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D S Fair

Publications and source records attributed to D S Fair.

At least 55 records · Page 3Linked to original sources
PubMed1985

Human alveolar macrophages synthesize factor VII in vitro. Possible role in interstitial lung disease.

Both fibrin and tissue macrophages are prominent in the histopathology of chronic inflammatory pulmonary disease. We therefore examined the procoagulant activity of freshly lavaged human alveolar macrophages in vitro. Intact macrophages (5 X 10(5) cells) from 13 healthy volunteers promoted clotting of whole plasma in a mean of 65 s. Macrophage procoagulant activity was at least partially independent of exogenous Factor VII as judged by a mean clotting time of 99 s in Factor VII-deficient plasma and by neutralization of procoagulant activity by an antibody to Factor VII. Immunoprecipitation of extracts of macrophages metabolically labeled with [35S]methionine by Factor VII antibody and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a labeled protein consistent in size with the known molecular weight of blood Factor VII, 48,000. The addition of 50 micrograms of unlabeled, purified Factor VII blocked recovery of the 48,000-mol wt protein. In addition, supernatants of cultured macrophages from six normal volunteers had Factor X-activating activity that was suppressed an average of 71% after culture in the presence of 50 microM coumadin or entirely by the Factor VII antibody indicating that Factor VII synthesized by the cell was biologically active. Endotoxin in vitro induced increases in cellular tissue factor but had no consistent effect on macrophage Factor VII activity. We also examined the tissue factor and Factor VII activities of freshly lavaged alveolar cells from nine subjects with clinical and/or histologic evidence of sarcoidosis. Four of the nine subjects expressed increased tissue factor and seven of nine had increased Factor VII activity over the normal range (P less than 0.01). We estimate the mean Factor VII associated with the cells of sarcoid patients to be 4.7 ng/10(6) cells (range 0.4-20) as compared to a mean of 0.74 ng/10(6) cells (range 0.2-2) for that of normal subjects. Along with previous data showing synthesis of plasminogen activator, these findings indicate that human alveolar macrophages normally synthesize and express measurable amounts of the initial enzymes of proteolytic reactions regulating both fibrin deposition and fibrin resorption. Abnormalities in Factor VII activity in a small group of patients with sarcoidosis raise the possibility that modulation of fibrin turnover by macrophages may contribute to the pathology of this and perhaps other interstitial lung diseases.

Blood Coagulation↗
PubMed1985

Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis.

Twenty-six patients with hereditary factor VII deficiency (VII:C less than 10%) were evaluated using a panel of three thromboplastins of varying species and tissue origin in both coagulant and chromogenic assay systems. Normal values for the coagulation and chromogenic assays were 104% +/- 7% and 108% +/- 21%, respectively. Factor VII antigen was measured by a specific radioimmunoassay (normal, 470 +/- 112 ng/mL). The patients were divided into two groups based on the factor VII:Ag assay results. Group 1, 18 patients, had decreased levels of factor VII:Ag and group 2, eight patients, had normal levels of factor VII:Ag. The two groups were further subdivided on the basis of discrepant factor VII:C levels in the chromogenic and coagulant assays. The number of observed patterns of functional factor VII:C activity suggests a high degree of complexity of factor VII and thromboplastin interaction. Whereas no clinical bleeding was reported in any of the nine black patients evaluated, all Caucasians (16) and one Hispanic presented with mild to severe bleeding. Patients with factor VII:C greater than 10% using a human thromboplastin had a negative bleeding history, regardless of the activity measured with other thromboplastins. Factor VII activity measured with a human thromboplastin appears to correlate best with the clinical picture.

Adolescent↗
PubMed1984

Quantitation of human protein S in the plasma of normal and warfarin-treated individuals by radioimmunoassay.

