The mutation in fibrinogen Bicêtre II (γ Asn308 →Lys) does not affect the binding of t-PA and plasminogen to fibrin

Grailhe, P.; Boyer-Neumann, Catherine; Haverkate, F.; Grimbergen, Jos; Larrieu, M J; Anglès-Cano, Eduardo

doi:10.1097/00001721-199304050-00003

Cited by 19 publications

(25 citation statements)

References 23 publications

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“…[18][19][20][21] Analysis of 2 dysfibrinogens with substitutions at ␥-chain residue 308, Asn308 to lysine and Asn308 to isoleucine, indicated that this residue has an important role in polymerization. [22][23][24][25] As is the case with most dysfibrinogens, these substitutions occurred in individuals who are heterozygous for the mutation. This complicates functional analysis because fibrinogen in these individuals is a complex mixture of normal and abnormal molecules.…”

Section: Introductionmentioning

confidence: 99%

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

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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Crystallographic structures indicate that ␥-chain residue Asn308 participates in D:D interactions and indeed substitutions of ␥Asn308 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: ␥Asn308 changed to lysine (␥N308K), isoleucine (␥N308I), and alanine (␥N308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa-catalyzed crosslinking 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 ␥N308K 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 ␥N308K. Factor XIIIa-catalyzed ␥-␥ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) ␥-␥ dimer formation of only ␥N308K 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. IntroductionFibrinogen is a 340-kDa glycoprotein that circulates in the blood at 5.88 to 11.76 M (2-4 g/L). It is composed of a pair of 3 polypeptide chains, A␣, B␤, and ␥, connected by 29 intrachain and interchain disulfide bonds. 1 The 6 chains are arranged into 3 globular nodules. The central E nodule contains the N-termini of all chains and the distal D nodules contain the C-termini of the ␤, ␥, and a short segment of the ␣ chains. Coiled coils composed of all 3 chains link the E and D nodules. 2 The C-terminal residues of A␣ chains form globular nodules that are associated with one another, as well as with the E nodule. 3 Recent crystallographic studies have provided high-resolution structures of the C-terminal region of the ␥ chain, which contains functional sites for fibrin polymerization, calcium binding, platelet aggregation, and factor XIII (FXIII) cross-linking. [4][5][6] During coagulation, thrombin cleaves fibrinogen, releasing fibrinopeptides A (FpA) and B (FpB) from the N-termini of the A␣ and B␤ chains, respectively, and converting fibrinogen to fibrin monomers (for reviews, see Blomback 1 and Doolittle 2 ). Fibrin monomers polymerize spontaneously through a 2-step process, with a second step being calcium-dependent. 7,8 The first step begins with the release of FpA. This exposes the "A" site, which interacts with the "a" site in the D nodule of another molecule, leading to the formation of half-staggered, double-stranded protofibrils. 9,10 Within protofibrils, individual fibrin mol...

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Section: Introductionmentioning

confidence: 99%

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

Okumura

Gorkun²,

Terasawa³

et al. 2004

Blood

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“…This variant, named fibrinogen Hillsborough, has characteristics similar to several known dysfibrinogens with mutations located in the C-terminal end of the ␥ chain. 7 Like fibrinogens Baltimore III (␥Asn308Ile) and Kyoto I, Matsumoto II, and Bicêtre II (␥Asn308Lys), [16][17][18][19][20][21][22] fibrinogen Hillsborough presents with a normal prothrombin time and aPTT but a prolonged TCT. Similarly, each of these mutations is associated with low functional but normal immunologic/gravimetric levels.…”

