Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Yoshida, Nobuhiko; Imaoka, Shingi; Hirata, Hajime; Matsuda, Michio; Asakura, Shinji

doi:10.1055/s-0038-1646313

Cited by 10 publications

(4 citation statements)

References 32 publications

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“…All the above variants were partially degraded to fragments D2, D3 and smaller degradation products in the presence of calcium, but digestion in the presence of EDTA or EGTA showed virtually no difference to normal fibrinogen. On the other hand, the same digestion pattern as with normal fibrinogen in the presence of calcium was observed with fibrinogen variants Kyoto I (␥ 308 Asn Ǟ Lys) (23) and Osaka III (␥ 275 Arg Ǟ His) (24), but they exhibited a faster conversion from D1 to D3 in the presence of EGTA. In the variant Kyoto I an additional plasmin cleavage site at position ␥ 308 Lys-309 Gly was proposed (23).…”

Section: Discussionsupporting

confidence: 56%

Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis

Stucki¹,

Schmutz²,

Schmid³

et al. 1999

Thromb Haemost

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SummaryFibrinogen St. Gallen I was detected in an asymptomatic Swiss woman. Routine coagulation tests revealed a prolonged thrombin and reptilase time. Functionally measured fibrinogen levels were considerably lower than those determined immunologically. Polymerization of fibrin monomers derived from purified fibrinogen was delayed in the presence of either calcium or EDTA. Normal fibrinopeptide A and B release by thrombin was established. An abnormal degradation of fibrinogen St. Gallen I by plasmin was observed. Fragment D1 of normal fibrinogen was fully protected against further proteolysis in the presence of 10 mM calcium, whereas fibrinogen St. Gallen I was partially further degraded to fragments D2 and D3. In the presence of 10 mM EDTA, the conversion of variant fragment D1 to D2 was accelerated whereas the degradation of fragment D2 to D3 was delayed in comparison to degradation of fragments D1 and D2 of normal fibrinogen. Three high-affinity calcium binding sites were found in both normal and variant fibrinogen. Mutation screening with SSCP analysis suggested a mutation in exon VIII of the → Val) as previously described in fibrinogen Baltimore I (4, 5). Dysfibrinogen St. Gallen I is characterized by delayed polymerization and abnormal fibrinogenolysis compared to normal fibrinogen. Computer modeling of the variant fibrinogen revealed structural changes that may explain its abnormal functional behaviour.γ-chain gene. Cycle sequencing of this gene portion revealed a single base substitution from G to T of the base 7527, leading to replacement of γ 292 glycine by valine. The same mutation has already been described for the fibrinogen variant Baltimore I. Molecular modeling was performed of a part of the γ-chain containing the mutation site, based on recently published X-ray crystal structures of human fibrinogen fragment D and of a 30 kD C-terminal part of the γ-chain. Significant structural alterations due to the substitution of glycine by valine at γ 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the γ-chain at 356 Lys and 302 Lys. A shift of γ 297 Asp that is involved in interactions of fragment D with the Gly-Pro-Arg-Pro-peptide was noted by molecular modeling. The latter observation is compatible with delayed polymerization of fibrin monomers.

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Section: Discussionsupporting

confidence: 56%

Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis

Stucki¹,

Schmutz²,

Schmid³

et al. 1999

Thromb Haemost

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“…On the other hand, only single y-chain bands of fibrinogens Haifa (275 Arg His) (27), Milano V (275 Arg -» Cys) (15), Bern I (337 Asn -> Lys) (14) and Osaka V (375 Arg -> Gly) (25) were noted on reduced SDS-polyacrylamide gels. Yoshida et al (34) suggested that the SDS-PAGE system of Laemmli (20) is uniquely capable of separat ing the normal and mutant y-chains and proposed that the latter method The arrows denote the substitutions in the five variants used in this study. The proposed calcium binding site is located between residues 311 and 336 (10) and the tentative carboxy ter minal polymerization site between residues 337-379 (11) should be included in the screening tests for abnormal fibrinogens.…”

