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Biomedical subjects

B Kudryk

Publications and source records attributed to B Kudryk.

At least 19 recordsLinked to original sources
PubMed2001

Genetic and immunological characterization of fibrinogen inclusion bodies in patients with hepatic fibrinogen storage and liver disease.

Fibrinogen storage in liver cells can occur under three different morphological inclusions. Type I contain all three fibrinogen chains (A alpha, B beta, and gamma) as well as D and E fragments, whereas type II and III lack B beta as well as D and E fragments. Patients with type I inclusions carry a point mutation (gamma 284 Gly-Arg). The mutation is not present in patients with type II and III inclusions. These results appear to suggest that the three various phenotypic expressions (i.e., morphological variants) reflect different genetical abnormalities of fibrinogen.

Fibrinogen↗
PubMed2001

A hamster antibody to the mouse fibrinogen gamma chain inhibits platelet-fibrinogen interactions and FXIIIa-mediated fibrin cross-linking, and facilitates thrombolysis.

Murine models employing genetically altered mice have the potential to provide important new information about the hemostatic system, but before such data can be extrapolated to humans it is necessary to define the similarities and differences between murine and human hemostasis. After establishing the similarities of murine fibrinogen to human fibrinogen in its pattern of proteolysis in response to plasmin and its cross-linking by factor XIIIa, we studied a new hamster monoclonal antibody (mAb) 7E9 that reacts with the gamma chain of mouse fibrinogen. This antibody inhibits platelet adhesion to fibrinogen, platelet-mediated clot retraction, platelet aggregation, and FXIIIa-mediated cross-linking of fibrin; it also facilitates tissue plasminogen activator (tPA)-mediated lysis of fibrin formed either in the absence or presence of platelets. These data provide evidence that the C-terminus of mouse fibrinogen gamma chain, like that of human fibrinogen, is involved in fibrinogen binding to platelets and FXIIIa-mediated cross-linking of fibrin. Our data raise the possibility that a therapeutic agent that targets the C-terminus of the gamma chain in human fibrinogen might have broad antithrombotic and profibrinolytic effects.

Animals↗
PubMed2000

Does prosthetic covering of nitinol stents alter healing characteristics or hemodynamics?

PURPOSE: To evaluate placement of polyester (Dacron) coverings on nitinol stents implanted in the canine aorta to determine the effect on cross-sectional lumen area, development of intimal hyperplasia, device endothelialization, and flow hemodynamics. METHODS: Ten polyester-covered and 10 uncovered nitinol stents (60-mm length, 10- or 12-mm diameter) were deployed percutaneously in the normal infrarenal aorta of 20 adult mongrel dogs using random assignment. Angiography, intravascular ultrasound (IVUS), and duplex ultrasound performed at device deployment and before explantation at 6 weeks were used to measure aorta/device diameter and cross-sectional area. Pressure-perfusion-fixed aortic segments were compared for surface endothelialization (CD31 staining) and for thickness of neointimal formation. RESULTS: All 20 endoluminal devices were accurately positioned in the infrarenal aorta without early or delayed evidence of device thrombosis, significant lumen narrowing, or device deformity. IVUS and duplex scanning identified no anatomical stenosis in either the covered or the bare devices by duplex ultrasound; peak systolic velocity measurements were similar (106+/-25 cm/s in the covered stent versus 96+/-25 cm/s for bare stents, p > 0.05). Mean neointimal thickness was significantly greater (p < 0.005) in the covered (326+/-145 microm) compared with the bare (219+/-62 microm) stents. Intima-to-media height ratios were greater in the covered stents (3.0+/-1.1 compared with 1.1+/-0.2, p < 0.003). Mean surface area endothelialization in the proximal, middle, and distal sections of each device was similar (p > 0.05) in covered (59%, 56%, and 69%) and bare (59%, 65%, and 53%) stents. CONCLUSIONS: Deployment and balloon dilation of a covered nitinol stent in a nondiseased canine aorta increased neointimal development compared with an uncovered stent, but overall lumen cross-sectional area was preserved. No differences in device patency, intradevice thrombus formation, flow hemodynamics, or luminal endothelialization were demonstrated, despite a thicker intradevice neointima induced by the polyester covering.

Alloys↗
PubMed1998

Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer.

