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B Kudryk

Publications and source records attributed to B Kudryk.

At least 37 records · Page 2Linked to original sources
PubMed1987

Endogenous forms of fibrinogen in Hep G2 cells.

The three polypeptide chains of fibrinogen, A alpha, B beta and gamma chain, are synthesized on separate polysomes. Fully formed fibrinogen is a six chain, disulfide-linked, dimeric molecule with a molecular weight of 340kDa. Previous pulse-chase studies with L-35 S methionine using the human hepatocellular carcinoma cell line, Hep-G2, showed that the three chains are not immediately disulfide-linked and that there exist intermediate precursors as well as pools of A alpha and gamma chains (J. Biol. Chem. 259, 10574-10581, 1984). In this study the endogenous levels of fibrinogen and its precursors are measured by two different methods; pulse and steady-state labelling with L-35 S-methionine and immunoblotting. In Hep-G2 cells intracellular fibrinogen-related antigen is primarily (30-53%) composed of an A alpha-gamma complex and, to a smaller degree, of fully-formed fibrinogen (13-33%). Furthermore, the Hep G2 cell also contains endogenous pools of free gamma chain (11-26%). Other fibrinogen precursors (namely, the B beta-A alpha, B beta-gamma complexes as well as the fibrinogen half-molecule) do not appear to accumulate intracellularly. Most, if not all, of these precursors occur as isoforms but this heterogeneity is not due to varying degrees of glycosylation. In all the intracellular fibrinogen forms identified thus far, free sulfhydryl groups, detected by 14C-iodoacetamide incorporation, occur only in the A alpha-gamma complex and the free gamma chains.

Carcinoma, Hepatocellular↗
PubMed1986

Fibrinogen Aarhus--a new case of dysfibrinogenemia.

Fibrinogen Aarhus was found in a woman with slightly prolonged whole blood clotting time. The thrombin induced clotting of plasma and purified fibrinogen was much prolonged. Kinetic analysis of FPA and FPB release revealed larger apparent Km and Vmax values for fibrinogen Aarhus than for normal fibrinogen. No clot formation of fibrinogen Aarhus was demonstrated in the presence of Batroxobin and the release of FPA was slower than normal. Upon addition of the clotting enzyme from Agkistrodon contortrix contortrix clotting did occur but the clotting time was much prolonged in comparison with normal fibrinogen. The turbidity of fibrin gels obtained from fibrinogen Aarhus was similar to normal fibrin gels at low thrombin concentrations. Increasing thrombin concentration resulted in appearance of degradation products in the fibrin gels from fibrinogen Aarhus and at the same time a relative increase in turbidity of the gels was observed. Possibly reasons for the slow release of fibrinopeptides, the delayed gelation, and susceptibility to degradation by thrombin are discussed.

Adult↗
PubMed1985

Radioimmunoimaging of venous thrombi using iodine-131 monoclonal antibody.

Murine monoclonal antibody (Mab) specific for the NH2-terminal region of human fibrin, but not cross-reactive with fibrinogen, was used in radioimmuno-imaging of fresh, induced venous thrombi in three dogs. Iodine-131-labeled Mab was injected intravenously, with iodine 125-labeled polyclonal murine gamma-G globulin (IgG) simultaneously injected as a control. Images were strongly positive at 24 and 48 hours in all three animals, with thrombus-to-blood and thrombus-to-muscle ratios of 8.4 and 228.0, respectively, for I-131-labeled Mab; these ratios for control IgG were 1.2 and 13.0. Radioimmunodetection of thrombi in vivo is feasible in dogs and may have clinical application since Mab is specific to human fibrin.

Animals↗
PubMed1984

Fibrinogen precursors. Order of assembly of fibrinogen chains.

Hep-G2 cells, incubated with L-[35S]methionine, incorporate radioactivity into fibrinogen and several fibrinogen-related compounds. Pulse-chase experiments indicate that several of these compounds are precursors of fibrinogen and that the cells contain intracellular pools of A alpha and gamma chains which participate in the assembly of fibrinogen. The rate of synthesis of the three component chains of fibrinogen is unequal with that of the B beta chain being less than that of the A alpha and gamma chains. The sequence of events which lead to the assembly of fibrinogen was deduced by determining the appearance of the radioactive chains in each of the fibrinogen precursors and in fibrinogen at various times during a pulse-chase incubation. Fibrinogen assembly commences while nascent incomplete B beta chains are attached at polysomes. Preformed A alpha and gamma chains, drawn from the intracellular pool, combine independently to the growing B beta chains. On completion of the nascent B beta chains, newly formed B beta-A alpha and B beta-gamma complexes are released from the polysomes and enter the luminal space of the endoplasmic reticulum. Later other A alpha and gamma chains are added by ordered disulfide interaction, leading to the eventual formation of dimeric fibrinogen.

