Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4157-4163 ◽  
Author(s):  
Nobuo Okumura ◽  
Oleg V. Gorkun ◽  
Fumiko Terasawa ◽  
Susan T. Lord
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Dimer Formation ◽  
Polymer Formation ◽  
Crystallographic Structures ◽  
Link Formation

Abstract Crystallographic structures indicate that γ-chain residue Asn308 participates in D:D interactions and indeed substitutions of γAsn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: γAsn308 changed to lysine (γN308K), isoleucine (γN308I), and alanine (γN308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of γN308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with γN308K. Factor XIIIa–catalyzed γ-γ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) γ-γ dimer formation of only γN308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.

Clottability and Cross-Linking Reactivity of Fibrin(ogen) Following Differential Release of Fibrinopeptides A and B

1976 ◽  
Vol 36 (03) ◽  
pp. 582-592 ◽  
Author(s):  
M Furlan ◽  
T Seelich ◽  
E. A Beck
Keyword(s):  
Early Stage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Prolonged Incubation ◽  
Fibrin Networks ◽  
Covalently Linked ◽  
Bothrops Atrox

SummaryHuman fibrinogen was treated at pH 6.0, 7.3 and 9.0 with thrombin, batroxobin (thrombinlike fraction of Bothrops atrox venom) or an extract of the venom from Ancistrodon contortrix contortrix. These three enzymes released the NH2-terminal fibrinopeptides A and B at different rates. Thrombin-free, preactivated factor XIII (factor XIIIa) was added to incubation mixtures to stabilize resulting fibrin(ogen) aggregates. Cross-linking of γ-chains and the size of covalently linked fibrin-fibrinogen oligomers were studied in an early stage of fibrinopeptide cleavage using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Batroxobin (pH 7.3) and thrombin (pH. 6.0) preferentially released fibrinopeptide A, and resulting fibrin aggregates became rapidly insoluble. However, when fibrinopeptide B was removed with the contortrix enzyme, soluble cross-linked oligomers appeared initially. The opaque fibrin clots, produced by thrombin (pH 6.0) or contortrix procoagulant fraction (pH 7.3), were found to be devoid of α-polymers even after prolonged incubation with factor XIIIa. Our data suggest that the solubility and opacity of fibrin networks are not primarily related to the type of the cross-link (γ-γ versus α-α interactions).

Factor XIIIa Cross-Linking of the Marburg Fibrin: Formation of αm·γn-Heteromultimers and the α-Chain–Linked Albumin·γ Complex, and Disturbed Protofibril Assembly Resulting in Acquisition of Plasmin Resistance Relevant to Thrombophila

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3282-3288 ◽  
Author(s):  
Teruko Sugo ◽  
Chizuko Nakamikawa ◽  
Mikihiro Takebe ◽  
Isao Kohno ◽  
Rudorf Egbring ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Caesarian Section ◽  
Tissue Type ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Pelvic Vein ◽  
Vein Thrombosis ◽  
Type Plasminogen Activator

The truncated Aα-chain of fibrinogen Marburg is partly linked with albumin by a disulfide bond. Based on the recovery of the first six amino acid residues assigned to the subunit polypeptides of fibrinogen (the Aα-and γ-chains) and albumin, 0.33 mol of albumin was estimated to be linked to 1 mol of the Marburg fibrinogen. When the Marburg fibrinogen was clotted with thrombin-factor XIIIa-Ca2+, various αmγnheteromultimers were produced, and part of the albumin was cross-linked to the γ-chain. Acid-solubilized Marburg fibrin monomer failed to form large aggregates that could be detected by monitoring turbidity at A350, but it was able to enhance tissue-type plasminogen-activator–catalyzed plasmin generation, though not as avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to be supported by normal factor XIIIa–catalyzed cross-linking of the fibrin γ-chains. However, the cross-linked Marburg fibrin, being apparently fragile and translucent, was highly resistant against plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the exact mechanisms are still unclear, the albumin-incorporated factor XIIIa–cross-linked Marburg fibrin seems to have undergone a critical structural alteration(s) to acquire resistance against plasmin. This aquisition of plasmin resistance may be contributed to the postoperative pelvic vein thrombosis and recurrent pulmonary embolisms in the patient after caesarian section for her first delivery at the age of 20 years.

Amine Inhibition of Fibrin Cross-Linking

1975 ◽  
Author(s):  
M. K. Dubiel ◽  
M. F. Scully ◽  
D. A. Lane ◽  
V. V. Kakkar
Keyword(s):  
Methyl Ester ◽  
Factor Xiii ◽  
Inhibitory Effect ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Amino Group ◽  
Dimer Formation ◽  
Polymer Formation ◽  
Glycine Methyl Ester

Various amines have been shown to possess an inhibitory effect on the enzymatic cross-linking of fibrin in vitro, as measured by fibrin isolubility in monochloroacetic acid (Lorand, L. et al., Biochemistry, 7, 1214, 1968). The amines were found to act as pseudo-donor substrates for the enzyme Factor XIIIa.We have measured γ dimer and α polymer formation in vitro, using 125I-fibrinogen as a tracer. A solution of 0.4% fibrinogen clotted with thrombin at pH 7.5 produced 50% of the γ dimers within 2 seconds and 65% of the oc polymers in approximately 15 minutes. In the presence of 5 mM isoniazid, glycine methyl ester, histamine and putrescine, 60-70% inhibition of α polymer formation was observed at 15 minutes, as compared with the control incubation mixture. There was minimal inhibition of γ dimer formation. Tyramine and histidine dihydrochloride had no inhibitory effect upon either γ dimer or α polymer formation.These results provide further evidence that a carboxylate anion in the vicinity of the amino group reduces inhibition of Factor XIII a and suggests that inhibition as measured by acid insolubility is also influenced by α polymer formation.

