Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 117
Filtrar
Más filtros

Bases de datos
Tipo del documento
Intervalo de año de publicación
1.
J Exp Med ; 127(3): 589-603, 1968 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-4169967

RESUMEN

The C'1a-fixing properties of purified rabbit IgM anti-benzenearsonate antibody were determined. When tested with sheep erythrocytes to which hapten had been coupled by diazo linkage, the number of C'1a molecules fixed was 21% of the number of IgM antibody molecules bound to the erythrocyte surface. This was not due to loss of C'1a-fixing capacity during the purification procedure. Preparative electrophoresis of the antibody concentrated C'1a-fixing molecules in the anodal region so that antibody fractions with greater C'1a-fixing capacity were obtained. The demonstration that C'1a fixation is a property of a subpopulation of IgM molecules provides evidence for previously unrecognized micro-chain heterogeneity.


Asunto(s)
Anticuerpos/análisis , Pruebas de Fijación del Complemento , gammaglobulinas/análisis , Animales , Arsenicales , Benceno , Cromatografía , Electroforesis Discontinua , Eritrocitos , Concentración de Iones de Hidrógeno , Sueros Inmunes , Inmunoelectroforesis , Isótopos de Yodo , Proteínas/análisis , Conejos , Ultracentrifugación
2.
J Clin Invest ; 89(5): 1382-7, 1992 May.
Artículo en Inglés | MEDLINE | ID: mdl-1569181

RESUMEN

Factor VIII East Hartford (FVIII-EH) procoagulant activity is reduced because the substitution of cysteine for arginine 1689 abolishes an essential Factor VIII light chain thrombin cleavage site. Incubation of FVIII-EH plasma with penicillamine or DTT causes a five- to sixfold increase in FVIII-EH VIII:C, at 80 and 1 mM, respectively. While there is no FVIII-EH light chain cleavage when thrombin is added in the presence of penicillamine or DTT, these reducing agents disrupt the FVIII-vWf complex. For example, the addition of 5 mM DTT to normal or FVIII-EH plasma causes a 50% reduction in Factor VIII binding to vWf. These observations suggested that DTT increases FVIII-EH VIII:C by partial dissociation of FVIII-EH from vWf. This was verified by showing that vWf-free FVIII-EH had VIII:C activity of 21 U/dl, while the starting plasma level was 2.5 U/dl. Removal of other FVIII-EH plasma proteins by agarose gel filtration had no effect on VIII:C activity. The demonstration that this mutant Factor VIII has cofactor function when separated from vWf indicates that the dissociation of Factor VIII from vWf is an essential effect of Factor VIII light chain cleavage at arginine-1689.


Asunto(s)
Coagulación Sanguínea , Factor VIII/genética , Factor de von Willebrand/fisiología , Cisteamina/farmacología , Ditiotreitol/farmacología , Activación Enzimática , Factor VIII/fisiología , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Relación Estructura-Actividad , Trombina/metabolismo
3.
J Clin Invest ; 83(6): 1978-84, 1989 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-2498393

RESUMEN

Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (less than 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid.


Asunto(s)
Antígenos/inmunología , Autoanticuerpos/fisiología , Sitios de Unión de Anticuerpos , Factor VIII/inmunología , Isoanticuerpos/fisiología , Fosfolípidos/metabolismo , Antígenos/metabolismo , Autoanticuerpos/análisis , Unión Competitiva , Pruebas de Coagulación Sanguínea , Ensayo de Inmunoadsorción Enzimática , Factor VIII/metabolismo , Humanos , Immunoblotting , Isoanticuerpos/análisis , Sustancias Macromoleculares , Fosfatidilserinas/metabolismo
4.
J Clin Invest ; 49(1): 87-95, 1970 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-5409811

