The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Susan T. Lord - One of the best experts on this subject based on the ideXlab platform.
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An engineered Fibrinogen Variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links.
Thrombosis and haemostasis, 2012Co-Authors: Rojin Park, Jong Rak Choi, Oleg V. Gorkun, Lifang Ping, Jaewoo Song, Joo-young Seo, Tae-youn Choi, Susan T. LordAbstract:A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular e-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 α chain can support FXIIIa-catalysed fibrin cross-linking.
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Fibrinogen Variant BβD432A has normal polymerization but does not bind knob “B”
Blood, 2008Co-Authors: Sheryl R. Bowley, Susan T. LordAbstract:Fibrinogen residue Bβ432Asp is part of hole “b” that interacts with knob “B,” whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BβD432A has normal polymerization, we hypothesized that Bβ432Asp is not critical for knob “B” binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BβD432A. Surprisingly, the structure (rfD-BβD432A+GH) showed the peptide GHRP was not bound to hole “b.” We then re-evaluated the polymerization of this Variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of γ-γ dimer formation for BβD432A were indistinguishable compared with normal Fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BβD432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than “B:b” interactions. We conclude that hole “b” and “B:b” knob-hole binding per se have no influence on fibrin polymerization.
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Polymerization-Defective Fibrinogen Variant gammaD364A Binds Knob “A” Peptide Mimic
Biochemistry, 2008Co-Authors: Sheryl R. Bowley, Nobuo Okumura, Betsy K. Merenbloom, Laurie Betts, Annie Heroux, Oleg V. Gorkun, Susan T. LordAbstract:Fibrin polymerization is supported in part by interactions called 'A:a'. Crystallographic studies revealed ?364Asp is part of hole 'a' that interacts with knob 'A' peptide mimic, GPRP. Biochemical studies have shown ?364Asp is critical to polymerization, as polymerization of Variants ?D364A, ?D364H, and ?D364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from ?D364A. Surprisingly, the structure (rfD-?D364A+GP) showed near normal 'A:a' interactions with GPRP bound to hole 'a' and no change in the overall structure of ?D364A. Of note, inspection of the structure showed negative electrostatic potential inside hole 'a' was diminished by this substitution. We examined GPRP binding to the ?364Asp Variants in solution by plasmin protection assay. We found no protection of either ?D364H or ?D364V but partial protection of ?D364A, indicating the peptide does not bind to either ?D364H or ?D364V and binds more weakly than normal to ?D364A. We also examined protection by calcium and found all Variants were indistinguishable from normal, suggesting the global structures of the Variants are not markedly different from normal. Our data imply that ?364Asp per se is not required for knob 'A' binding to hole 'a'; rather,more » this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.« less
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polymerization defective Fibrinogen Variant gammad364a binds knob a peptide mimic
Biochemistry, 2008Co-Authors: Sheryl R. Bowley, Nobuo Okumura, Betsy K. Merenbloom, Laurie Betts, Annie Heroux, Oleg V. Gorkun, Susan T. LordAbstract:Fibrin polymerization is supported in part by interactions called 'A:a'. Crystallographic studies revealed ?364Asp is part of hole 'a' that interacts with knob 'A' peptide mimic, GPRP. Biochemical studies have shown ?364Asp is critical to polymerization, as polymerization of Variants ?D364A, ?D364H, and ?D364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from ?D364A. Surprisingly, the structure (rfD-?D364A+GP) showed near normal 'A:a' interactions with GPRP bound to hole 'a' and no change in the overall structure of ?D364A. Of note, inspection of the structure showed negative electrostatic potential inside hole 'a' was diminished by this substitution. We examined GPRP binding to the ?364Asp Variants in solution by plasmin protection assay. We found no protection of either ?D364H or ?D364V but partial protection of ?D364A, indicating the peptide does not bind to either ?D364H or ?D364V and binds more weakly than normal to ?D364A. We also examined protection by calcium and found all Variants were indistinguishable from normal, suggesting the global structures of the Variants are not markedly different from normal. Our data imply that ?364Asp per se is not required for knob 'A' binding to hole 'a'; rather,more » this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.« less
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Probing the gamma2 calcium-binding site: studies with gammaD298,301A Fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site.
