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Biomedical subjects

B Furie

Publications and source records attributed to B Furie.

At least 145 records · Page 8Linked to original sources
PubMed1984

Comparison of the native prothrombin antigen and the prothrombin time for monitoring oral anticoagulant therapy.

We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.

Administration, Oral↗
PubMed1983

Immunoassays of human prothrombin species which correlate with functional coagulant activities.

Specific immunoassays have been developed for forms of human prothrombin that vary in their degree of carboxylation. Human abnormal (des-gamma-carboxyl) prothrombin was isolated in 18% yield from the plasma of a patient treated with warfarin. The purified protein migrated as a single band in electrophoresis and contained an average of three gamma-carboxyglutamic acid residues per molecule. A specific antibody subpopulation was isolated from rabbit anti-abnormal prothrombin antiserum by using affinity chromatography. These antibodies, which bound to abnormal prothrombin but which cross-reacted minimally with prothrombin, were used to establish an immunoassay specific for abnormal prothrombin. In parallel, a specific antibody subpopulation, anti-prothrombin: Ca(II), was isolated from rabbit anti-prothrombin antiserum by conformation perturbation affinity chromatography. This antibody, which bound prothrombin but minimally cross-reacted with abnormal prothrombin, was used to establish a specific immunoassay for native prothrombin. An anti-prethrombin 1 subpopulation bound abnormal prothrombin and prothrombin equivalently and was used for an immunoassay that measured total prothrombin. These assays permit the quantitation of abnormal prothrombin and prothrombin in plasma and serum. The level of native prothrombin antigen correlates precisely with the functional prothrombin activity. These assays provide an example of the use of specific antibodies against functionally important antigenic surfaces to monitor properties of coagulation proteins with the precision and reliability of immunoassy.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies.

Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.

Animals↗
PubMed1982

Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases.

Computer-generated molecular models of the trypsin-like domains of blood coagulation factor IXa, Factor Xa, and thrombin have been prepared. These hypothetical models are based upon the sequence homology of the blood coagulation enzymes with the pancreatic serine proteases and the known three-dimensional structure of the pancreatic serine proteases. The internal structures and active sites of these enzymes are highly conserved. The high degree of substrate specificity which characterizes the blood coagulation enzymes appears to be defined not entirely by the active site, but by the unique molecular surface surrounding the active site of each enzyme. Several regions which demonstrate high sequence variability among these enzymes likely participate in forming the putative extended substrate binding sites.

Amino Acid Sequence↗
PubMed1982

Studies on a family with combined functional deficiencies of vitamin K-dependent coagulation factors.

Two siblings with m ild hemorrhagic symptoms had combined functional deficiencies of vitamin K-dependent clotting factors. Prothrombin (0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) were most severely affected. Antigenic amounts of affected coagulation factors were normal and normal generation of thrombin activity occurred in the patients' plasmas after treatment with nonophysiologic activators that do not require calcium for prothrombin activation. Hepatobilary disease, malabsorptive disorders, and plasma warfarin were not present. Both parents had normal levels of all coagulation factors. The patients' plasmas contained prothrombin that reacted both with antibody directed against des-gamma-carboxyprothrombin and native prothrombin. Crossed immunoelectrophoresis of patients' plasmas and studies of partially purified patient prothrombin suggested the presence of a relatively homogeneous species of dysfunctional prothrombin, distinct from the heterologous species found in the plasma of warfarin-treated persons. These studies are most consistent with a posttranslational defect in hepatic carboxylation of vitamin K-dependent factors. This kindred uniquely possesses an autosomal recessive disorder of vitamin K-dependent factor formation that causes production of an apparently homogeneous species of dysfunctional prothrombin; the functional deficiencies in clotting factors are totally corrected by oral or parenteral administration of vitamin K1.

Adolescent↗
PubMed1982

Production of autoantibodies by human-human hybridomas.

Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.

Anemia, Hemolytic, Autoimmune↗
PubMed1981

Gamma-carboxyglutamic acid.

Gamma-carboxyglutamic acid is an amino acid with a dicarboxylic acid side chain. This amino acid, with unique metal binding properties, confers metal binding character to the proteins into which it is incorporated. This amino acid has been discovered in blood coagulation proteins (prothrombin, Factor X, Factor IX, and Factor VII), plasma proteins of unknown function (Protein C, Protein S, and Protein Z), and proteins from calcified tissue (osteocalcin and bone-Gla protein). It has also been observed in renal calculi, atherosclerotic plaque, and the egg chorioallantoic membrane, among other tissues. Gamma-carboxyglutamic acid is synthesized by the post-translational modification of glutamic acid residues. This reaction, catalyzed by a hepatic carboxylase, requires reduced vitamin K, oxygen, and carbon dioxide. The function of gamma-carboxyglutamic acid is uncertain. In prothrombin gamma-carboxyglutamic acid residues bound to metal ions participate as an intramolecular non-covalent bridge to maintain protein conformation. Additionally, these amino acids participate in the calcium-dependent molecular assembly of proteins on membrane surfaces through intermolecular bridges involving gamma-carboxyglutamic acid and metal ions.

