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)

It is well established that cultured endothelial cells are induced to generate tissue factor activity when incubated with either endotoxin or thrombin. In this study a perfusion system was used on 3-4 cm long human saphenous veins. The veins were perfused with thrombin (2.5 U/ml), endotoxin (30 ng/ml) or just medium for 3 h at 37 degrees C. After the perfusion, the veins were treated with collagenase, and EC were collected and subjected to tissue factor activity measurements. Some perfused veins were examined for tissue factor activity on the vessel wall by allowing factor VII and factor X to interact with the lumen of the intact vessels, followed by quantitation of generated factor Xa in a chromogenic assay. No formation of tissue factor activity could be found after perfusion in either collagenase-dissolved endothelial cells or in the coupled chromogenic assay for tissue factor activity performed in the lumen of the vessel. Our data strongly suggest that endothelial cells in intact endothelium may behave quite differently from isolated endothelial cells stimulated in cell cultures.
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PMID:Lack of ability to synthesize tissue factor by endothelial cells in intact human saphenous veins. 213 38

Components of coagulation and fibrinolysis reactions were identified in situ by immunohistochemical staining in fresh frozen sections of small cell carcinoma of the lung tissue. Tumor cells stained positively for tissue factor, a protein that is capable of activating the extrinsic pathway of coagulation (the components of which have been seen within small cell carcinoma of the lung [SCCL] tissue), and for proteins C and S antigens. Fibrin was seen in a focal distribution at the host-tumor interface, indicating that thrombin had acted upon the fibrinogen found throughout the tumor stroma. Staining with a neoepitope-specific antibody, which does not discriminate between fibrinogen fragment D and fibrin fragment D-dimer, was similar to that of the fibrin antibody. High molecular weight urokinase-type and tissue-type plasminogen activators were seen in vascular endothelium, but neither existed within the tumor. Low molecular weight urokinase was found in rare isolated foci of tumor cells primarily adjacent to areas of necrosis. Plasminogen activator inhibitor-3 occurred in tumor cell cytoplasmic blebs and in necrotic tumor cells, but plasminogen activator inhibitors 1 and 2 were not seen. Our data suggest a mechanism for thrombin generation and fibrin formation within SCCL tissues that could support cell proliferation, stroma formation, and preservation. These features could be conductive to perpetuation of this tumor and conceivably could form the basis of the beneficial effects of antithrombotic therapy seen in SCCL.
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PMID:Abnormal regulation of coagulation/fibrinolysis in small cell carcinoma of the lung. 215 29

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.
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PMID:Mammalian cell transient expression of tissue factor for the production of antigen. 216 68

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions.
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PMID:Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression. 216 19

Endothelial cells play an important role in the regulation of thrombosis. Normal resting (i.e. unstimulated) endothelial cells exhibit antithrombotic activity. This property is due to an active participation of endothelial cells in the inhibition of platelet adhesion and aggregation, in the inhibition of thrombin generation, in the direct inactivation of thrombin, and in clot lysis through the fibrinolytic system. When endothelial cells are stimulated by cytokines such as tumor necrosis factor (TNF) or interleukin 1 (IL-1), they may switch to an active procoagulant state. On the one hand, thrombin generation can be regulated on the endothelial cell surface by thrombomodulin, which allows the activation by thrombin of protein C which subsequently acquires and expresses potent anticoagulant properties. On the other hand, after activation, the same endothelial cell can express tissue factor on its surface, which will lead to the triggering of the coagulation cascade resulting in the generation of thrombin. TNF has been shown both to induce tissue factor gene expression and to suppress transcription of the thrombomodulin gene in endothelial cells. Many cytokines induce tissue factor gene expression and procoagulant activity in the monocyte/macrophage lineage; they also stimulate adhesion of leukocytes to endothelial cells. Cytokines such as IL-1 or TNF can thus be characterized as important intercellular messengers during the onset of coagulation. The role of these compounds can be schematized as: 1) agents of the stimulation of endothelial cells by leukocytes, 2) agents of stimulation of leukocytes by endothelial cells, 3) localization of the coagulation response through the initiation of endothelial cell-leukocyte interactions. Pharmacological modulation of these responses is possible along two pathways: 1) inhibition of the activation of endothelial cells or leukocytes responsible for cytokine release, 2) inhibition of the cytokine-induced cellular activation responsible for potentiation of procoagulant activity.
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PMID:Modulation by cytokines of leukocyte-endothelial cell interactions. Implications for thrombosis. 217 56

