant throughout the tumor connective tissue, but staining for fibrin and D-dimer cross-linked sites of fibrin was restricted to areas adjacent to macrophages, indicating that thrombin was generated in association with tumor macrophages but not with tumor cells. By contrast, tumor cells stained diffusely for urokinase-type plasminogen activator and focally for thrombomodulin. These findings contrast with those reported previously for small cell carcinoma of the lung and suggest that coagulation activation in adenocarcinoma and squamous cell carcinoma of the lung may occur indirectly through activation of certain host cells such as macrophages. By contrast, tumor cell plasminogen activator may mediate certain aspects of the malignant phenotype in these tumor types.
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PMID:Coexisting macrophage-associated fibrin formation and tumor cell urokinase in squamous cell and adenocarcinoma of the lung tissues. 191 76

We studied the changes of coagulation and fibrinolysis in bronchoalveolar lavage (BAL) and plasma obtained serially at intervals after the onset of adult respiratory distress syndrome (ARDS). BAL procoagulant activity was increased at 3 days and tended to decrease thereafter. Tissue factor associated with factor VII was the major BAL procoagulant. Fibrinopeptide A was increased, indicating increased thrombin-mediated conversion of fibrinogen to fibrin. Fibrinolytic activity was usually undetectable in BAL at 3 days post-ARDS and remained depressed for up to 14 days despite unchanged concentrations of urokinase and variably detectable tissue plasminogen activator. Depressed fibrinolytic activity was associated with increased antiplasmin activity and plasminogen activator inhibitor 1 (PAI-1) while PAI-2 concentrations approximated those of control samples and did not change during evolving ARDS. Evidence of systemic coagulopathy and increased systemic fibrin degradation were commonly found in serial ARDS plasma samples, consistent with accelerated vascular and/or extravascular fibrin deposition in these patients. The data indicate that intra-alveolar as well as systemic derangements of fibrin turnover are common features of evolving ARDS. Concurrent local abnormalities of both coagulation and fibrinolytic pathways favor persistence of alveolar fibrin for up to 14 days after clinical recognition of ARDS.
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PMID:Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. 192 57

Procoagulant activity may persist during coronary thrombolysis and result in either delay in the time to recanalization or recurrent thrombosis. Although heparin and aspirin form the mainstay of current therapy, recurrent thrombosis occurs despite adjunctive heparin therapy during thrombolysis. Newer agents that inhibit thrombin by antithrombin III-independent mechanisms, or that inhibit earlier steps in the coagulation cascade, have been shown to be effective in the experimental preparation of coronary thrombolysis. Because heparin-antithrombin III is a relatively inefficient inhibitor of thrombin bound to fibrin, agents such as hirudin or small peptide inhibitors of the thrombin-active site appear to be more effective inhibitors of clot-associated thrombin activity. Inhibition of early steps in the coagulation cascade with the inhibitor of tissue factor-factor VIIa complex, or with activated protein C, also appears to be an effective anticoagulant strategy. In experimental preparations all of these agents have shown superiority in preventing recurrent thrombosis compared with heparin, and in some cases they appear to accelerate the rate of clot lysis.
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PMID:Role of new anticoagulants as adjunctive therapy during thrombolysis. 199 Jul 82

Uremia is associated with bleeding diathesis. Platelet adhesion to the subendothelium is inhibited by a factor in uremic plasma that may play a role in the disturbed hemostasis of uremic patients. In the formation of the hemostatic plug, platelet adherence is followed by stimulus-induced platelet aggregation and reinforcement by thrombin-generated fibrin. To study these processes in uremic blood, a newly developed thrombosis model was used. Perfusates anticoagulated with low-molecular-weight heparin were circulated over a matrix of stimulated cultured endothelial cells. By stimulation of the endothelial cells, tissue factor was synthesized and deposited in the matrix. When this tissue factor rich-matrix was exposed to flowing blood, local thrombin was formed via activation of the extrinsic coagulation pathway. With this system, platelet adhesion, thrombin-dependent platelet activation, and fibrin formation can all be studied at the same surface. In addition to an adhesion defect, decreased aggregate formation was also found in uremic perfusates. Normal platelets in uremic plasma showed similar results, which indicates that a factor in uremic plasma caused this adhesion and aggregation defect. Platelet aggregation in the system was dependent on endogenously formed thrombin. Fibrinopeptide A generation, however, was normal in uremic perfusates; therefore, uremic plasma has a normal capacity to form thrombin. Resuspension of washed uremic platelets in control plasma did not reverse the aggregation defect in perfusions. In contrast, aggregometer studies with isolated uremic platelets could not detect an abnormal response to threshold concentrations of exogenous thrombin. Thus, uremic toxin(s) cause defective aggregate formation in flow, but not necessarily in the aggregometer. This apparent discrepancy may be due to the higher shear forces in the flow system, which may prevent aggregate formation that is allowed in the aggregometer. Another explanation, that uremic platelets are less responsive to locally formed thrombin than they are to exogenously added thrombin, seems less likely.
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PMID:Defects in platelet adhesion and aggregate formation in uremic bleeding disorder can be attributed to factors in plasma. 202 8

The potential importance of pleural fibrin deposition in the pathogenesis of pleural injury is supported by both clinical and experimental observations. We hypothesized that the local equilibrium between procoagulant and fibrinolytic activities is disrupted to favor fibrin deposition in exudative pleuritis. To test this hypothesis, we characterized procoagulant and fibrinolytic activities in pleural exudates from patients with pneumonia, lung cancer, or empyema and transudates from patients with congestive heart failure. Procoagulant activity was generally increased in exudative processes and was due mainly to tissue factor. All effusions contained antithrombin III and inhibited factor Xa and thrombin, but endogenous prothrombinase or thrombin activities were variably detected. Pleural fluid fibrinolytic activity was increased in congestive heart failure and was due to both tissue plasminogen activator and urokinase. Depressed fibrinolytic activity was found in pleural exudates despite increased concentrations of plasminogen, mainly glu-1-plasminogen, and was due to inhibition of plasminogen activation by plasminogen activator inhibitors 1 and 2 and of plasmin, in part by alpha 2-antiplasmin. Concentrations of PAI-1 in exudative pleural fluids were increased up to 913-fold, compared with normal pooled plasma. Exudative pleural effusions are characterized by increased procoagulant and depressed fibrinolytic activity, favoring fibrin deposition in the pleural space. The balance of these activities is reversed and favors fibrin clearance in congestive heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Abnormalities of pathways of fibrin turnover in the human pleural space. 206 28

The mechanism of coagulation activation in renal cell carcinoma was investigated using immunohistochemical techniques applied to fresh frozen sections of resected primary tumors. Tissue factor antigen was detected in the endothelium of vascular channels within the tumors. Fibrinogen and factor V were distributed diffusely in the perivascular tumor connective tissue. Fibrin was readily detected in a linear pattern along the edges of nodules of viable tumor indicating that thrombin had formed from the interaction of coagulation factors demonstrated previously in renal cell carcinoma tissue. Tissue plasminogen activator was detected in the endothelium of blood vessels in the vicinity of the tumor and urokinase in areas of necrosis but neither were associated with viable tumor cells. These results indicate that thrombin is formed locally in renal cell carcinoma tissue that transforms fibrinogen to fibrin. There also appears to be a net deficit in fibrinolysis in situ in this tumor. We postulate that these conditions might contribute to stabilization and progression of renal cell carcinoma and that clinical trials of antithrombotic agents are justified in this tumor type.
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PMID:Fibrinogen-fibrin transformation in situ in renal cell carcinoma. 211 16


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