1-Carboxyglutamic Acid↗
PubMed1981

Acquired vitamin K-dependent carboxylation deficiency in liver disease.

gamma-Carboxyglutamic acid residues on prothrombin are synthesized from glutamic acid on a prothrombin precursor in the liver through a vitamin K-dependent carboxylase. In the absence of vitamin K or in the presence of vitamin K antagonists, an inert form of prothrombin - abnormal prothrombin - circulates in the blood. We have developed specific immunoassays for native and abnormal human prothrombin. The prothrombin concentration in our normal subjects was 108 +/- 19 microgram per milliliter. The abnormal-prothrombin concentration varied over four orders of magnitude between the limits of detection in normal plasma and the level in patients with cirrhosis (0 to 5 microgram per milliliter), acute hepatitis (0 to 33 microgram per milliliter), or vitamin K deficiency (32 to 100 microgram per milliliter) and in those treated with sodium warfarin (12 to 65 microgram per milliliter). These studies indicate that abnormal prothrombin is not a component of normal plasma but appears in a variety of hepatic and nutritional disorders characterized by impaired hepatic vitamin-K-dependent carboxylation.

Adult↗
PubMed1981

Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids.

Hybridomas the produce anti-DNA autoantibodies were prepared from spleen cells of unimmunized MRL/1 mice, a strain that spontaneously develops severe systemic lupus erythematous (SLE). Reactivities of these monoclonal antibodies with a wide range of polynucleotides prompted tests of their reactions with phospholipids which, like polynucleotides, contain diester-linked phosphate groups in their backbones. In competitive radioimmunoassays, cardiolipin, phosphatidic acid, and phosphatidyl glycerol blocked the binding of these hybridoma antibodies to denatured DNA. These phospholipids also specifically inhibited the reaction between a hybridoma antibody and a site-specific anti-idiotypic antibody. The antinuclear reaction of one of these antibodies was specifically inhibited by cardiolipin. This same antibody prolonged the activated partial thromboplastin time in a manner characteristic of a lupus anticoagulant, presumably by binding to phospholipid in the test system. The polyspecific reactivity of a single molecular species of lupus autoantibody suggests that some of the diverse serological abnormalities of SLE may be a result of the binding of certain autoantibodies to a phosphodiester-containing epitope that is present in diverse biological molecules.

Animals↗
PubMed1981

A computer-generated three-dimensional model of the B chain of bovine alpha-thrombin.

A computer graphic molecular display system has been used to construct a three-dimensional model of the B chain of bovine thrombin. The model is derived from the bovine alpha-chymotrypsin structure as determined by X-ray crystallographic studies. The amino acid sequence of bovine thrombin has been substituted for that of alpha-chymotrypsin, preserving the beta-barrel structure and maximizing homology of the amino acid sequence of the two proteins. With the exception of an area in the vicinity of the specificity binding pocket, most of the changes observed in thrombin occur on the surface of the molecule. The most notable changes observed in the model are the increases on the surface of positively charged (arginine and lysine) and negatively charged (glutamate and aspartate) residues. A glutamate replaces methionine 192 near the entrance to the specificity binding pocket. The nature of this site was further altered by the substitution of an aspartate for serine 189 and an alanine for serine 190. The structure of the resulting specificity binding pocket is consistent with that of serine proteases, which have trypsin-like substrate specificity. The computer graphics molecular display system has been used to insert models of synthetic thrombin inhibitors into the active site of the thrombin B chain model. With the model, it has been possible to correlate the interaction of thrombin with the observed binding constants of two inhibitors of trypsin-like serine proteases, p-amidinophenylmethylsulfonylfluoride (Ki = 1.27 x 10(-6) M) and m-[m-(trifluoromethyl)phenoxypropoxy]benzamidine (KD = 2.9 x 10(-6) M).

Amino Acids↗
PubMed1980

Thrombotic stroke associated with elevated plasma factor VIII.

A previously healty 43-year-old woman developed bilateral middle cerebral artery occlusion, thrombocytopenia, and multiple thrombi of arteries in the spleen. No underlying cause of her thrombotic disease was detected except for a factor VIII level that was elevated fivefold. An etiological relationship between the cerebral occlusive disease and elevated factor VIII level is suggested. We recommend that factor VIII levels be assayed in patients with stroke when an underlying disorder is not identified.

Adult↗
PubMed1980

Zymogens and cofactors of blood coagulation.

Blood coagulation is a system in which a series of zymogens of serine proteases are sequentially activated. In this regard, there is little fundamental difference between coagulation and the activation of the homologous pancreatic zymogens. There are, however, several aspects unique to coagulation which are discussed in detail. These are (1) the requirement for a high-molecular-weight protein or lipoprotein cofactor for optimal reaction rates, (2) the requirement for membranes or a membrane-like surface which further distinguishes this system; (3) a metal ion requirement for most reactions (in contrast to the pancreatic serine proteases) relating to the content of the newly described amino acid gamma-carboxyglutamic acid in the four vitamin K-dependent proteins, regarding which recent data relating to the metal binding sites on prothrombin are discussed in detail; and (4) the uniqueness of the initiating reactions in comparison to those which activate the pancreatic zymogens, insofar as no enzyme corresponding to enterokinase has been identified. The implications of this phenomenon are analyzed with particular attention to the potential role of the endogenous activity of certain zymogens in initiating coagulation. The article deals finally with the specific problems attendant on analyzing a system in which many serine proteases lacking absolute specificity are generated and regulated.

1-Carboxyglutamic Acid↗