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ABSTRACT: A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.
Blood 03/2001; 97(4):973-80. · 9.90 Impact Factor
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ABSTRACT: A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.
Thrombosis and Haemostasis 09/2000; 84(2):263-70. · 5.04 Impact Factor
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ABSTRACT: We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1alpha (IL-1alpha) in the human hepatoma cell line HepG2. PMA, serum, and IL-1alpha induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1alpha) increase in PAI-1 mRNA, peaking after approximately 4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1alpha on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the -489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate-response element (TRE; between -58 and -50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1alpha. In line with these findings, IL-1alpha poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)1,2 activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1alpha. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 alpha only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1alpha in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.
Arteriosclerosis Thrombosis and Vascular Biology 02/1999; 19(1):39-46. · 6.37 Impact Factor
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ABSTRACT: Experiments were designed to clarify the role of c-Jun/c-Fos and of putative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA and protein levels prior to PAI-1 induction. This induction of PAI-1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of c-Jun and c-Fos in basal and PMA-stimulated transcription of the PAI-1 gene is demonstrated by our finding that antisense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown that two TREs between positions -58 and -50 and between -79 and -72 of the PAI-1 promoter are essential for basal and PMA-induced PAI-1 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -79 and -72 shows very strong PMA induction of an unknown factor, which is not related to c-Jun or c-Fos. The TRE binding between positions -58 and -50 forms two complexes, both containing c-Jun protein. The faster migrating complex primarily contains c-Jun homodimers. The amount of the faster migrating complex is enhanced more than 30-fold in PMA-treated cells, due to a strongly increased binding of c-Jun homodimers and, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissociation experiments suggest that the c-Jun/c-Fos heterodimers bind with much lower affinity compared to binding of c-Jun homodimers. Together with the finding that both antisense c-jun and antisense c-fos oligodeoxynucleotides reduced the amount of c-Jun homodimer, we conclude that binding of c-Jun homodimer to the TRE at positions -58 to -50 is important in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.
European Journal of Biochemistry 11/1996; 241(2):393-402. · 3.58 Impact Factor
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ABSTRACT: We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.
Biochemical Journal 09/1995; 310 ( Pt 1):171-6. · 4.90 Impact Factor
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ABSTRACT: The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor-coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein-sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.
Blood 12/1994; 84(9):2984-91. · 9.90 Impact Factor
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ABSTRACT: The aim of this study was to investigate the interactions of t-PA and plasminogen with fibrin derived from an abnormal fibrinogen detected in a 40-year-old male patient who had had an episode of thrombophlebitis with pulmonary embolism. An abnormal fibrinogen was diagnosed on the basis of prolonged thrombin and reptilase times also detected in two other family members. Fibrinogen purified from plasma, in the presence of protease inhibitors, by glycine precipitations, gel filtration and affinity chromatography, was devoid of plasminogen, fibronectin, and vWf. SDS-PAGE analysis according to Laemmli under reducing conditions, showed an abnormal gamma chain (approximately 50% of the total) migrating in a more anodic position (M(r) 48 kDa). By PCR amplification and DNA sequencing, the abnormality was identified as an Asn308-->Lys mutation of the gamma chain. Since such a mutation constitutes a new plasmin cleavage site as first reported for fibrinogen Kyoto I, it may modify interactions of plasminogen and t-PA with carboxy-terminal lysine residues. Ligand-binding studies were therefore performed using intact and plasmin-degraded fibrin surfaces obtained from the abnormal fibrinogen. The plasminogen and t-PA binding isotherms obtained with the abnormal fibrinogen were similar to the control. Moreover, the stimulation by fibrin of plasminogen activation by t-PA was not different from the control. These results suggest (i) that the lysine 308 residue may not be exposed to plasmin cleavage in fibrin, and (ii) that the thrombotic accident of the propositus cannot be explained by an abnormality of the plasminogen/t-PA binding to fibrin.
Blood Coagulation and Fibrinolysis 11/1993; 4(5):679-87. · 1.24 Impact Factor
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ABSTRACT: The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C-->T) in the A alpha-chain gene, resulting in the amino acid substitution A alpha 554 Arg-->Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (A alpha 554 Arg-->Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals.
Journal of Clinical Investigation 04/1993; 91(4):1637-43. · 15.39 Impact Factor
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ABSTRACT: In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461-610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino-terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.
Blood 11/1992; 80(8):1972-9. · 9.90 Impact Factor
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ABSTRACT: In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.
Journal of Clinical Investigation 08/1992; 90(1):238-44. · 15.39 Impact Factor
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ABSTRACT: The molecular defects in two congenital abnormal fibrinogens, IJmuiden and Nijmegen, were determined by sequence analysis of genomic DNA amplified by the polymerase chain reaction. Both fibrinogens were heterozygous, IJmuiden having a B beta Arg14----Cys substitution and Nijmegen having a B beta Arg44----Cys substitution. Clotting induced by thrombin or Reptilase was impaired in both fibrinogens, indicating defective fibrin polymerization. Immunoblot analysis of both purified fibrinogens demonstrated that some of the abnormal molecules were linked by disulfide bonds to albumin. In addition, abnormal high molecular weight fibrinogen complexes with Mrs between 600,000 and 700,000 were present. Fibrinogen-albumin and high molecular weight complexes were also detected in the patients' plasmas. Quantitative analysis demonstrated that of the total plasma fibrinogen in the IJmuiden patient, 20% was linked to albumin and 10% was present as high molecular weight complexes. In plasma Nijmegen, 13% was linked to albumin and 15% was present as high molecular weight complexes. These results demonstrate that the additional abnormal cysteine in fibrinogens IJmuiden and Nijmegen resulted in the formation of disulfide-linked complexes with other proteins, predominantly albumin. We also found that a significant fraction of the abnormal fibrinogen molecules contained free sulfhydryl groups. These findings complicate interpretation of functional studies of these altered fibrinogens.
Proceedings of the National Academy of Sciences 05/1992; 89(8):3478-82. · 9.68 Impact Factor
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ABSTRACT: The aim of this study was to investigate the interactions of t-PA and plasminogen with fibrin derived from an abnormal fibrinogen detected in a 40-year-old male patient who had had an episode of thrombophlebitis with pulmonary embolism. An abnormal fibrinogen was diagnosed on the basis of prolonged thrombin and reptilase times also detected in two other family members. Fibrinogen purified from plasma, in the presence of protease inhibitors, by glycine precipitations, gel filtration and affinity chromatography, was devoid of plasminogen, fibronectin, and vWf. SDS-PAGE analysis according to Laemmli under reducing conditions, showed an abnormal ? chain (~50% of the total) migrating in a more anodic position (M(r) 48 kDa). By PCR amplification and DNA sequencing, the abnormality was identified as an Asn308 ? Lys mutation of the ? chain. Since such a mutation constitutes a new plasmin cleavage site as first reported for fibrinogen Kyoto I, it may modify interactions of plasminogen and t-PA with carboxy-terminal lysine residues. Ligand-binding studies were therefore performed using intact and plasmin-degraded fibrin surfaces obtained from the abnormal fibrinogen. The plasminogen and t-PA binding isotherms obtained with the abnormal fibrinogen were similar to the control. Moreover, the stimulation by fibrin of plasminogen activation by t-PA was not different from the control. These results suggest (i) that the lysine 308 residue may not be exposed to plasmin cleavage in fibrin, and (ii) that the thrombotic accident of the propositus cannot be explained by an abnormality of the plasminogen/t-PA binding to fibrin.
4(5):679-687.
