Biomedical subjects
N L Esmon
Publications and source records attributed to N L Esmon.
Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro.
Endotoxemia is frequently associated clinically with disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in vivo is unclear. Modulation of tissue factor (TF) and thrombomodulin (TM) expression on the endothelial surface may be relevant pathophysiologic mechanisms. Stimulation of human umbilical vein endothelial cells with endotoxin (1 microgram/ml) increased surface TF activity from 1.52 +/- 0.84 to 11.89 +/- 8.12 mU/ml-10(6) cells at 6 h (n = 11) which returned to baseline by 24 h. Repeated stimulation at 24 h resulted in renewed TF expression. Endotoxin (1 microgram/ml) also caused a decrease in TM expression to 55.0 +/- 6.4% of control levels at 24 h (n = 10) that remained depressed at 48 h. Both effects were dose and serum dependent. A temporary rise in TF expression accompanied by a sustained fall in TM expression comprise a shift in the hemostatic properties of the endothelium that would favor intravascular coagulation and may contribute to the pathogenesis of DIC in gram-negative septicemia.
Analysis of protein C and protein S in disease states.
Protein C and protein S are two vitamin K dependent plasma proteins which function in concert to serve as a natural anticoagulant. Deficiencies of both proteins are associated with thrombotic disease. Assay procedures employing monoclonal antibodies are discussed and compared to previous assay procedures. The assays are effective in patients undergoing heparin or oral anticoagulant therapy and are not influenced by inhibitory factors generated during intravascular coagulation. These assays should aid in our understanding of the role of protein C and protein S in thrombotic disease.
Protein C, isolation and potential use in prevention of thrombosis.
Protein C and protein S serve as natural anticoagulants. Deficiencies of these proteins are often associated with recurrent deep vein thrombosis and coumarin induced skin necrosis. These two proteins function by selectively inactivating factors Va and VIIIa, two of the "cofactors" of blood coagulation. Hence, inhibition of coagulation by this pathway complements the better known inhibition mediated by the antithrombin III-heparin system. These observations suggest that protein C and/or activated protein C may prove useful in controlling thrombosis and/or DIC. We have developed a Ca2+ dependent monoclonal antibody which allows the rapid isolation of human protein C. This rapid isolation has allowed us to demonstrate that activated protein C can protect baboons from the lethal effects of E. coli/endotoxin and that protein C supplementation can minimize fibrinogen consumption following tissue factor infusion into dogs.
Interaction of thrombin with thrombomodulin.
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Complex interaction between factor Va-light chain and thrombomodulin in the regulation of protein C activation.
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Distribution of the thrombomodulin antigen in the rabbit vasculature.
The purpose of this study was to determine the distribution of the thrombomodulin (TM) antigen in the rabbit vasculature in vivo. Acetone-fixed, frozen sections of various tissues (brain, heart, aorta, lung, liver, spleen, kidney, small intestine, large intestine, skeletal muscle, bone, and skin) were stained by indirect immunofluorescence with goat affinity-purified antibodies to TM. Both large and small vessels (including capillaries) had demonstrable TM antigen localized to the endothelium while vascular smooth muscle and connective tissue cells were negative. The parenchyma of the organs examined were also negative. Based on antibody dilutions and brightness of staining, there is an indication that lung, followed by heart and intestinal vessels, has the highest concentration of antigen; kidney glomerular capillary loops have the least. These results also indicate that TM is an excellent cell type-specific marker of endothelial cells as the endothelium of all vessels and vascular beds was positive.
Localization of thrombomodulin antigen in rabbit endothelial cells in culture. An immunofluorescence and immunoelectron microscope study.
A total of 13 rabbit endothelial cell lines were examined for the presence, distribution, and localization of the thrombomodulin (TM) antigen. Three endothelial cell lines were derived from aorta, 5 lines from brain microvessels, and 5 lines from fat microvessels. Immunofluorescence demonstrated the presence of the TM antigen in all cell endothelial lines examined, at all passage levels, and at all stages of growth i.e., log phase, confluent, postconfluent stationary. The percentage of positive cells in a given culture and the distribution of the TM antigen within the cells, as well as on their surfaces, was a cell line characteristic. The intercellular variation in the expression of the TM antigen was insignificant in some cell lines but was striking in others. These characteristics were clonable. Immunofluorescence indicated and immunoperoxidase electron microscopy confirmed the localization of the TM antigen to the outer cell surface and in the Golgi-endoplasmic reticulum (GERL) complex.
The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells.
Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TM antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.
Effects of thrombomodulin and coagulation Factor Va-light chain on protein C activation in vitro.