RESUMEN

The importance of antigen site density has been studied by means of a model passive hemagglutination system using human red cells coupled with sulfanilic acid groups. Relative site numbers were estimated from the covalent linkage of sulfanilic acid-(35)S to red cell membrane protein and the effective antigen site number was determined with (125)I-labeled rabbit IgG antisulfanilic acid. Cells which had fewer than 20,000 antigen sites per cell were not agglutinated. As greater numbers of sulfanilic groups were coupled to the red cells, the agglutination titers increased to maximum values with red fanilic groups were coupled to the red cells, the agglutination titers of purified IgM antibody were 10-20 times greater than IgG antibody when preparations with the same protein concentration were compared, but this difference was not noted when IgG antibody was measured by antiglobulin reactions. These findings emphasize the need to consider differences in antigen site density when comparing blood group systems. They are consistent with the hypothesis that those blood group antigens which have a very low site number will not be detected by IgG antibodies in saline hemagglutination determinations.


Asunto(s)
Antígenos , Eritrocitos/inmunología , Hemaglutinación , Compuestos de Anilina , Animales , Anticuerpos/análisis , Sitios de Unión , Electroforesis , Humanos , Sueros Inmunes , Inmunoglobulina G , Isótopos de Yodo , Métodos , Ácidos Sulfónicos , Isótopos de Azufre
5.
J Clin Invest ; 50(9): 1840-6, 1971 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-5105661

RESUMEN

The importance of antigen site density has been studied by means of a model passive hemolysis system using red cells coupled with sulfanilic acid groups. Relative site numbers were estimated from the covalent linkage of sulfanilic acid-(35)S to red cell membrane protein, and the effective antigen site number was determined with (125)I-labeled rabbit IgG anti-sulfanilic acid (anti-S). Immune hemolysis was demonstrated for red cells which had greater than a threshold number of antigen sites, the value of which was different for normal human cells (80,000 sites/cell), cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH) (40,000 sites/cell), and sheep red blood cells (RBC) (15,000 sites/cell). Cells with antigen site densities below these values did not hemolyze when tested with 1 mg/ml purified rabbit IgM anti-S. 2-8 times greater antigen site densities were required to obtain hemolysis with IgG anti-S. Above the threshold value, hemolysis titers were proportional to the antigen site number until maximal values were obtained. The greater hemolytic efficiency of IgM antibody was demonstrated in this system, and it was established that the magnitude of the difference was related to the test cell antigen site density. These data, taken with previously reported hemagglutination studies, have been used to develop a general classification of immune hemolysis and hemagglutination based on antigen site density and antibody class. It is suggested that the heterogeneity of blood group systems is caused by differences in the site separation of erythrocyte membrane antigens.


Asunto(s)
Antígenos/análisis , Sitios de Unión , Eritrocitos/inmunología , Hemólisis , Compuestos de Anilina , Animales , Antígenos de Grupos Sanguíneos , Hemaglutinación , Hemoglobinuria Paroxística/sangre , Humanos , Inmunoglobulina G , Isótopos de Yodo , Modelos Biológicos , Conejos , Ácidos Sulfónicos , Isótopos de Azufre
6.
J Clin Invest ; 62(5): 1048-52, 1978 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-81838

RESUMEN

A fluid-phase immunoradiometric assay has been developed which identifies an antigen on the Factor VIII (antihemophilic factor) procoagulant protein. This sensitive and quantitative assay is not influenced by levels of Favor VIII-related antigen (von Willebrand factor) or other plasma proteins. There is a close correlation of procoagulant activity and immunologically detectable protein in normal and von Willebrand's disease plasmas. In contrast, several different patterns have been identified in hemophilic plasmas. Neither procoagulant activity nor procoagulant antigen is detectable in plasmas from patients with severe classic hemophilia. Patients with mild and moderate hemophilia have either comparable plasma concentrations of procoagulant activity and procoagulant antigen or relatively greater levels of immunologically detectable protein.