Biochemistry, 2007Co-Authors: Michael S. Kostelansky, Oleg V. Gorkun, Lifang Ping, Karim C. Lounes, Sarah K Dickerson, Susan T. LordAbstract:To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the Fibrinogen Variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A Fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the Variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A Fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal Fibrinogen. Notably, the influence of calcium on polymerization was the same for the Variant and for normal Fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.
Miha Furlan - One of the best experts on this subject based on the ideXlab platform.
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Fibrinogen st gallen i gamma 292 gly val evidence for structural alterations causing defective polymerization and Fibrinogenolysis
Thrombosis and Haemostasis, 1999Co-Authors: B Stucki, Bernhard Lämmle, Peter Schmutz, Luzius Schmid, Andre Haeberli, Miha FurlanAbstract:Fibrinogen 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 gamma-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 gamma 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 gamma-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 gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 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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Fibrinogen St. Gallen I (gamma 292 Gly--> Val): evidence for structural alterations causing defective polymerization and Fibrinogenolysis.
Thrombosis and haemostasis, 1999Co-Authors: B Stucki, Bernhard Lämmle, Peter Schmutz, Luzius Schmid, Andre Haeberli, Miha FurlanAbstract:Fibrinogen 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 gamma-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 gamma 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 gamma-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 gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 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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Fibrinogen Claro — Another dysfunctional Fibrinogen Variant with γ 275 Arginine → Histidine substitution
Thrombosis research, 1996Co-Authors: Colette Steinmann, Myriam Jungo, Eugene A. Beck, Bernhard Lämmle, Miha FurlanAbstract:Abstract Dysfunctional Fibrinogen Variants have been discovered by prolongation of thrombin and reptilase clotting times and a discrepancy of the functionally and immunologically determined Fibrinogen concentration. More than 250 Variants have been described (1). Majority of the abnormal Fibrinogen Variants have a structural defect near or at the thrombin cleavage site in the Aα-chain (2,3). Removal of fibrinopeptide A is required to expose the amino-terminal polymerization site (4,5). Most molecular defects in abnormal Fibrinogen Variants with normal fibrinopeptide A release but impaired fibrin monomer polymerization have been localized within residues γ 275 to γ 375 (1) containing the putative carboxy terminal polymerization site γ 337–379 (6,7). In the present report we describe another congenitally abnormal Fibrinogen that was found in a 42-year-old asymptomatic woman and her son. A single amino acid substitution γ 275 Arg → His was found in this dysfunctional Fibrinogen Variant.
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Fibrinogen claro another dysfunctional Fibrinogen Variant with γ 275 arginine histidine substitution
Thrombosis Research, 1996Co-Authors: Colette Steinmann, Myriam Jungo, Eugene A. Beck, Bernhard Lämmle, Miha FurlanAbstract:Abstract Dysfunctional Fibrinogen Variants have been discovered by prolongation of thrombin and reptilase clotting times and a discrepancy of the functionally and immunologically determined Fibrinogen concentration. More than 250 Variants have been described (1). Majority of the abnormal Fibrinogen Variants have a structural defect near or at the thrombin cleavage site in the Aα-chain (2,3). Removal of fibrinopeptide A is required to expose the amino-terminal polymerization site (4,5). Most molecular defects in abnormal Fibrinogen Variants with normal fibrinopeptide A release but impaired fibrin monomer polymerization have been localized within residues γ 275 to γ 375 (1) containing the putative carboxy terminal polymerization site γ 337–379 (6,7). In the present report we describe another congenitally abnormal Fibrinogen that was found in a 42-year-old asymptomatic woman and her son. A single amino acid substitution γ 275 Arg → His was found in this dysfunctional Fibrinogen Variant.
