Fibrinogen residue γAla341 Is Necessary for Calcium Binding and ‘A-a’ interaction.

Abstract Abstract 1154 Fibrinogen, a 340-kDa glycoprotein has essential roles in blood coagulation and platelet aggregation. Fibrinogen is a complex molecule consisting of 3 pairs of Aα, Bβ, and γ chains intertwined to form a tri-nodular molecule with 2 terminal D regions and a central E region. The fibrinogen γ-nodule, a part of D region, has several important sites relating to fibrinogen function, which are the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of those sites and conserved between all available species, is altered in two variant fibrinogens: fibrinogen Seoul (γAla341Asp) (Song et al., Clin Appl Thromb Hemost 2006) and fibrinogen Tolaga Bay (γAla341Val) (Davis et al., Thromb Haemost 2007). Fibrinogen Seoul showed hypodysfibrinogenemia, and fibrinogen Tolaga Bay hypofibrinogenemia. We have expressed these two variant fibrinogens in CHO cells, purified them from the culture media and performed biochemical tests to elucidate their function. Thrombin-catalyzed kinetics of FpA release was not different (p-value, 0.3, n=3) from normal fibrinogen. Average specificity constant, kcat/Km for FpA was 7.4±1.8, 5.7±1.8, and 8.1±1.2 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. However, FpB release from both variants was slower than that of normal (p-value, 0.005 in One Way ANOVA; p-value, 0.006 and 0.002 for normal vs γAla341Val, and normal vs γAla341Asp, respectively in multiple comparison). Average specificity constant for FpB was 3.6±1.1, 1.7±0.4, and 1.4±0.3 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. We measured fibrin polymerization by turbidity with the final thrombin and fibrinogen concentration being 0.2mg/mL and 0.1U/mL, respectively. At 10mM calcium we saw no turbidity rising with either variant. Both variants showed impaired polymerization with a longer lag period and a slower Vmax than normal fibrinogen at physiologic 1mM calcium. Lag period, which reflects protofibril formation, for normal, γAla341Val, and γAla341Asp was 110±30, 2,500±150, and 1,200±40, respectively (mean±SD, sec). Vmax, which reflects lateral aggregation, was 81±26, 15±3, and 3±1 (mean±SD, 10-5s-1) for normal, γAla341Val, and γAla341Asp. With the FXIIIa cross-linking, measured by SDS-PAGE, we found that γ and α chain cross-linking was delayed in both variants. We tested the calcium binding and the functionality of ‘a’ polymerization site with the plasmin protection assay. Both variants were not protected from plasminolysis in the presence of 1mM calcium or 0.55mM GPRP, indicative of impaired binding of calcium and knob ‘A’. Given these results, both fibrinogen Seoul and Tolaga Bay likely have a conformational change in their calcium and GPRP binding sites resulting in the impaired fibrin polymerization. In conclusion, we think fibrinogen residue γAla341 is important for calcium binding, ‘A-a’ interactions and the conformation of the γ-nodule. Disclosures: No relevant conflicts of interest to declare.

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Calcium-Binding Site β2, Adjacent to the “b” Polymerization Site, Modulates Lateral Aggregation of Protofibrils during Fibrin Polymerization†,‡

Biochemistry ◽

10.1021/bi0359978 ◽

2004 ◽

Vol 43 (9) ◽

pp. 2475-2483 ◽

Cited By ~ 18

Author(s):

Michael S. Kostelansky ◽

Karim C. Lounes ◽

Li Fang Ping ◽

Sarah K. Dickerson ◽

Oleg V. Gorkun ◽

...

Keyword(s):

Binding Site ◽

Calcium Binding ◽

Calcium Binding Site ◽

Fibrin Polymerization

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Characterization Of An Acquired IgG Autoantibody To Bβ and γ Chains Of Fibrinogen Resulting In Delayed Fibrin Polymerization and Severe Bleeding

Blood ◽

10.1182/blood.v122.21.2362.2362 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2362-2362

Author(s):

Premal Lulla ◽

Swapan Dasgupta ◽

Lawrence Rice ◽

John J. McCarthy ◽

Perumal Thiagarajan

Keyword(s):