Thrombin-catalyzed, cross-linked fibrin (XLF) formation is a characteristic histopathological finding in many human and experimental tumors and is thought to be of importance in the local host defense response. Although the pathogenesis of tumor-associated fibrin deposition is not entirely clear, several tumor procoagulants have been described as likely primary stimuli for the generation of thrombin (and XLF) in the tumor microenvironment (TME). In a previous study of a variety of human tumors we have shown that tissue factor (TF) is the major procoagulant. However, the relative contribution to fibrin deposition in the TME of tumor cell TF and host cell TF (eg, macrophage-derived) was not established. In addition, recent evidence has implicated TF in the regulation of the synthesis of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells. In the current study we used in situ techniques to determine the cellular localization of XLF, TF, VEGF, and an alternative tumor procoagulant, so-called cancer procoagulant (CP), a cysteine protease that activates clotting factor X. In lung cancer we have found XLF localized predominantly to the surface of tumor-associated macrophages, as well as to some endothelial cells and perivascular fibroblasts in the stromal area of the tumors co-distributed with TF at the interface of the tumor and host cells. Cancer pro-coagulant was localized to tumor cells in several cases but not in conjunction with the deposition of XLF. TF and VEGF were co-localized in both lung cancer and breast cancer cells by in situ hybridization and immunohistochemical staining. Furthermore, a strong relationship was found between the synthesis of TF and VEGF levels in human breast cancer cell lines (r2 = 0.84; P < 0.0001). Taken together, these data are consistent with a highly complex interaction between tumor cells, macrophages, and endothelial cells in the TME leading to fibrin formation and tumor angiogenesis.

Blood Coagulation↗
PubMed1998

Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading.

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.

Antibodies, Monoclonal↗
PubMed1995

Secretion of biologically active recombinant fibrinogen by yeast.

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation.

Animals↗
PubMed1993

Fibrin--recombinant human factor XIII a-subunit association.

The association of factor XIII a-subunits with fibrin was characterized using recombinant human placental factor XIII (rFXIII) and native fully hydrated fibrin clots formed from purified fibrinogen and thrombin. Binding was assessed using small columns containing fibrin and perfusing them with radioiodinated rFXIII. Results show that thrombin activation of rFXIII led to fibrin binding. The association was partially reversible since much of the bound enzyme could be removed by percolating clots with more buffer. Binding was blocked by antibody directed against the COOH-terminal part of fibrinogen A alpha-chain (A alpha 389-402) and also by the COOH-terminal A alpha-chain peptide fragment A alpha 241-476 (Hi2-DSK).

Antigen-Antibody Reactions↗
PubMed1993

Symmetrical disulfide bonds are not necessary for assembly and secretion of human fibrinogen.

Human fibrinogen is a dimer, and each half-molecule is composed of three different polypeptides (A alpha, B beta, and gamma). The two half-molecules are joined together at the amino-terminal ends by three symmetrical disulfide bonds between adjacent A alpha chains, at position 28, and between adjacent gamma chains at gamma 8 and 9. To determine the role of these disulfide bonds in fibrinogen assembly and secretion, site-directed mutagenesis was used to change cysteines of A alpha 28 and gamma 8 and 9 to serine, the mutant chains were coexpressed with normal fibrinogen chains in COS cells, and assembly and secretion of fibrinogen were measured. Elimination of the symmetrical disulfide bonds did not affect assembly of the chains and dimeric fibrinogen was secreted. Analysis by plasmin digestion indicated that the secreted mutant fibrinogens have a similar structure to normal fibrinogen. Our results indicate that other domains of fibrinogen participate in dimer formation and that the three symmetrical disulfide bonds are not crucial for assembly and secretion of fibrinogen.

Animals↗
PubMed1992

Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.

Alkylation↗
PubMed1991

Accessibility of epitopes on fibrin clots and fibrinogen gels.

Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241-476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4-2/xl-f, anti gamma 392-406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15-21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1-reactive epitopes. An antibody that does not recognize fibrin (antibody 1-8C6, antiB beta 1-21) predictably did not bind to fibrin clots and bound to 35% of the 1-8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

Antibodies, Monoclonal↗
PubMed1991

Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces.

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.

Adsorption↗
PubMed1990

Fibrin and the vessel wall.