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Binding of calcium ions and their effect on clotting of Fibrinogen Milano III, a Variant with truncated A alpha-chains.
Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 1996Co-Authors: Miha Furlan, Colette Steinmann, Myriam Jungo, Bernhard LämmleAbstract:Calcium ions are known to be required for normal polymerisation of fibrin monomers. Normal human Fibrinogen has three high-affinity calcium binding sites. Two of these are located in the D-domains whereas the third binding site was tentatively assigned either to the E-domain or to the C-terminal part of the A alpha-chain. Furthermore, binding of calcium to the low-affinity binding sites (n > or = 10) facilitates fibrin monomer polymerisation. In several abnormally clotting Fibrinogen Variants, the polymerisation defect was partially normalised following addition of calcium ions. In this study, we show normal binding of calcium to Fibrinogen Milano III, a homozygous Fibrinogen Variant with truncated A alpha-chains (A alpha 452 Gly-Pro-Asp-->Trp-Ser-Stop). These results confirm that the C-terminal parts of the A alpha-chains beyond residue 451 Ile are not involved in calcium binding. The thrombin time was severely prolonged and the final clot turbidity was strongly reduced in Fibrinogen Milano III. Moreover, calcium ions did not significantly improve the abnormal clotting behavior of this dysFibrinogen. The polymerisation defect in Fibrinogen Milano III appears to be due to truncated A alpha-chains as well as to the disulphide-linked albumin.
Jan E. Dyr - One of the best experts on this subject based on the ideXlab platform.
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a novel Fibrinogen Variant liberec dysFibrinogenaemia associated with γ tyr262cys substitution
European Journal of Haematology, 2008Co-Authors: Roman Kotlín, Alžběta Sobotková, Jiří Suttnar, Peter Salaj, L. Walterová, Tomáš Riedel, Zuzana Reicheltová, Jan E. DyrAbstract:Objective: A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysFibrinogenaemia was suspected. Patients and methods: The patient from Liberec (Czech Republic) had a low Fibrinogen plasma level as determined by Clauss method, normal Fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysFibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, Fibrinogen clottability measurement and scanning electron microscopy were performed. Results: DNA sequencing showed the heterozygous Fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of Fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control. Conclusion: A case of dysFibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel Fibrinogen Variant (γ Y262C) that has been named Liberec.
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A novel Fibrinogen Variant – Liberec: dysFibrinogenaemia associated with γ Tyr262Cys substitution
European journal of haematology, 2008Co-Authors: Roman Kotlín, Alžběta Sobotková, Jiří Suttnar, Peter Salaj, L. Walterová, Tomáš Riedel, Zuzana Reicheltová, Jan E. DyrAbstract:Objective: A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysFibrinogenaemia was suspected. Patients and methods: The patient from Liberec (Czech Republic) had a low Fibrinogen plasma level as determined by Clauss method, normal Fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysFibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, Fibrinogen clottability measurement and scanning electron microscopy were performed. Results: DNA sequencing showed the heterozygous Fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of Fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control. Conclusion: A case of dysFibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel Fibrinogen Variant (γ Y262C) that has been named Liberec.