Asunto(s)
Factor VIII/inmunología , Precursores de Proteínas/inmunología , Antígenos/análisis , Epítopos , Factor VIII/análisis , Hemofilia A/sangre , Humanos , Precursores de Proteínas/análisis , Radioinmunoensayo , Enfermedades de von Willebrand/sangre
7.
J Clin Invest ; 60(5): 1070-9, 1977 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-908750

RESUMEN

Although human antibodies to Factor VIII inactivate its procoagulant activity, they do not form immunoprecipitates when tested with this antigen. To understand this observation, we have examined the interaction of normal human Factor VIII with four high-titer human anti-Factor VIII, two from transfused hemophiliacs and two "spontaneous" antibodies from nonhemophilic individuals. An estimate of the size of complexes formed by these antibodies has been obtained by agarose gel filtration of mixtures of anti-Factor VIII with cryoprecipitate. Complexed anti-Factor VIII was detected by the method of Allain and Frommel: acid dissociation of complexes at pH 3.5. Complexed anti-Factor VIII was detected in column fractions eluting between the void volume and those which correspond to the elution volume of human IgG. In contrast, Factor VIII procoagulant activity was restricted to void volume fractions when separations were carried out in antigen excess, and free anti-Factor VIII was limited to late-eluting fractions when separations were carried out in antibody excess. A small proportion of the complexed anti-Factor VIII was present in void volume fractions; the quantity was directly related to the ratio of antibody to antigen.Thus, although some complexed anti-Factor VIII is detected in void volume fractions, as would be expected for complexes formed with a very large plasma protein, most immune complexes elute in fractions that indicate interaction with a smaller antigen. These findings suggest that human anti-Factor VIII inactivates procoagulant activity by forming a complex with a small, apparently univalent, component of Factor VIII. This property may prevent immunoprecipitate formation.


Asunto(s)
Anticuerpos/metabolismo , Complejo Antígeno-Anticuerpo/análisis , Factor VIII/inmunología , Cromatografía en Gel , Frío , Hemofilia A/inmunología , Humanos , Pruebas de Precipitina
8.
J Clin Invest ; 52(11): 2757-64, 1973 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-4583980

RESUMEN

Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system.


Asunto(s)
Antígenos , Endotelio/metabolismo , Factor VIII/biosíntesis , Animales , Células Cultivadas , Cicloheximida/farmacología , Factor VIII/análisis , Fibroblastos , Técnica del Anticuerpo Fluorescente , Humanos , Radioisótopos de Yodo , Leucina/metabolismo , Métodos , Músculo Liso/metabolismo , Pronasa/metabolismo , Biosíntesis de Proteínas , Conejos/inmunología , Radioinmunoensayo , Ácido Tricloroacético , Tritio , Tripsina/metabolismo
9.
J Clin Invest ; 53(5): 1375-84, 1974 May.
Artículo en Inglés | MEDLINE | ID: mdl-4596507

RESUMEN

Tissue localization of antihemophilic factor (AHF, factor VIII) antigen and fibrinogen by immunofluorescent microscopy was determined in 146 specimens of normal and diseased kidneys. AHF antigen was present in the endothelial cells of glomeruli, peritubular capillaries, arteries, and veins of normal kidneys; a distribution similar to that in other tissues. In scleroderma and malignant hypertension, deposition of AHF antigen and fibrinogen was limited to the markedly thickened endothelial layers of arteries. More extensive intense deposition of both AHF antigen and fibrinogen in glomeruli and in arterial walls were present in hyperacute renal homograft rejection, hemolyticuremic syndrome, postpartum renal failure, and in some cases of acute homograft rejection. In contrast, deposition of fibrinogen was observed in glomerular epithelial cresents in severe proliferative glomerulonephritis, but AHF deposition was not present in these lesions. Glomerular deposition of fibrinogen without increased AHF standing was also detected in renal tissue from patients with anaphylactoid purpura nephritis and in recurrent macroscopic hematuria with focal glomerulonephritis. Increased staining of peritubular capillaries with anti-AHF was seen in diseased kidneys irrespective of etiology. Immunofluorescent localization of AHF, a participant in the intrinsic coagulation pathway, offers a new way by which to analyze the mechanisms responsible for fibrinogen deposition in disease.