The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 988-995 ◽  
Author(s):  
Robert A. S. Ariëns ◽  
Helen Philippou ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel ◽  
David A. Lane ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Factor Xiii ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Similar Rate ◽  
Cross Linking ◽  
Fibrinopeptide A ◽  
Thrombin Cleavage

Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links

2013 ◽  
Vol 109 (02) ◽  
pp. 199-206 ◽  
Author(s):  
Lifang Ping ◽  
Jaewoo Song ◽  
Joo-Young Seo ◽  
Tae-Youn Choi ◽  
Jong-Rak Choi ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fibrin Clot ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Acceptor Site ◽  
Double Mutation ◽  
Cross Links ◽  
Α Chain

SummaryA fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular ε -lysyl-γ -glutamyl covalent cross-links between α chains to form α polymers and between γ chains to form γ dimers. In a previous study we characterised fibrinogen Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens: AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens from the culture media and performed biochemical tests to see how the introduced changes affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin polymerisation of all variants was impaired with the double mutation being the most impaired. In contrast, sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analysis showed α polymer formation with all three engineered proteins. This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in the a chain can support FXIIIa-catalysed fibrin cross-linking.

scholarly journals Factor XIIIa Cross-Linking of the Marburg Fibrin: Formation of αm·γn-Heteromultimers and the α-Chain–Linked Albumin·γ Complex, and Disturbed Protofibril Assembly Resulting in Acquisition of Plasmin Resistance Relevant to Thrombophila

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3282-3288 ◽  
Author(s):  
Teruko Sugo ◽  
Chizuko Nakamikawa ◽  
Mikihiro Takebe ◽  
Isao Kohno ◽  
Rudorf Egbring ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Caesarian Section ◽  
Tissue Type ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Pelvic Vein ◽  
Vein Thrombosis ◽  
Type Plasminogen Activator

Abstract The truncated Aα-chain of fibrinogen Marburg is partly linked with albumin by a disulfide bond. Based on the recovery of the first six amino acid residues assigned to the subunit polypeptides of fibrinogen (the Aα-and γ-chains) and albumin, 0.33 mol of albumin was estimated to be linked to 1 mol of the Marburg fibrinogen. When the Marburg fibrinogen was clotted with thrombin-factor XIIIa-Ca2+, various αmγnheteromultimers were produced, and part of the albumin was cross-linked to the γ-chain. Acid-solubilized Marburg fibrin monomer failed to form large aggregates that could be detected by monitoring turbidity at A350, but it was able to enhance tissue-type plasminogen-activator–catalyzed plasmin generation, though not as avidly as the normal control, indicating that the double-stranded protofibrils had, to some extent, been constructed. This idea seems to be supported by normal factor XIIIa–catalyzed cross-linking of the fibrin γ-chains. However, the cross-linked Marburg fibrin, being apparently fragile and translucent, was highly resistant against plasmin, and its subunit components were considerably retained for 48 hours as noted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the exact mechanisms are still unclear, the albumin-incorporated factor XIIIa–cross-linked Marburg fibrin seems to have undergone a critical structural alteration(s) to acquire resistance against plasmin. This aquisition of plasmin resistance may be contributed to the postoperative pelvic vein thrombosis and recurrent pulmonary embolisms in the patient after caesarian section for her first delivery at the age of 20 years.

scholarly journals Damage to actin filaments by glutaraldehyde: protection by tropomyosin.

1981 ◽  
Vol 90 (2) ◽  
pp. 459-466 ◽  
Author(s):  
S S Lehrer
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Actin Filaments ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Cross Link ◽  
Reaction Conditions ◽  
Cross Links ◽  
Link Formation

Reaction of F-actin and the F-actin-tropomyosin complex with 20 mM glutaraldehyde for 19-22 h at 0 degrees C and 25 degrees C results in extensively cross-linked filaments, as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Electron micrographs show shorter, more irregular filaments for glutaraldehyde-treated F-actin in the absence of tropomyosin as compared to the presence of tropomyosin or untreated controls. There was a 40% drop in viscosity of glutaraldehyde-treated F-actin solutions but a 90% increase in viscosity for the glutaraldehyde-treated F-actin-tropomyosin complex in solution, as compared to the untreated controls, indicating different effects of cross-linking. SDS gels indicate that intrasubunit cross-links are introduced into F-actin and that when tropomyosin is present, intramolecular cross-link formation is inhibited. Inhibition of the salt-induced G leads to F polymerization results when intramolecular cross-links are introduced into G-actin under similar or milder reaction conditions. These data indicate that, under conditions for which extensive F-actin filament cross-linking (fixing) occurs, the filaments become damaged due to the concurrent formation of intrasubunit cross-links that cause local depolymerization and distortion and that tropomyosin protects against this damage.

The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 988-995 ◽  
Author(s):  
Robert A. S. Ariëns ◽  
Helen Philippou ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel ◽  
David A. Lane ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Factor Xiii ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Similar Rate ◽  
Cross Linking ◽  
Fibrinopeptide A ◽  
Thrombin Cleavage

Abstract Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

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