Section: Discussionmentioning

confidence: 99%

“…A reduced protection by Ca2+ of the y-chain against plasmic degradation was not only observed in fibrinogens Vlissingen (deletion of y 319 Asn and 320 Asp) (24) and Osaka V (y 375 Arg Gly) (25) with defective calcium binding, but also in fibrinogens Haifa (y 275 Arg His) (27) and Bern I (y 337 Asn -h> Lys) (41) with normal calcium binding. On the other hand, degradation of Dj to D3 in the presence of 5 mM Ca2+ was absent in fibrinogens Osaka III (34), Saga (y 275 Arg -> His) (42) and Kyoto I (y 308 Asn -> Lys) (26) as in normal fibrinogen, though an accelerated to D3 con version by plasmin was observed in the absence of calcium (in 10 mM EGTA). Furthermore, plasmic digestion of fragment Dj to D3 from fibrinogen Kyoto III (y 330 Asp Tyr) (40) was apparently delayed in 10 mM EGTA.…”

Section: Discussionmentioning

confidence: 99%

Normal Binding of Calcium to Five Fibrinogen Variants with Mutations in the Carboxy Terminal Part of the γ-Chain

et al. 1996

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SummaryCalcium ions are known to accelerate polymerization of fibrin monomers. Each of the two carboxy terminal domains of normal fibrinogen contains one high-affinity calcium binding site that seems to be situated close to the polymerization site in the γ-chain. Most hitherto described functionally defective fibrinogen variants showed impaired clot formation. Since the tightly bound calcium ions may influence the conformation of the polymerization site, the question arises whether the abnormal clotting of a dysfibrinogen might be due to defective calcium binding. We investigated binding of calcium to fibrinogen and the effect of calcium on the clotting properties of five heterozygous fibrinogen variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinogens has one single amino acid substitution in the carboxy-terminal part of the γ-chain: fibrinogen Claro (γ 275 Arg ⟶ His), Milano V (γ 275 Arg Cys), Milano I (γ 330 Asp ⟶ Val), Bern I (γ 337 Asn Lys), and Milano VII (γ 358 Ser Cys). The shortest thrombin clotting time and the earliest onset of turbidity increase were observed in fibrinogen γ 358 Ser ⟶ Cys; both parameters were little affected by calcium concentration. In the variant γ 337 Asn ⟶ Lys, the thrombin time was abnormally prolonged at 0.01 mM Ca2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen γ 330 Asp ⟶ Val was barely improved at increasing calcium concentrations. Both variants with the substitution of γ 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and the lowest turbidity of fibrin clots at any Ca2+ concentration used. High affinity calcium binding was found to be normal in all five fibrinogen variants studied, suggesting that their abnormal clotting was not due to defective binding of calcium. The γ-chain in the fragment D1 derived from the variant γ 337 Asn ⟶ Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas fragments D1 from the other four γ-chain variants as well as from normal fibrinogen were protected against plasmic degradation in the presence of 1 mM Ca2+.

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“…The relative migration rate of a protein in a polyacrylamide gel containing SDS depends on its molecular size, shape, 28 and binding affinity for SDS. 29 The latter factor was proposed to be responsible for shifts in electrophoretic mobility of the fibrinogen ␥-chain that have been observed in several fibrinogen mutants [30][31][32][33][34][35] because of the introduction of a more basic or more hydrophobic amino acid. 36 Fibrinogen Milano XII showed abnormal migration under nonreducing conditions but normal migration under reducing conditions.…”

Section: Discussionmentioning

confidence: 99%

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Bolliger-Stucki

Lord

Furlan

2001

Blood

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Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B␤ 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B␤-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B␤-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...

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Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Cited by 10 publications

References 32 publications

Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis

Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis

Normal Binding of Calcium to Five Fibrinogen Variants with Mutations in the Carboxy Terminal Part of the γ-Chain

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

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