Protein C activation by thrombin is significantly accelerated by the endothelial cell surface protein thrombomodulin, Factor Va, or its light chain. In this study we have compared the activation of protein C in the presence of either cofactor and examined the possibility that thrombomodulin and Factor Va-light chain act together to regulate protein C activation by thrombin. At all concentrations of protein C used, thrombomodulin was 20 times more efficient than Factor Va-light chain in accelerating protein C activation by thrombin. Protein C treated with chymotrypsin to remove the amino-terminal 41 amino acids that contain the gamma-carboxyglutamyl residues was activated by the thrombin-thrombomodulin complex at an identical rate to native protein C, whereas the modified protein C was activated by Factor Va-light chain and thrombin at only 5% of the rate obtained by using native protein C. Increasing concentrations of Factor Va-light chain, greater than or equal to 30 nM, inhibited thrombin-thrombomodulin catalyzed protein C activation with complete inhibition observed at 90 nM Factor Va-light chain. On the other hand, increasing thrombomodulin concentrations did not inhibit protein C activation by Factor Va-light chain and thrombin. These reactions in solution mimic, in part, those obtained on endothelial cells where protein C lacking the gamma-carboxyglutamyl domain is activated poorly and Factor Va-light chain at concentrations greater than 50 nM inhibited the activation of native protein C. The results of this study suggest that thrombomodulin and Factor Va-light chain may act in concert to regulate protein C activation by thrombin.
Thrombomodulin blocks the ability of thrombin to activate platelets.
When thrombin is complexed to the endothelial cell surface receptor thrombomodulin, it loses its procoagulant activities in that it no longer clots fibrinogen or activates factor V. Studies were initiated to determine if complex formation also blocks thrombin's other major procoagulant function, the activation of platelets. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release [14C] serotonin. Binding studies using 125I-labeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. When thrombomodulin is added after thrombin has bound to the platelets, the thrombin rapidly redistributes onto the thrombomodulin. These data suggest that in addition to its other anticoagulant effects, thrombomodulin may also act to inhibit and/or reverse platelet activation by thrombin.
Proteolytic formation and properties of gamma-carboxyglutamic acid-domainless protein C.
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Structural changes required for activation of protein C are induced by Ca2+ binding to a high affinity site that does not contain gamma-carboxyglutamic acid.
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Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation.
Protein C is activated rapidly when thrombin binds to a specific cell surface cofactor protein, thrombomodulin. Studies were initiated to determine the influence of thrombin-thrombomodulin complex formation on the substrate specificity of thrombin. When thrombin binds to thrombomodulin, the resultant complex retains less than 1% of the fibrinogen clotting activity of free thrombin. Permanent alteration of the thrombin molecule is not involved since full clotting activity is regenerated by incubation of the complex with excess diisopropyl phosphothrombin. Unlike the activation of protein C by the thrombin-thrombomodulin complex which is dependent on Ca2+, inhibition of fibrinogen clotting activity is not dependent on the presence of divalent metal ions. Formation of the thrombin-thrombomodulin complex also inhibits thrombin activation of factor V. Despite these changes in macromolecular substate specificity, no significant change in the hydrolysis of the synthetic substrates p-tosyl-L-arginine methyl ester and N alpha-benzoyl-L-arginine ethyl ester is detected upon formation of the thrombin-thrombomodulin complex. Formation of this complex results in a slight increase in the Km (from 9.0 +/- 0.4 to 10.2 +/- 0.6 microM) and Vmax (from 230 +/- 10 to 270 +/- 10 mol/s/mol of thrombin) for the specific thrombin substrate H-D-Phe-Pip-Arg-p-nitroanilide. These studies suggest that thrombomodulin has two distinct anticoagulant functions: 1) to inhibit the ability of thrombin to clot fibrinogen and to activate factor V; and 2) to accelerate the formation of the anticoagulant, activated protein C.
Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C.
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Different mechanisms for the modulation of TL antigens on murine lymphoid cells.
An indirect radioimmunoassay has been used to study the antibody-induced changes in expression of TL antigens on mouse thymus and leukemia cells. The results obtained indicate that the incubation of TL+ cells in relatively high concentrations of anti-TL antisera results in a detectable, but not major, loss in the quantity of antigen detected in the plasma membranes. This decrease is not inhibited by azide. Although the antigen loss seems to coincide with the appearance of patching of membrane TL antigens on leukemia cells observed in indirect immunofluorescence microscopy, this is not the case when thymocytes are used as targets. Fab fragments prepared from anti-TL antibodies can also induce a quantitative decrease in antigen expression. The major mechanism involved in membrane antigen loss seems to be endocytosis, although some shedding may also occur. Resistance to immune cytolysis (antigenic modulation) occurs during incubation in anti-TL antibodies before either endocytosis or antigen redistribution has progressed enough to be directly responsible. Since antigenic modulation does not represent a depletion in TL antigens being expressed on the cell surfaces, another mechanism must account for the failure of guinea pig complement to achieve cell lysis.
An indirect radioimmunoassay for thymus leukemia (TL) antigens.
An indirect radioimmunassay for thymus leukemia (TL)2 antigens has been developed and its specificity documented. The assay makes use of anti-TL antibodies produced in congenic mice (A-Tla(b)) and radioiodinated purified rabbit anti-mouse IgG. Using this assay, differences can be detected in the amounts of antigen expressed on thymocytes of the three known phenotypes (TL.1,2,3;TL.2;TL-) of inbred mouse strains. Significant differences are also detected in comparison of the thymocytes from homozygous TL.1,2,3 mice (A-Tla(a)) and heterozyhotes from Tla(a) and Tla(b) parents. Optimum conditions for the assay have been established. Its ability to detect antigens on glutaraldehyde-fixed cells and the binding of noncytolytic antibodies on both viable and fixed cells are documented. The assay has also been used to quantitate the changes in TL antigen expression on cells incubated in anti-TL antisera under conditions of antigenic modulation.