Carcinoma, Hepatocellular↗
PubMed1984

Specificity of a monoclonal antibody for the NH2-terminal region of fibrin.

A monoclonal antibody (MAb/T2G1s) was prepared by fusion using spleen cells from mice immunized with the NH2-terminal CNBr fragment of human fibrin II, the so-called (T)N-DSK [(A alpha 17-51, B beta 15-118, gamma 1-78)2]. In competition experiments, this antibody reacted with (T)N-DSK as well as peptide B beta 15-42 which can be obtained from (T)N-DSK by digestion with plasmin. Little or no reaction was observed with intact fibrinogen, the NH2-terminal CNBr fragments from fibrinogen (N-DSK) or fibrin I [(B)N-DSK], respectively, as well as peptide B beta 1-42. These results suggest that MAb/T2G1s is directed to an epitope on the B beta chain in fibrin II but not in fibrinogen or fibrin I. As such, MAb/T2G1s differs completely from another antibody (MAb/1-8C6)--also specific for the NH2-terminal region of the B beta chain--which was recently described [Kudryk et al. (1983) Molec. Immun. 20, 1191-1200].

Animals↗
PubMed1983

Intracellular assembly of human fibrinogen.

Hep-G2 cells, pulse-labeled with L-[35S]methionine, incorporate radioactivity within 2 min into precursor forms of fibrinogen and into fibrinogen. Pulse-labeled intracellular fibrinogen is first composed of radioactive B beta chains, followed by nascent A alpha chains. Radioactive gamma chains accumulate in the cells and later contribute, via intermediate forms, to the assembly of fibrinogen. Following a pulse-chase incubation with L-[35S]methionine, the radioactive composition of newly secreted fibrinogen also reflects the fact that there is a large intracellular pool of gamma chains.

Carcinoma, Hepatocellular↗
PubMed1983

Thrombin activity during hemodialysis; evaluated by the fibrinopeptide A assay. A comparison between a high and a low heparin dose regime.

Ten patients participated in this study which evaluated the effect of two heparin dose regimes, a high dose regime (mean dose 6750 IU) and a low dose regime (mean dose 3750 IU), on the thrombin activity, achieved during a 4-hour-dialysis. The thrombin activity was measured by use of the FPA assay. Heparin was administered as bolus dose followed by a constant rate infusion which was discontinued 1.5-2 hours prior to the end of the dialysis. Both dose regimes inhibited thrombin activity equally effectively as long as heparin was administered. In the high dose regime, the FPA levels remained unchanged until the end of the dialyses in all patients. In the low dose regime, six patients had the same FPA values at the end of the heparin infusion and at the end of the dialysis. In the remaining four patients much higher FPA levels were achieved at the end of the dialysis. No serious bleeding or clotting complications occurred, and all dialyses were uneventful.

Adult↗
PubMed1983

A monoclonal antibody with ability to distinguish between NH2-terminal fragments derived from fibrinogen and fibrin.

A stable hybridoma secreting homogeneous antibody (immunoglobulin class IgG2a) has been prepared by fusion using cells of immunoglobulin non-secreter myeloma (P3X63Ag8.653) and spleen cells of mice which had previously been immunized with the NH2-terminal CNBr fragment of human fibrinogen, the so-called N-DSK [(A alpha 1-51, B beta 1-118, gamma 1-78)2]. In competitive ELISA or radioimmunoassay this antibody (MAb/1-8C6) cross-reacted with intact fibrinogen, N-DSK, a des fibrinopeptide A (des FPA) variant of N-DSK, the so-called (B)N-DSK, as well as the intact B beta chain (B beta 1-118) obtained from N-DSK. Also, and mot importantly, cross-reactivity was observed with fibrinogen-free ethanol extracts of plasma obtained from patients known to contain high levels of fibrinogen or fibrin degradation products. In vitro thrombin digestion of any of these competitors resulted in complete loss of cross-reactivity. MAb/1-8C6 did not react with the A alpha or gamma-chains of N-DSK, free fibrinopeptide B(FPB), free B beta 15-42, as well as equimolar mixtures of the latter two peptides. These results suggest that MAb/1-8C6 may be to an epitope in or around the thrombin-susceptible B beta 14 Arg-25 Gly bond. Furthermore, due to its reactivity with patient plasma extracts, this antibody may be useful in clinical investigations dealing with fibrino(geno)lysis.