Asunto(s)
Factor VIII/análisis , Fibrinógeno/análisis , Riñón/análisis , Arterias/análisis , Biopsia , Capilares/análisis , Glomerulonefritis/patología , Síndrome Hemolítico-Urémico/patología , Humanos , Hipertensión Maligna/patología , Riñón/irrigación sanguínea , Enfermedades Renales/patología , Glomérulos Renales/análisis , Trasplante de Riñón , Microscopía Fluorescente , Púrpura/patología , Esclerodermia Sistémica/patología , Trasplante Homólogo , Venas/análisis
10.
J Clin Invest ; 89(5): 1375-81, 1992 May.
Artículo en Inglés | MEDLINE | ID: mdl-1569180

RESUMEN

We have recently identified the molecular defect responsible for cross-reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time-dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII-EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.


Asunto(s)
Cisteamina/farmacología , Factor VIII/genética , Coagulación Sanguínea/efectos de los fármacos , Activación Enzimática , Factor VIII/fisiología , Humanos , Mutación , Oxidación-Reducción , Compuestos de Sulfhidrilo/farmacología , Reactivos de Sulfhidrilo/farmacología , Trombina/metabolismo
11.
J Clin Invest ; 60(2): 390-404, 1977 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17621

RESUMEN

In normal plasma, the ratio of the procoagulant activity of factor VIII (VIII(AHF)) to that of the von Willebrand factor activity (ristocetin cofactor, VIII(VWF)) or factor VIII antigen (VIII(AGN)) is approximately 1, but ratios > 1 (e.g., VIII(AHF) > VIII(VWF) or VIII(AGN)) may be observed in some patients with von Willebrand's disease and in the "late" posttransfusion plasmas of patients with this disorder. The lability of VIII(AHF) was studied by incubating plasma, diluted 1:10 in imidazole buffer pH 7.1, for 6 h at 37 degrees C. With normal plasmas, 77+/-12% (SD) of the original VIII(AHF) activity remained after incubation. VIII(AHF) was labile (e.g., 35-55% residual activity) in the "late" posttransfusion plasmas (VIII(AHF) >> VIII(VWF)) of a patient with von Willebrand's disease, but not in the "early" posttransfusion plasmas (VIII(AHF) approximately VIII(VWF)). VIII(AHF) was also labile in the (base-line) plasmas of three patients with von Willebrand's disease in whom the ratios of VIII(AHF) to VIII(VWF) were 4.4 to 8.1, but not in the plasmas of four other patients in whom the ratio was approximately 1. The electrophoretic mobility of factor VIII antigen was increased in two of the three patients with labile VIII(AHF). In both of these patients, and in the late posttransfusion plasmas, labile VIII(AHF) activity could be stabilized by the addition of purified von Willebrand factor (lacking VIII(AHF) activity) or by hemophilic plasma, but not by plasmas of patients with severe von Willebrand's disease. Thus, VIII(VWF) may serve to stabilize VIII(AHF) and this might explain the posttransfusion findings in von Willebrand's disease.


Asunto(s)
Factores de Coagulación Sanguínea , Factor VIII , Enfermedades de von Willebrand/sangre , Factor de von Willebrand , Factores de Coagulación Sanguínea/aislamiento & purificación , Transfusión Sanguínea , Estabilidad de Medicamentos , Factor VIII/aislamiento & purificación , Factor VIII/metabolismo , Hemofilia A/sangre , Humanos , Concentración de Iones de Hidrógeno , Inmunoelectroforesis Bidimensional , Cinética , Unión Proteica , Enfermedades de von Willebrand/terapia , Factor de von Willebrand/aislamiento & purificación
12.
J Clin Invest ; 52(11): 2708-16, 1973 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-4542944