Protein S was purified from human plasma and used to raise monospecific antibodies. A double antibody equilibrium radioimmunoassay was constructed and met all the requirements for a sensitive, accurate and specific determination of Protein S. The sensitivity of the assay was between 8-250 ng of Protein S/ml and the coefficient of variation was 2-6% within assays and 13-19% between assays. Complement C4b-binding protein or Protein C did not effect the measurement of Protein S. Complete competition with parallel slopes of inhibition was seen among purified Protein S and plasma from normal and warfarin-treated individuals indicating immunochemical identity and validating the measurement of Protein S in plasma. The concentration of Protein S in normal plasma (n=24) was 23.4 +/- 4.42. micrograms/ml and in plasma from warfarin-treated individuals (n=24) was 12.6 +/- 3.92 micrograms/ml. Thus, a common feature of individuals undergoing warfarin therapy is the reduction of approximately 50% in the concentration of each of the vitamin K-dependent proteins.

Antibodies, Monoclonal↗
PubMed1984

Monocytes can be induced by lipopolysaccharide-triggered T lymphocytes to express functional factor VII/VIIa protease activity.

In the present study we demonstrate that human monocytes can be induced by the model stimulus, lipopolysaccharide (LPS), to produce and assemble on their surface functional Factor VII/VIIa. This protease was not induced in relatively purified monocytes alone following exposure to LPS; but was induced in the presence of Leu-3a positive helper/inducer T cells. The Factor VII/VIIa protease activity represented 35-40% of the potential initiating activity for the extrinsic coagulation pathway and was demonstrated using functional coagulation assays, as well as in amidolytic assays for the activation of Factor X. This activity of cell-bound Factor VII/VIIa appeared to involve a tight adduct of calcium. The identity of the Factor X-activating protease as Factor VII/VIIa was confirmed by the capacity of antibody specific for Factor VII/VIIa to neutralize the cell-bound protease. Further propagation of the extrinsic pathway following generation of Factor Xa required addition of exogenous Factor Va. These results expand the repertoire of proteases that have been identified with appropriately triggered cells of the monocyte/macrophage series, and suggest that initiation and propagation of the extrinsic coagulation protease network on induced monocytes involves not only expression of the initiating cofactor molecule, tissue factor, but also production of Factor VII and its organization into the molecular assembly. Thus, in the absence of exogenous Factor VII/VIIa a directly proteolytic effector cell can be generated. Further molecular assembly of the extrinsic pathway on the monocyte surface sequentially expands the proteolytic capacity of this response. The synthesis and assembly of the extrinsic activation complex by the monocyte and its derived progeny, the macrophage, provides a mechanism by which coagulation is initiated under T cell instruction at sites of immunologic responses.

Antibodies↗
PubMed1984

Monocyte procoagulant activity in Whipple's disease.

We have studied the expression of procoagulant activity by the circulating mononuclear cells of four patients with Whipple's disease. There was a spontaneous expression of procoagulant activity in two patients with active untreated Whipple's disease. This activity was shown to originate in the monocyte fraction of the mononuclear cells and was demonstrated to cleave prothrombin directly. This prothrombinase activity was not Factor Xa, because it was not neutralized by anti-Factor X serum and was not inhibited by an established panel of Factor Xa inhibitors. The prothrombinase activity was not expressed by the monocytes of these patients following 8 weeks of antibiotic therapy, by which time the patients' symptoms resolved, and was not found in two patients previously treated for Whipple's disease who were in clinical remission or in normal subjects. Serial studies in one patient with active disease showed that monocytes failed to express increased prothrombinase within 2 weeks of antibiotic therapy. A second procoagulant activity was produced in response to endotoxin (LPS) by cells from controls and patients with Whipple's disease and was identified as thromboplastin. These observations suggest that circulating monocytes of patients with active Whipple's disease are endogenously stimulated to express prothrombinase activity, which may contribute, at least in part, to the pathophysiology of this condition.

Adult↗
PubMed1984

Human hepatoma cells secrete single chain factor X, prothrombin, and antithrombin III.

The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single-chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.

Antithrombin III↗
PubMed1983

Synthesis and secretion of the fibrinolytic components, including alpha 2-antiplasmin, by a human hepatoma cell line.