Fibrin is a major component of atherosclerotic plaques, and there may also be situations in which intravascular fibrin is formed in contact with the endothelium. The studies to be presented describe the distribution of fibrinogen/fibrin I, fibrin II, and fragments D and D-dimer in normal vessels and atherosclerotic plaques of increasing severity and also describe some functional effects of fibrin on normal endothelium. Immunohistochemical studies using three specific monoclonal antibodies with the avidin-biotin complex immunoperoxidase technique demonstrated that little fibrinogen/fibrin I or fibrin II and no D/D-dimer were detected in normal aortas. In early lesions and in fibrous plaques, fibrinogen/fibrin I and fibrin II were distributed in long threads and around vessel wall cells. D/D-dimer was not seen in early lesions. In advanced plaques all three molecular forms were detected in areas of loose connective tissue, in thrombi, and around cholesterol crystals. Thus increased fibrin formation and degradation may be associated with progression of atherosclerotic disease. Additionally, the presence of fibrin II around vessel wall cells suggests that these cells may be involved in the fbgn to fibrin transition within the vessel wall. The second aspect of the work to be presented concerns effects of fibrin on vascular endothelium. Fibrin formed on the surface of cultured human umbilical vein endothelial cells stimulated production of prostacyclin and tissue plasminogen activator by the cells in a time- and dose-dependent manner. Stimulation of prostacyclin was completely inhibited by indomethacin and partially inhibited by actinomycin D, cycloheximide, and trifluoperazine, while stimulation of t-PA synthesis was completely inhibited by actinomycin D and cycloheximide and partially inhibited by cytochalasin D, vinblastine, and trifluoperazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Use of a synthetic homologue of human fibrinopeptide A for production of a monoclonal antibody specific for the free peptide.

It has been shown that epitopes reactive with one group of rabbit antibodies to human fibrinopeptide A (hFPA, A alpha 1-16) are included in its COOH-terminal region (A alpha 7-16). It was further established that Asp-7, Phe-8, and Arg-16 contribute to immunoreactivity and that intact fibrinogen and hFPA-containing fragments react poorly with such antibodies. The purpose of this investigation was to prepare a synthetic peptide corresponding to A alpha 7-16 and use it for generation of FPA-specific monoclonal antibodies (MoAbs). Such probes would allow for development of assays that could measure hFPA directly in plasma. In our approach, an ovalbumin-conjugate of the hFPA homologue served as immunogen. Mouse spleen cells were fused with the immunoglobulin nonsecretor myeloma (P3X63Ag8.653). A hybridoma (8C2-5) has been isolated that secretes an antibody (MoAb/8C2-5) with the following characteristics: (a) IgG1, kappa isotype; (b) equilibrium dissociation constant of 1.5 +/- 0.2 x 10(7) L/mol with the [125I]-labeled N-tyrosyl derivative of hFPA [( 125I] Tyr-hFPA) as ligand; (c) reacts with hFPA and dog FPA (dFPA) but not with the des Arg (A alpha 1-15) or shorter peptides; (d) does not react with intact fibrinogen or A alpha-chain of human or dog origin; (e) does not react with the elastase-generated hFPA-containing peptide A alpha 1-21. Enzyme-based immunoassays (EIAs) have been developed for measuring plasma hFPA levels in the range 3 x 10(-8) to 5 x 10(-7) mol/L. Since it has already been shown by a number of investigators that hFPA levels in patients with overt defibrination fall into this range, we propose that the MoAb/8C2-5-based assays may serve as useful clinical tools in screening patients at risk of thrombosis. The 8C2-5 antibody may also be helpful in studies dealing with congenital dysfibrinogenemias, particularly in identifying heterozygous propositi with amino acid substitutions at any position within the A alpha 7-16 region. Finally, due to its cross-reactivity with dFPA, assays using this antibody should also be valuable in the canine experimental thrombosis model studies.

Animals↗
PubMed1989

Immunohistochemical characterization of fibrin(ogen)-related antigens in human tissues using monoclonal antibodies.

A new approach to study the distribution of fibrin(ogen)-related antigens was investigated using three different monoclonal antibodies (MAbs) and the avidin-biotin complex immunoperoxidase technique. MAb I8C6 recognizes B beta 1-42 peptide and can react with either fibrinogen or fibrin I; MAb T2G1 recognizes B beta 15-42 peptide and detects fibrin II but does not cross-react with fibrinogen; MAb GC4 reacts with Fragments D/DD derived from plasmin degradation of fibrinogen or fibrin but not with intact fibrinogen. The method can be applied to frozen or Bouin's fixed paraffin-embedded tissues obtained at biopsy, surgery, and autopsy. The distribution of the three antigens observed with the three MAbs was compared with that obtained with a polyclonal antiserum to fibrinogen and with the more conventional histochemical stains used in pathology to demonstrate fibrin deposits in tissues (Lendrum and PTAH). The staining observed with the three monoclonals clearly detected three different populations of fibrin(ogen)-related antigen in the tissues examined. The staining with MAb T2G1 specifically detected fibrin II with greater sensitivity than did conventional stains. The results of this study suggest that this method allows the molecular form of fibrin(ogen)-related deposits in tissues to be determined and this information may help to elucidate the role of fibrin in various disease states, such as atherosclerosis and renal disease, and in tumor growth and metastasis.

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antibody to human cross-linked fibrin.

Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.

Antibodies, Monoclonal↗