RESUMEN

In a previous paper, we showed that the abnormality of ristocetin-induced platelet aggregation in platelet-rich plasma in 10 patients with von Willebrand's disease could be corrected by a factor in normal plasma that was present in the same fractions as factor VIII procoagulant activity (antihemophilic factor, AHF, VIII(AHF)) when prepared by chromatography on Bio-Gel 5 M (Bio-Rad Laboratories, Richmond, Calif.). This observation suggests that patients with this disorder are deficient in a plasma factor, associated with the factor VIII molecule, that is necessary for normal platelet function. In the present paper, we describe, an assay for this factor, the von Willebrand factor (VIII(VWF)), based on the observation that a log-log relationship exists between the amount of ristocetin-induced aggregation of washed, normal platelets and the concentration of normal plasma present in the test system. We assayed the activity of VIII(VWF) as well as antihemophilic factor procoagulant activity (VIII(AHF)) and factor VIII antigen (VIII(AGN)) in 15 patients with von Willebrand's disease and 20 normal subjects. A highly significant correlation (r approximately 0.80) between VIII(VWF) and both VIII(AHF) was found in normal subjects and in patients with von Willebrand's disease. This finding, in addition to the observation that agarose gel chromatography fractions that have VIII(AHF) procoagulant activity also have VIII(VWF) activity, strongly suggests that the von Willebrand factor is associated with the factor VIII molecule. VIII(VWF) in normal plasma was not inhibited by human anti-VIII, and VIII(VWF) levels were normal in hemophilic plasma. Thus, the VIII(VWF) site on the factor VIII molecule appears to be different from that determining VIII(AHF). Finally, the activity of VIII(VWF) appeared to correlate better with the bleeding time than either VIII(AHF) or VIII(AGN). This suggests that VIII(VWF) assayed in this study may be the "anti-bleeding factor" that is deficient in von Willebrand's disease. These findings are consistent with a decreased synthesis of the factor VIII molecule in von Willebrand's disease and suggest the possibility of additional abnormalities of the site on the molecule that determines the activity of VIII(VWF).


Asunto(s)
Antígenos/análisis , Factor VIII/análisis , Adhesividad Plaquetaria , Enfermedades de von Willebrand/sangre , Adulto , Animales , Anticuerpos , Plaquetas/efectos de los fármacos , Cromatografía , Factor VIII/aislamiento & purificación , Femenino , Hemofilia A/sangre , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Adhesividad Plaquetaria/efectos de los fármacos , Conejos/inmunología , Ristocetina/farmacología
13.
J Clin Invest ; 57(6): 1618-25, 1976 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-1084352

RESUMEN

Epinephrine infusion causes variable increases in the components of the Factor VIII (antihemophilic factor) complex in patients with von Willebrand's disease. The increase in antihemophilic factor procoagulant activity was greater than that of Factor VIII-related antigen and von Willebrand factor activity in two patients with von Willebrand's disease. Similar increases in the three individual factors were demonstrated in two other patients. A 4-10-fold increase in Factor VIII-related properties was identified in each of these individuals after infusion. One patient has been studied with very severe von Willebrand's disease; none of the Factor VIII-related properties increased despite two infusions of epinephrine. Bleeding times were normalized or remained normal in the two patients whose von Willebrand factor activity was greater than 25 U/100 ml. It remained prolonged in those three patients whose von Willebrand factor activity levels remained below that concentration. The increase in procoagulant activity was transient in all patients and t 1/2 values were estimated to be between 0.8 and 3.4 h.


Asunto(s)
Epinefrina/farmacología , Factor VIII , Enfermedades de von Willebrand/sangre , Adulto , Antígenos , Coagulación Sanguínea/efectos de los fármacos , Epinefrina/administración & dosificación , Factor VIII/inmunología , Femenino , Humanos , Infusiones Parenterales , Masculino , Peso Molecular , Enfermedades de von Willebrand/inmunología , Factor de von Willebrand
14.
J Clin Invest ; 52(11): 2737-44, 1973 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-4201264

RESUMEN

The tissue localization of antihemophilic factor (AHF, Factor VIII) has been determined by immunofluorescent studies using monospecific rabbit antibody to human AHF. Specific staining demonstrating AHF antigens has been identified in endothelial cells of a wide range of human tissues. The staining pattern was observed in endothelial cells of arteries, capillaries, and veins as well as the cells lining hepatic and splenic sinusoids. Specific fluorescence was limited to these endothelial cells in sections of kidney, liver, spleen, lymph node, cardiac and smooth muscle, thyroid, umbilical cord, and skin. Absorption studies established that the staining was specific for cells in which there were proteins that had AHF antigens. The demonstration of fluorescence within the cytoplasm of endothelial cells suggests that these cells synthesize proteins that have AHF antigens.