Antibodies, Monoclonal↗
PubMed1982

Measurement of fibrinogen in dog liver cell fractions by electroimmunoassay and radioimmunoassay.

Dog liver was fractionated and the amount of fibrinogen, in the various cell fractions (organelles) involved in the secretory process, was determined both by the Laurell electroimmunoassay and by radioimmunoassay. An unequal distribution of fibrinogen was noted for each cell fraction after an appropriate correction was made. This involved the estimation of the level of contamination of each cell fraction by soluble (blood) fibrinogen. It was observed that significant amounts of added 125I-labeled fibrinogen could be found in each organelle following cell fractionation. This was especially true for the rough endoplasmic reticulum which contained as much as 31% of the added tracer. Both the smooth endoplasmic reticulum and the Golgi fractions contained much lower amounts of added tracer. Using this correction factor, it was found that the rough endoplasmic reticulum contains about 0.1-0.2 micrograms fibrinogen per mg organelle protein. On the other hand, the smooth endoplasmic reticulum and Golgi fractions contained much more fibrinogen. The fibrinogen concentration ranges found for the latter two organelles were 1.9-5.0 and 1.5-5.7 micrograms fibrinogen per mg organelle protein, respectively.

Animals↗
PubMed1982

Measurement in human blood of fibrinogen/fibrin fragments containing the B beta 15-42 sequence.

This paper describes a radioimmunoassay for the B beta 15-42 peptide derived from human fibrinogen or fibrin. Iodinated B beta 15-42 is bound by specific antiserum and binding can be completely inhibited by excess of the non-iodinated B beta 15-42, B beta 1-42 or B beta 1-118. These peptides cannot be distinguished in this assay. Furthermore, fibrinogen can also completely inhibit binding of iodinated B beta 15-42 peptide. Due to the cross-reaction with fibrinogen, clinical blood samples require a processing step prior to their use in this radioimmunoassay. Ethanol precipitation allows for fibrinogen removal and a near quantitative recovery of both added B beta 15-42 peptide as well as of the endogenous blood peptide(s) containing the B beta 15-42 sequence. The mean level of B beta 15-42 immunoreactive material in normal individuals was found to be 0.41 pmol/ml while that in one group of patients was 20-40 times this value.

Adult↗
PubMed1981

Measurement of fibrinopeptide A in the evaluation of heparin activity and fibrin formation during hemodialysis.

In order to monitor heparin activity during hemodialysis, were evaluated three commonly used methods; measurement of whole blood activated coagulation time (WBACT), whole blood thrombin time (WBTT) and heparin concentration in plasma, determined with a chromogenic substrate. Studies were performed on six regular dialysis patients during 4-hour dialysis sessions, employing three different heparin regimens; a single intravenous loading dose only, priming of the dialyzer with heparin followed by a heparin infusion and a pharmaco-kinetic model. Efficacy of heparinization was assessed by determination of fibrinopeptide A (FPA) which is a peptide split product of the fibrinogen molecule formed during thrombin-induced conversion to fibrin. There was a linear correlation (r = 0.95) between FPA-production in the dialyzer and the FPA content of the blood at the inlet to the dialyzer; the slope of the correlation line indicates that at least 65% of FPA formed in the dialyzer is disposed during passage through the patient. Considerably higher production of FPA was noted when the heparin concentration was below 0.5 IU/ml than at a higher level. This was a common finding at the end of a dialysis, regardless of regimen. No consumption of antithrombin (AT III) occurred during a dialysis with any of the heparin regimens. Good correlations were found between WBACT, WBTT and heparin concentration. Heparin activity during a dialysis may be monitored with any of these three methods with equal reliability. However, from a practical point of view, WBACT appears most attractive because of its simplicity. FPA generation, frequency of visible clots in the dialyzer and hemorrhagic manifestations were essentially the same for each of the heparin dose regimens. The simple administration of a single loading dose was as safe as the more complicated infusion technique.

Female↗
PubMed1975

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.

Amino Acid Sequence↗