Conflicts Of Interest ◽

Specific Binding ◽

Protein A ◽

Severe Bleeding ◽

Fibrinogen Concentration ◽

Dependent Manner ◽

Thrombin Time ◽

Igg Antibody ◽

Concentration Dependent Manner ◽

Fibrin Polymerization

Abstract A 88 year-old woman was referred to our center for 3 weeks of gastrointestinal bleeding and a spontaneous right rectus femoris hematoma. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and reptilase time (RT) were markedly prolonged (Table 1). Assays for coagulation factors V, VIII, IX and X were normal. A fibrinogen concentration of < 50 mg/dl was measured by Clauss method while fibrinogen concentration measured by radial immunodiffusion was 180 mg/dl. Following infusions of cryoprecipitate there was accelerated clearance of fibrinogen as measured by the Clauss method. An inhibitor of fibrinogen function was suspected.Table 1TestTime (sec)1:1 mix (sec)Normal Range (sec)PT>902212-15aPTT70.73923-36TT>120>12015-19Reptilase Time>120>12014.8-20.4 When the patient’s plasma was tested towards immobilized fibrinogen in ELISA plates, a concentration dependent specific binding of IgG was seen while under similar conditions control IgG had no significant binding (Figure 1). The IgG antibody was polyclonal comprising of both kappa and lambda subtypes. The IgG fraction was purified on a Protein A column and the isolated IgG fraction prolonged the thrombin time of normal plasma in a concentration dependent manner. The effect of the isolated IgG fraction on fibrin polymerization was measured by monitoring the absorbance at λ390 in a spectrophotometer. The IgG inhibited fibrin polymerization in a concentration dependent manner. Control IgGs had no significant effect on polymerization under these conditions (Figure 2). The immunologic specificity of antibody was tested in immunoblots of fibrinogen subjected to SDS-PAGE. The patients IgG reacted with fibrinogen in unreduced buffers. Under reducing conditions, the IgG reacted with Bβ and γ chains of fibrinogen while the control IgG did not (Figure 3). The patient was treated with rituximab and steroids. After four, weekly doses of rituximab, fibrinogen levels by Clauss method normalized (450 mg/dl). There was no detectable antibody to fibrinogen and no further bleeding was noted. Conclusion This is a description of a rare case of a polyclonal autoantibody to Bβ and γ chains of fibrinogen that inhibited fibrin polymerization leading to a severe bleeding diathesis. Disclosures: No relevant conflicts of interest to declare.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650587 ◽

1996 ◽

Vol 76 (03) ◽

pp. 377-383 ◽

Cited By ~ 6

Author(s):

Miha Furlan ◽

Bettina Stucki ◽

Colette Steinmann ◽

Myriam Jungo ◽

Bernhard Lämmle

Keyword(s):

Calcium Ions ◽

Calcium Binding ◽

Single Amino Acid ◽

Thrombin Time ◽

Terminal Part ◽

Calcium Binding Site ◽

Fibrin Polymerization ◽

High Affinity ◽

Carboxy Terminal ◽

Fibrin Monomers

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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A functional calcium-binding site in the metalloprotease domain of ADAMTS13

Blood ◽

10.1182/blood-2008-03-144683 ◽

2009 ◽

Vol 113 (5) ◽

pp. 1149-1157 ◽

Cited By ~ 31

Author(s):

Michelle D. Gardner ◽

Chan K. N. K. Chion ◽

Rens de Groot ◽

Anuja Shah ◽

James T. B. Crawley ◽

...

Keyword(s):

Binding Site ◽

Active Site ◽

Calcium Binding ◽

Major Influence ◽

Calcium Binding Site ◽

High Affinity ◽

Fold Reduction ◽

Reported Study ◽

Specificity Constant ◽

Von Willebrand

Abstract ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with KD(app) of 80 μM (± 15 μM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the KD(app) for Ca2+ of ADAMTS13, and for D187A and E212A also reduced the Vmax to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed an approximately 13-fold reduction in specificity constant, kcat/Km, contributed by changes in both Km and kcat. These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.

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Fibrinogen Bastia (γ 318 Asp → Tyr) a Novel Abnormal Fibrinogen Characterized by Defective Fibrin Polymerization

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614892 ◽

1999 ◽

Vol 82 (12) ◽

pp. 1639-1643 ◽

Cited By ~ 9

Author(s):

Karim Chabane Lounes ◽

Claudine Soria ◽

Antoine Valognes ◽

Marie France Turchini ◽

Jaap Koopman ◽

...

Keyword(s):

Calcium Ions ◽

Clinical Symptoms ◽

Degradation Products ◽

Base Substitution ◽

Chain Gene ◽

Fibrinogen Concentration ◽

Clotting Time ◽

Terminal Part ◽

Fibrin Polymerization ◽

Chain Sequence

SummaryA new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the γ-chain since by SDS-PAGE performed according to the method of Laemmli two γ-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia γ-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the γ-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G → T) in the exon VIII of the γ chain gene, resulting in the amino acid substitution 318 Asp (GAC) → Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the γ-chain.

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