Asunto(s)
Antígenos/análisis , Endotelio/inmunología , Factor VIII , Animales , Aorta/inmunología , Técnica del Anticuerpo Fluorescente , Cabras/inmunología , Humanos , Sueros Inmunes , Inmunodifusión , Inmunoelectroforesis , Riñón/inmunología , Hígado/inmunología , Ganglios Linfáticos/inmunología , Músculos/inmunología , Miocardio/inmunología , Vena Porta/inmunología , Conejos/inmunología , Piel/inmunología , Bazo/inmunología , Glándula Tiroides/inmunología , Ombligo/inmunología
15.
J Clin Invest ; 52(6): 1427-34, 1973 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-4634046

RESUMEN

The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant.


Asunto(s)
Antígenos/análisis , Hemofilia A/inmunología , Linfocitos/inmunología , Animales , Factores de Coagulación Sanguínea/análisis , Células Cultivadas , Centrifugación por Gradiente de Densidad , Reacciones Cruzadas , Factor VIII/análisis , Humanos , Isótopos de Yodo , Lectinas/farmacología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Linfocitos/efectos de los fármacos , Métodos , Filtros Microporos , Mycobacterium tuberculosis , Placenta/inmunología , Proteínas/farmacología , Conejos/inmunología , Radioinmunoensayo , Factores de Tiempo , Tritio
16.
J Clin Invest ; 93(6): 2497-504, 1994 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-8200986

RESUMEN

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Factor VIIIa/antagonistas & inhibidores , Animales , Factor VIIIa/metabolismo , Factor X/farmacología , Polarización de Fluorescencia , Humanos , Ratones
17.
Nat Biotechnol ; 15(10): 971-5, 1997 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9335047

RESUMEN

Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.


Asunto(s)
ADN Complementario/biosíntesis , Factor VIII/biosíntesis , Glándulas Mamarias Animales/metabolismo , Leche/química , Porcinos/genética , Animales , Animales Modificados Genéticamente , Coagulación Sanguínea/efectos de los fármacos , Dimerización , Factor VIII/genética , Factor VIII/farmacología , Femenino , Regulación de la Expresión Génica/genética , Hemofilia A/tratamiento farmacológico , Hemofilia A/economía , Humanos , Ratones , Proteínas de la Leche/genética , Proteínas Recombinantes/biosíntesis , Trombina
18.
Methods Enzymol ; 222: 169-76, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-8412792

RESUMEN

Immunopurification and characterization of dysfunctional factor VIII-like molecules in CRM-positive and CRM-reduced hemophilia A permit correlation of structural changes with molecular defects. The technique described here is sufficiently sensitive to characterize the molecular mass and enzymatic fragments of the factor VIII chains in patients with as little VIII: Ag as 0.05 units/ml. Specific abnormalities have been identified in 5 of the first 24 samples tested. In each case, the mutation responsible for factor VIII dysfunction has been determined by sequencing a part of the abnormal gene. Mutations have been identified that abolish critical thrombin cleavage sites or which generate new N-glycosylation sites. The technique provides a useful approach to the study factor VIII structure-function relationships, and it has the potential to clarify further the molecular basis of factor VIII procoagulant activity.