Atherosclerotic lesions have been reported to contain herpes simplex virus (HSV) genomic material. This and other evidence suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV lesions manifest histologically marked fibrin deposition in microvessels. Our laboratory tested in vitro whether HSV infection would cause human umbilical vein endothelial cells to become procoagulant and attract inflammatory cells. Early infection of human endothelial cells with HSV-1 alters the surface conformation as detected by merocyanine 540 staining. The efficiency of prothrombinase complex assembly increases, resulting in a two- to threefold accelerated rate of thrombin generation on the cell surface of virally infected endothelium. HSV infection of endothelium results in a marked increase in thrombin-induced platelet adhesion with a concomitant decrease in prostacyclin secretion in response to thrombin. Viral infection enhances coagulation by decreasing endothelial thrombomodulin expression and subsequent activation of protein C. Viral infection also induces tissue factor in human endothelial cells within 4 hours of infection. Not only does the endothelial monolayer become procoagulant when infected with HSV, it also becomes a more adherent surface for granulocytes. Resting and stimulated granulocyte adherence is enhanced twofold on virally infected endothelium. Enhanced adhesion is accompanied by excessive granulocyte-mediated lysis of 51Cr-labeled HSV-infected endothelium and endothelial cell detachment from its substrate. Exaggerated endothelial detachment correlated with poor binding of infected endothelial cells to substratum matrix proteins. Resuspended virus-infected cells bound significantly less well to tissue culture containers coated with fibronectin, laminin, and type IV collagen. HSV-infected endothelium alters the anticoagulant properties of the endothelium causing it to become procoagulant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proinflammatory and procoagulant effects of herpes simplex infection on human endothelium. 219 Jun 48

Nephrotic syndrome (NS) is associated with several disorders of hemostasis: thrombocytosis and platelet hyperaggregability; increased plasma levels of factors V and VIII, and of fibrinogen with blood hyperviscosity; decreased plasma levels of natural anticoagulants: free protein S, and antithrombin III compensated by increased levels of alpha 2-macroglobulin; lowered fibrinolytic activity. Intensity of hypercoagulability is related to the degree of hypoalbuminemia; however, the role of hypercoagulability in the increased incidence of thromboembolic events, including renal vein thrombosis, is not proved. Clotting disorders are due to urinary losses of anticoagulants or to increased liver synthesis of procoagulants stimulated by hypoalbuminemia. Moreover, changes in clotting factors levels may be due to intravascular thrombin formation (marked by increased plasma levels of fibrinopeptide A). During active phases of glomerulonephritides (GN) with NS, thrombin formation might in fact arise in glomeruli, following activation of the glomerular hemostasis system. Isolated glomeruli from human crescentic GN, rabbit nephrotoxic GN and rat HgCl2 autoimmune GN produce excessive amounts of procoagulant (tissue factor) activity (PCA). Sequential studies of the self-limited HgCl2 GN showed that glomerular PCA, proteinuria and glomerular fibrin deposits peaked concomitantly at the acme of the disease, suggesting that immunologically mediated glomerular damage had triggered the extrinsic coagulation pathway.
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PMID:Coagulation factors in nephrotic syndrome. 225 77

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.
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PMID:Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl. 226 68

The role of factor VIIa in haemostasis has been studied using a canine model of factor VIII deficiency. Highly purified human factor VIIa was administered to dogs at a dosage of 0.5 microgram/kg. At selected times pre- and post-infusion, haemostasis was evaluated by the cuticle bleeding time. Plasma was collected for the assay of various parameters, including fibrinopeptide A (FPA) as a marker for thrombin generation in vivo. Factor VIIa infusion resulted in a 6-fold increase of factor VII clotting activity with a t1/2 of 2 h. FPA levels, which were 1.4 ng/ml before infusion, did not increase significantly in haemophilic dogs. In normal dogs, however, FPA levels rose to a mean value of 190 ng/ml 30 min post-infusion. It appeared that thrombin generation by factor VIIa infusion had occurred mainly via the intrinsic, factor VIII-dependent pathway. In factor VIII-deficient dogs, factor VIIa infusion did not correct cuticle bleeding, but an inconsistent haemostatic effect was observed 15-30 min post-infusion. Similar results were obtained in haemophilic dogs with circulating antibodies against factor VIII. The haemostatic effectivity could not be improved by increasing the factor VIIa dosage up to 40-fold. Although these data suggest that the extrinsic, factor VII-dependent factor X activation provides only a minor pathway of thrombin generation in vivo, it is possible that the suboptimal haemostatic effect noted may be promoted in bleeding situations where tissue factor availability is less limited. As such, factor VIIa may prove useful in the treatment of haemophilia A patients with acquired inhibitors to factor VIII.
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PMID:The role of factor VII in haemostasis: infusion studies of factor VIIa in a canine model of factor VIII deficiency. 227 19

Expression of tumor necrosis factor (TNF alpha), tissue factor (TF), and interleukin 1-beta (IL-1 beta) mRNA was evaluated in monocytes isolated from patients infected with human immunodeficiency virus (HIV). There was a significant depression (66%) of the induced level of TF mRNA expression in response to lipopolysaccharide. Conversely, the response of TNF alpha and IL-1 beta, following LPS induction, was "normal." TF mRNA reduction was also observed to a lesser degree in AIDS-related complex patients (20%) but not in asymptomatic seropositives. TF is necessary for initiation of the coagulation protease cascade, leading to thrombin production and fibrin deposition, which play a role in inflammatory responses. Its selective reduction may be a factor in the diminished resistance to secondary infections observed in AIDS. Further, since the TF defect increases as patients progress toward AIDS, it may serve as a marker for disease progression.
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PMID:A selective defect in tissue factor mRNA expression in monocytes from AIDS patients. 229 2


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