The capacity of the human hepatoma cell line Hep G2 to synthesize and secrete components of the fibrinolytic system was examined. Although fibrinogen, plasminogen, and alpha 2-antiplasmin were present in culture supernatants, histidine-rich glycoprotein was not detected. Albumin was monitored for comparative purposes and was also secreted. Intracellular levels of the fibrinolytic proteins were not detected, suggesting that once synthesized, these proteins were rapidly secreted by the cell. The fibrinogen, plasminogen, alpha 2-antiplasmin, and albumin secreted by the cells were immunochemically identical to their plasma counterparts. The concentration of these four molecules increased exponentially with time in the culture medium when normalized to total cellular protein. The kinetics of their secretion were similar, but the amounts of each protein differed. On day 8 the culture medium contained 29.6, 0.64, 0.39, and 0.59 micrograms/ml albumin, fibrinogen, plasminogen, and alpha 2-antiplasmin, respectively, whereas the time required for doubling the concentrations of the proteins in the medium ranged from 2.20 to 2.49 days. A detailed characterization of alpha 2-antiplasmin demonstrated that this inhibitor was synthesized by the cells. The molecular weight of intrinsically labeled alpha 2-antiplasmin was 69,000. The secreted inhibitor was biologically active, since it could inhibit plasmin cleavage of a chromogenic substrate, and this inhibition was neutralized by monospecific antibodies to alpha 2-antiplasmin. In addition, intrinsically labeled alpha 2-antiplasmin and the plasma molecule behaved in an identical manner with respect to their capacity to form a complex with plasmin. These studies suggest that the Hep G2 cell line may provide a model for assessing the regulation of synthesis and secretion of components of the human fibrinolytic system.

Albumins↗
PubMed1983

Human hepatoma cell line plasminogen activator.

A human hepatoma cell line, Hep G2, which synthesizes and secretes several components of the fibrinolytic system, was examined for the capacity to produce plasminogen activator (PA). PA activity was found to accumulate in medium conditioned by the cells but was not detected in cell extracts. Analysis of Hep G2 conditioned medium by SDS-polyacrylamide gel electrophoresis revealed multiple PAs of Mr 100,000, 60,000, and 52,000 daltons. In addition, a heterogeneous band of activity was present between the 100,000 and 60,000 dalton PAs. All detectable PA activity in conditioned medium fractionated at a single position after isoelectric focusing. The isoelectric point of this material (pI 8.6) was identical to that of purified urokinase. Anti-urokinase IgG, but not anti-tissue activator IgG, neutralized the activity of these PAs. Treatment of conditioned medium with 25 mM diisopropylfluorophosphate for 5 hr at 37 degrees C inactivated the 52,000 and 60,000 dalton PAs but had no effect on the 100,000 dalton PA or the heterogeneous zone of activity. The possibility that the absence of detectable PA activity in the cell extracts resulted from the presence of fibrinolytic inhibitors was considered. Addition of cell extract to purified urokinase resulted in a concentration-dependent reduction of urokinase-mediated fibrinolytic activity, consistent with the presence of such inhibitors. Acidification of the cell extracts to inactivate the inhibitor(s) revealed the presence of a latent, cell-associated PA activity. Thus, in addition to the other major components of the fibrinolytic system, Hep G2 cells produce multiple forms of a urokinase-like PA. Detection of these PAs may be obscured by the presence of cellular inhibitors.

Carcinoma, Hepatocellular↗
PubMed1983

Quantitation of factor VII in the plasma of normal and warfarin-treated individuals by radioimmunoassay.

Highly purified single-chain factor VII was isolated from plasma and used to generate monospecific antibodies. A double-antibody equilibrium radioimmunoassay was constructed. The assay was tested for and met all the criteria required for a specific, sensitive, and accurate determination of factor VII in plasma. The range of sensitivity of the assay was between 1 and 500 ng factor VII/ml, and the coefficient of variation was 1%-3% within assay and 12%-16% between assays. Pure factor VII and plasmic factor VII from normal, warfarin-treated, and hereditary deficient individuals inhibited competition assays with parallel slopes, indicating the expression of similar epitopes by these molecules and validating the measurement of this protein in plasma. The concentration of factor VII in normal plasma (n = 41) was 470 +/- 112 ng/ml, and the measurement of factor VII antigen correlated with activity (r = 0.82). Factor VII concentration in the plasma of individuals on warfarin therapy (n = 24) was 238 +/- 73 ng/ml. Factor VII activity was about 38% of normal and correlated less well with factor VII antigen (r = 0.53). The specific activity of these molecules was 78% of normal (p less than 0.01), suggesting the presence of nonfunctional or partially functional molecules in the circulation of individuals undergoing drug therapy. Analysis of two hereditary deficient patients revealed that, while there were significant levels of factor VII protein, the procoagulant activity was less than 2%, indicating a discordant relationship of these parameters in individuals expressing the deficient factor VII phenotype.