Asunto(s)
Factor VIII/genética , Hemofilia A/genética , Secuencia de Bases , ADN/genética , ADN/aislamiento & purificación , Electroforesis Discontinua/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Factor VIII/química , Factor VIII/aislamiento & purificación , Glicosilación , Hemofilia A/sangre , Humanos , Immunoblotting/métodos , Técnicas de Inmunoadsorción , Indicadores y Reactivos , Mutación , Ácidos Nucleicos Heterodúplex/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Trombina/metabolismo
19.
Am J Med ; 91(5A): 40S-44S, 1991 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-1746596

RESUMEN

Future progress in our ability to treat acquired factor VIII (FVIII) inhibitors must be based on advances in knowledge of both the FVIII molecule and the nature of the human immune response. New therapeutic approaches to patients with acquired FVIII inhibition likely will emphasize modifications of the immune response. This concept holds considerable promise, because studies have characterized the critical steps leading to tolerance of self-antigens. Development of FVIII inhibitors represents a loss of self-tolerance, which any successful therapy must restore. Conceivably, restoration of self-tolerance can be accomplished in many ways: prevention of antigen binding to helper T lymphocytes, deletion of self-antigen-reactive T cells, inhibition of major histocompatibility complex (MHC) recognition, or enhancement of the antigen-specific suppressor T lymphocyte population. Recent data have demonstrated that highly specific methods can suppress ongoing immune responses against defined autoantigens. Antibodies that inhibit T-cell activation, peptides that block self-antigen binding, and antibodies that inhibit MHC recognition all have been successful in modifying experimentally induced autoimmune diseases. Whether any of these immunotherapeutic approaches will be effective in the treatment of acquired FVIII inhibition remains to be determined. Until data from animal model systems establish the feasibility of immune intervention, scrutiny of other new therapeutic approaches to patients with spontaneous inhibitors will continue to be important. Administration of FVIII-bypassing procoagulant proteins shows promise, as does removal of inhibitors by affinity reagents, such as FVIII peptides containing relevant epitopes (antigenic sites). Farther on the horizon is development of recombinant FVIII molecules so modified as to remove antigenic determinants while preserving procoagulant function. Articles in this supplement summarize several avenues for treatment of patients with acquired FVIII inhibitors. Alternatives include treatment with sufficient human or porcine FVIII to offset inhibitors, use of materials that reestablish hemostasis even though FVIII levels are not increased (the so-called FVIII-bypassing agents), manipulation of immune responses through physical depletion of inhibitor by plasmapheresis or affinity chromatography, and administration of intravenous immunoglobulin or immunosuppressive cytotoxic drugs. Thus, the heterogeneous clinical presentation is paralleled by the wide range of available therapeutic approaches.


Asunto(s)
Autoanticuerpos/análisis , Enfermedades Autoinmunes/terapia , Factor VIII/inmunología , Humanos , Inmunoterapia , Linfocitos T/inmunología
20.
Am J Med ; 62(3): 452-8, 1977 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-300225

RESUMEN

The clinical and laboratory findings in a patient with uncontrolled gastrointestinal bleeding secondary to combined hemostatic defects (von Willebrand's disease and hemorrhagic telangiectasia) are described. Evidence for von Willebrand's disease was found in five family members, but no other affected relative was found to have hemorrhagic telangiectasia. Complete assestivity, factor VIII antigen and von Willebrand factor levels. The patient described also was evaluated for her response to transfusion utilizing these same measurements. Previous reports of the coexistence of hemostatic defects with hereditary hemorrhagic telangiectasia are reviewed. The importance of complete hemostatic evaluation of patients with mucocutaneous bleeding is stressed in light or current knowledge of the diagnostic specificity of available laboratory tests.


Asunto(s)
Hemorragia Gastrointestinal/etiología , Telangiectasia Hemorrágica Hereditaria/complicaciones , Enfermedades de von Willebrand/complicaciones , Pruebas de Coagulación Sanguínea , Factor VIII/análisis , Femenino , Hemorragia Gastrointestinal/sangre , Humanos , Persona de Mediana Edad , Linaje , Recurrencia , Telangiectasia Hemorrágica Hereditaria/genética , Enfermedades de von Willebrand/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA