Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization

Reber, P. U.; Furlan, Miha; Rupp, C; Kehl, Maria; Henschen, Agnes; Pm, Mannucci; Beck, EA

doi:10.1182/blood.v67.6.1751.1751

Cited by 40 publications

(16 citation statements)

References 14 publications

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“…16 For isoelectric focusing fibrinogen was first reduced in 0.1 mol/L dithiothreitol, 8 mol/L urea, 5 mmol/L Tris/HCl, pH 8.0, and the chains focused over a pH range of 4 to 8 in 7% acrylamide gels in 8 mol/L urea, 0.5% Triton X-100. 17 Ion-exchange high-performance liquid chromatography (HPLC) was performed on a 10-cm CX-300 column (Brownlee, Santa Clara, CA) in 8 mol/L urea, 10 mmol/L phosphate buffer, pH 6.5, and 0.1% mercaptoethanol. Gradient elution was with this same buffer made 0.2 mol/L with respect to NaCl.…”

Section: Fibrinogen Purification and Protein Analysismentioning

confidence: 99%

Fibrinogen Brescia

Brennan¹,

Wyatt²,

Medicina³

et al. 2000

The American Journal of Pathology

101

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The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass inclusions. The hepatic inclusion bodies were weakly periodic acid-Schiff diastase-positive, and on immunoperoxidase staining reacted specifically with anti-fibrinogen antisera. Coagulation investigations revealed low functional and antigenic fibrinogen together with a prolonged thrombin time of 37 seconds (normal, 17 to 22 seconds) suggestive of a hypodysfibrinogenemia. DNA sequencing of all three fibrinogen genes showed a single heterozygous mutation of GGG (Gly)-->CGG (Arg) at codon 284 of the gamma-chain gene. However, examination of purified fibrinogen chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, and isoelectric focusing, failed to show any evidence of the mutant gamma(Br) chain in plasma fibrinogen. This finding was substantiated by electrospray ionization mass spectrometry, which showed only a normal gamma (and Bbeta) chain mass, but a large increase in the portion of their disialo isoforms. We speculate that misfolding of the variant protein causes hepatic retention and the subsequent hypofibrinogenemia, and that the functional defect (dysfibrinogenemia) results from hypersialylation of otherwise normal Bbeta and gamma chains consequent to the liver cirrhosis. These conclusions were supported by studies on six other family members with hypofibrinogenemia, and essentially normal clotting times, who were heterozygous for the gamma284 Gly-->Arg mutation.

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Section: Fibrinogen Purification and Protein Analysismentioning

confidence: 99%

Fibrinogen Brescia

Brennan¹,

Wyatt²,

Medicina³

et al. 2000

The American Journal of Pathology

101

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“…Analysis by SDS–PAGE (7.5% reducing and 4% non‐reducing) was essentially as described by Laemmli (1970). For isoelectric focusing, fibrinogen was first reduced in 0.1 m dithiothreitol, 8 m urea and 5 m m tris‐HCl, pH 8.0, and the chains were focused, over a pH range of 4–8, in 7% acrylamide gels in 8 m urea and 0.5% Triton X‐100 ( Reber et al , 1986 ).…”

Section: Methodsmentioning

confidence: 99%

Defective fibrinogen polymerization associated with a novel γ279Ala→Asp mutation

Brennan

Wyatt

Ockelford³

et al. 2000

Br J Haematol

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A woman with menorrhagia was investigated for a suspected fibrinogen mutation when coagulation tests revealed prolonged thrombin (55 s) and reptilase (43 s) times together with a functional and an antigenic fibrinogen concentration of 0.7 and 2.8 mg/ml respectively. Heterozygosity for a gamma-chain mutation was suggested by a doublet gamma band on SDS-PAGE and an increased negative charge was observed on isoelectric focusing of HPLC-isolated gamma-chains. Electrospray ionization mass spectrometry revealed a gamma-chain mass of 48 411 Da, which was 20 Da more than the control value of 48 391 Da. Because the normal and variant gamma-chains were not resolved, this implied a 40-Da increase in 50% of the gamma-molecules. An increased negative charge and a 44-Da increase in mass was verified when DNA sequencing showed heterozygosity for an Ala (GCC)-->Asp (GAC) substitution at codon 279 of the gamma-gene. Fibrin polymerization curves indicated a delay in the onset, and a decrease in the rate, of polymerization. Examination of crystal structures showed that the adjacent Tyr-gamma280 side chain is involved in bonding across the D-D interface, and from the proximity of the gamma279Ala-->Asp mutation it would appear that this perturbs the end-to-end DD interactions between fibrin units of the growing polymer.

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“…By crystal structure studies, the critical residues involved in the a site are shown to be γ Gln‐329, γ Asp‐330, and γ Asp‐364 10,11. In fact, replacements at any of these three positions are known in association with defective fibrin polymerization, that is, γ Gln‐329 to Val (Nagoya38), γ Asp‐330 to Val (Milano I39) or Tyr (Kyoto III40), and γ Asp‐364 to His (Matsumoto I41) or Val (Melun I42). Besides these three positions in the γ‐chain, γ Arg‐375 may also be involved in the stabilization of the γ‐chain hole by providing the guanidino group that forms a hydrogen bond with γ Asp‐364 and a salt link with the carboxyl group of γ Asp‐297 43.…”

Section: Defects In Fibrin Gel Formationmentioning

confidence: 99%

Hereditary Disorders of Fibrinogen

Matsuda

Sugo

2001

Annals of the New York Academy of Sciences

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Fibrinogen, a 340‐kDa plasma protein, is composed of two identical molecular halves each consisting of three non‐identical Aα‐, Bβ‐ and γ‐chain subunits held together by multiple disulfide bonds. Fibrinogen is shown to have a trinodular structure; that is, one central nodule, the E domain, and two identical outer nodules, the D‐domains, linked by two coiled‐coil regions. After activation with thrombin, a pair of binding sites comprising Gly‐Pro‐Arg is exposed in the central nodule and combines with its complementary binding site a in the outer nodule of another molecules. By using crystallographic analysis, the α‐amino group of αGly‐1 is shown to be juxtaposed between γAsp‐364 and γAsp‐330, and the guanidino group of αArg‐3 between the carboxyl group of γAsp‐364 and γGln‐329 in the a site. Half molecule‐staggered, double‐stranded protofibrils are thus formed. Upon abutment of two adjacent D domains on the same strand, D‐D self association takes place involving Arg‐275, Tyr‐280, and Ser‐300 of the γ‐chain on the surface of the abutting two D domains. Thereafter, carboxyl‐terminal regions of the α‐chains are untethered and interact with those of other protofibrils leading to the formation of thick fibrin bundles and networks. Although many enigmas still remain concerning the exact mechanisms of these molecular interactions, fibrin assembly proceeds in a highly ordered fashion. In this review, these molecular interactions of fibrinogen and fibrin are discussed on the basis of the data provided by hereditary dysfibrinogens on introducing representative molecules at each step of fibrin clot formation.

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Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization

Cited by 40 publications

References 14 publications

Fibrinogen Brescia

Fibrinogen Brescia

Defective fibrinogen polymerization associated with a novel γ279Ala→Asp mutation

Hereditary Disorders of Fibrinogen

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