Section: Discussionmentioning

confidence: 99%

Fibrinogen Hillsborough: a novel γGly309Asp dysfibrinogen with impaired clotting

Mullin

Brennan

Ganly

et al. 2002

Blood

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We present a novel ␥-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/ mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfatepolyacrylamide gel electrophoresis revealed a ␥-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal ␥ chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)3GAC (Asp) at codon 309 of the ␥ chain gene. This Gly3 Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue ␥309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. ␥Gly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting ␥D domains of condensing fibrin monomers during polymerization. IntroductionFibrinogen is a plasma glycoprotein that consists of 2 sets of 3 polypeptide chains, A␣, B␤ and ␥, which combine to form a symmetrical, trinodular molecule with 2 terminal D domains and a central E domain, denoted D-E-D. The E domain consists of the N-termini of all 6 chains, whereas the D domains consist of the C-termini of the B␤ and ␥ chains. Conversion of soluble fibrinogen into insoluble fibrin occurs when the serine protease thrombin removes 2 fibrinopeptides from the N-termini of the A␣ and B␤ chains, in the E domain. The new N-terminal GPR sequences exposed by release of these peptides noncovalently interact with polymerization sites in the D domains of other fibrin molecules to form half-staggered, double-stranded protofibrils. These protofibrils grow linearly and laterally to form the final fibrin clot structure. During this polymerization process, activated factor XIII, a transglutaminase, cross-links the chains, thus stabilizing the overall clot structure.The ␥ chain contains several sites that are crucial for normal fibrin polymerization. These sites include: (1) a high-affinity Ca 2ϩ binding site, which is located between residues ␥318 and 324, 1 (2) the "a" polymerization site, which after fibrinopeptide release interacts with the newly exposed GPR sequence of the "A" site, to promote D:E interactions, (3) the D:D interface, where abutting D domains interact during protofibril formation, and (4) a crosslinking site, where activated factor XIII catalyzes the formation of covalent ␥ glu...

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“…Fibrinogen was purified from fresh-frozen human plasma supplemented with proteinase inhibitors (100 k.i.u./ml aprotinin, 2 mmol/l phenylmethysulphonyl fluoride PMSF, 1 Amol/l H-D-Phe-Pro-Arg-CH 2 CI, 200 mmol/l 6-Ahx, 10 Amol/l p-nitrophenyl-pV-guanidinobenzoate, and 4 mmol/l benzamidine, final concentrations), as previously described [25] with minor modifications [26]. In brief, the procedure included two glycine precipitations of proteins remaining in the soluble phase of plasma precipitated twice with BaSO 4 , followed by chromatography on a Sepharose 6B column in 50 mmol/l phosphate buffer, pH 7.4, containing 0.5 mol/l NaCl, 2 mmol/l EDTA and all aforementioned inhibitors except 6-Ahx.…”

Section: Protein Purification and Radioiodinationmentioning

confidence: 99%

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

Domínguez¹,

Montes²,

Páramo³

et al. 2002

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Binding of plasminogen to fibrin and cell surfaces is essential for fibrinolysis and pericellular proteolysis. We used surface plasmon resonance and enzyme kinetic analyses to study the effect of two mAbs (A10.2, CPL15) on plasminogen binding and activation at fibrin surfaces. A10.2 is directed against the lysine-binding site (LBS) of kringle 4, whereas CPL15 recognises a region in kringle 1 outside the LBS. In the presence of CPL15 and A10.2 mAbs, binding of plasminogen (K d = 1.16 F 0.22 Amol/l) to fibrin was characterised by a mAb concentration-dependent bell-shaped isotherm. A progressive increase in the concentration of mAbs at the surface was also detected, and reached a plateau corresponding to the maximum of plasminogen bound. These data indicated that at low mAb concentration, bivalent plasminogen -mAb -plasminogen ternary complexes are formed, whereas at high mAb concentration, a progressive shift to monovalent plasminogen -mAb binary complexes is observed. Plasmin formation in the presence of mAbs followed a similar bell-shaped profile. Monovalent Fab fragments of mAb A10.2 showed no effect on the binding of plasminogen, confirming the notion that a bivalent mAb interaction is essential to increase plasminogen binding and activation at the surface of fibrin. D

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The mutation in fibrinogen Bicêtre II (γ Asn308 →Lys) does not affect the binding of t-PA and plasminogen to fibrin

Cited by 19 publications

References 23 publications

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

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

Fibrinogen Hillsborough: a novel γGly309Asp dysfibrinogen with impaired clotting

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

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