Animals↗
PubMed1982

Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase.

Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated

Animals↗
PubMed1981

Immunochemical mapping of the conformation of human fibrinogen. The gamma 95-264 segment in inaccessible to antibody in native fibrinogen but progressively exposed by plasmic cleavage.

The accessibility of the gamma 95-264 sequence to specific antibody probes in the native fibrinogen molecule and its plasmic cleavage fragments have been investigated. The gamma 95-264 segment was generated by cyanogen bromide cleavage of the gamma chain and isolated by gel filtration and ion exchange chromatography. Rabbit antisera to this peptide and to gamma chain recognized at least five antigenic loci uniformly distributed throughout this segment. In primary binding assays, antibodies to gamma 95-264 bound gamma 95-264, free gamma chain, and fibrinogen fragment D, but not native fibrinogen. Also, gamma 95-264 was bound by antibodies to gamma chain and fibrinogen fragment D, but not by antibodies generated to native fibrinogen. Thus, the gamma 95-264 sequence was not accessible to antibody in the native structure. In competitive equilibrium radioimmunoassays, neither native fibrinogen nor highly soluble fibrinogen fraction I-9 inhibited the binding of gamma 95-264 by its antiserum or anti-gamma chain. With plasmic cleavage, however, the gamma 95-264 sequence became accessible to antibody and the series of fragments D greater than Y greater than D:E = X describes the relative reactivity of the gamma chain sequence in fibrinogen degradation products. Differential expression of gamma 95-264 antigenic loci was also observed with D fragments differing in molecular weight. Plasmic cleavage of cross-linked and noncross-linked fibrin generated D fragments which did not express gamma 95-264 as well as fibrinogen D derivatives, indicating that the D domains of fibrinogen and fibrin are immunochemically distinguishable. These findings indicate that the central segment of the gamma chain is inaccessible to antibody in native fibrinogen, but that proper surface orientation is achieved upon plasmic degradation.

Antibodies↗
PubMed1981

Plasma lipoprotein induction and suppression of the generation of cellular procoagulant activity in vitro: two procoagulant activities are produced by peripheral blood mononuclear cells.

In the process of analyzing the effects of lipoproteins on functions of lymphoid cells, it was observed that physiological concentrations of isolated human plasma lipoproteins possess varying capacities to rapidly enhance the expression of procoagulant activity of human peripheral blood mononuclear cells in vitro. In a strict dose-dependent fashion, very low density lipoprotein, intermediate density lipoprotein, and high density lipoprotein enhanced both the surface expression by viable cells and the total cellular content of procoagulant activity during a 6-h incubation. Very low density lipoprotein induced a maximal 6.7-fold increase in the expression of a thromboplastin activity, which was consistent with tissue factor, in that it was dependent on Factors VII, X, and II. Both intermediate density lipoprotein and high density lipoprotein induced approximately a 12-fold increase of a different procoagulant activity which appears to be a direct prothrombin activator. This prothrombinase was calcium dependent and was inhibited by 2.5 mM diisopropylfluorophosphate, but was not neutralized by anti-Factor X antibodies or by inhibitors of Factor Xa. In contrast to the other lipoprotein density classes, low density lipoprotein did not stimulate procoagulant activity, but instead actively suppressed the generation of the two procoagulant activities induced by the stimulatory lipoproteins. Suppression by low density lipoprotein was clearly evident at molar ratios of low density lipoprotein to stimulatory lipoproteins of 1:3 or less. Reconstitution of all lipoproteins to physiological concentrations was not stimulatory as a consequence of the suppressive effects of low density lipoprotein. These data indicate that isolated plasma lipoproteins are capable of regulating the expression of two different procoagulant activities of peripheral blood mononuclear cells in vitro. The possibility that these interactions may be implicated in the association between certain types of hyperlipoproteinemias and thromboembolic disease merits study.

Blood Coagulation Factors↗