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a novel Fibrinogen Variant praha i hypoFibrinogenemia associated with γ gly351ser substitution
European Journal of Haematology, 2007Co-Authors: Roman Kotlín, Jiří Suttnar, Peter Salaj, Tomáš Riedel, Martina Chytilová, Jiří Šantrůček, Pavel Klener, Jan E. DyrAbstract:Objectives: A 25-yr-old man from Prague had abnormal bleeding after several surgical operations with low Fibrinogen level and hypoFibrinogenemia was suspected. Patients and methods: The patient, 25 yr-old male had a low Fibrinogen concentration as determined by the thrombin time and immunoturbidimetrical method. His 48-yr-old mother presented with normal coagulation tests, normal Fibrinogen level and reported no history of bleeding. To identify the genetic mutation responsible for this hypoFibrinogen, genomic DNA extracted from the blood was analyzed. Fibrin polymerization measurement, kinetics of fibrinopeptide release, Fibrinogen clottability measurement, mass spectroscopy, and scanning electron microscopy were performed. Results: DNA sequencing showed heterogeneous Fibrinogen γG351S mutation in the propositus. The mutant chain was found not to be expressed to the circulation by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Scanning electron micrographs of the patient's fibrin clot as well as kinetics of fibrinopeptide release and fibrin polymerization were found to be normal. Conclusion: A case of hypoFibrinogenemia γG351S was found by routine coagulation testing and was genetically identified.
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A novel Fibrinogen Variant – Praha I: hypoFibrinogenemia associated with γ Gly351Ser substitution
European journal of haematology, 2007Co-Authors: Roman Kotlín, Jiří Suttnar, Peter Salaj, Tomáš Riedel, Martina Chytilová, Jiří Šantrůček, Pavel Klener, Jan E. DyrAbstract:Objectives: A 25-yr-old man from Prague had abnormal bleeding after several surgical operations with low Fibrinogen level and hypoFibrinogenemia was suspected. Patients and methods: The patient, 25 yr-old male had a low Fibrinogen concentration as determined by the thrombin time and immunoturbidimetrical method. His 48-yr-old mother presented with normal coagulation tests, normal Fibrinogen level and reported no history of bleeding. To identify the genetic mutation responsible for this hypoFibrinogen, genomic DNA extracted from the blood was analyzed. Fibrin polymerization measurement, kinetics of fibrinopeptide release, Fibrinogen clottability measurement, mass spectroscopy, and scanning electron microscopy were performed. Results: DNA sequencing showed heterogeneous Fibrinogen γG351S mutation in the propositus. The mutant chain was found not to be expressed to the circulation by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Scanning electron micrographs of the patient's fibrin clot as well as kinetics of fibrinopeptide release and fibrin polymerization were found to be normal. Conclusion: A case of hypoFibrinogenemia γG351S was found by routine coagulation testing and was genetically identified.
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Surface plasmon resonance analysis of immobilized Fibrinogen and fibrin and their interaction with thrombin and Fibrinogen
Biomedical Sensors Fibers and Optical Delivery Systems, 1999Co-Authors: Jan E. Dyr, Markéta Jiroušková, Jitka Rysava, Ivo Tichy, Petr Tobiska, Radan Slavik, Jiri Homola, Jiri SuttnarAbstract:The exploitation of surface plasmon resonance optical sensor for the study of the interaction of immobilized Fibrinogen and fibrin monomer with soluble Fibrinogen and thrombin is reported. Soluble Fibrinogen was mostly reversible, the bound thrombin could be inhibited by milimolar concentration of phenylmethylsulphonyl fluoride (PMSF). At lease three sets of different thrombin binding sites were found. There was a residual fraction of thrombin bound to washed fibrin (ogin) (to about a five to ten percent of fibron monomer units) suggesting that a known naturally occurring Fibrinogen Variant differing in the gamma chain was the target. Surface bound Fibrinogen was converted by thrombin to fibrin monomer that interacted with Fibrinogen in solution. At low fibrin monomer surface density the second layer was formed that contained about the same amount of protein as the first layer, at higher fibrin monomer concentration less than one molecule of Fibrinogen per molecule of fibrin monomer was captured. Starting with surface-bound Fibrinogen and alternating addition of thrombin and Fibrinogen a fibrin network of predetermined composition, size, and arrangement could be formed.
Bernhard Lämmle - One of the best experts on this subject based on the ideXlab platform.
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Fibrinogen st gallen i gamma 292 gly val evidence for structural alterations causing defective polymerization and Fibrinogenolysis
Thrombosis and Haemostasis, 1999Co-Authors: B Stucki, Bernhard Lämmle, Peter Schmutz, Luzius Schmid, Andre Haeberli, Miha FurlanAbstract:Fibrinogen 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 gamma-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 gamma 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 gamma-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 gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 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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Fibrinogen St. Gallen I (gamma 292 Gly--> Val): evidence for structural alterations causing defective polymerization and Fibrinogenolysis.
Thrombosis and haemostasis, 1999Co-Authors: B Stucki, Bernhard Lämmle, Peter Schmutz, Luzius Schmid, Andre Haeberli, Miha FurlanAbstract:Fibrinogen 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 gamma-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 gamma 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 gamma-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 gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 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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Fibrinogen Claro — Another dysfunctional Fibrinogen Variant with γ 275 Arginine → Histidine substitution
Thrombosis research, 1996Co-Authors: Colette Steinmann, Myriam Jungo, Eugene A. Beck, Bernhard Lämmle, Miha FurlanAbstract:Abstract Dysfunctional Fibrinogen Variants have been discovered by prolongation of thrombin and reptilase clotting times and a discrepancy of the functionally and immunologically determined Fibrinogen concentration. More than 250 Variants have been described (1). Majority of the abnormal Fibrinogen Variants have a structural defect near or at the thrombin cleavage site in the Aα-chain (2,3). Removal of fibrinopeptide A is required to expose the amino-terminal polymerization site (4,5). Most molecular defects in abnormal Fibrinogen Variants with normal fibrinopeptide A release but impaired fibrin monomer polymerization have been localized within residues γ 275 to γ 375 (1) containing the putative carboxy terminal polymerization site γ 337–379 (6,7). In the present report we describe another congenitally abnormal Fibrinogen that was found in a 42-year-old asymptomatic woman and her son. A single amino acid substitution γ 275 Arg → His was found in this dysfunctional Fibrinogen Variant.
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Fibrinogen claro another dysfunctional Fibrinogen Variant with γ 275 arginine histidine substitution
Thrombosis Research, 1996Co-Authors: Colette Steinmann, Myriam Jungo, Eugene A. Beck, Bernhard Lämmle, Miha FurlanAbstract:Abstract Dysfunctional Fibrinogen Variants have been discovered by prolongation of thrombin and reptilase clotting times and a discrepancy of the functionally and immunologically determined Fibrinogen concentration. More than 250 Variants have been described (1). Majority of the abnormal Fibrinogen Variants have a structural defect near or at the thrombin cleavage site in the Aα-chain (2,3). Removal of fibrinopeptide A is required to expose the amino-terminal polymerization site (4,5). Most molecular defects in abnormal Fibrinogen Variants with normal fibrinopeptide A release but impaired fibrin monomer polymerization have been localized within residues γ 275 to γ 375 (1) containing the putative carboxy terminal polymerization site γ 337–379 (6,7). In the present report we describe another congenitally abnormal Fibrinogen that was found in a 42-year-old asymptomatic woman and her son. A single amino acid substitution γ 275 Arg → His was found in this dysfunctional Fibrinogen Variant.
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Binding of calcium ions and their effect on clotting of Fibrinogen Milano III, a Variant with truncated A alpha-chains.
Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 1996Co-Authors: Miha Furlan, Colette Steinmann, Myriam Jungo, Bernhard LämmleAbstract:Calcium ions are known to be required for normal polymerisation of fibrin monomers. Normal human Fibrinogen has three high-affinity calcium binding sites. Two of these are located in the D-domains whereas the third binding site was tentatively assigned either to the E-domain or to the C-terminal part of the A alpha-chain. Furthermore, binding of calcium to the low-affinity binding sites (n > or = 10) facilitates fibrin monomer polymerisation. In several abnormally clotting Fibrinogen Variants, the polymerisation defect was partially normalised following addition of calcium ions. In this study, we show normal binding of calcium to Fibrinogen Milano III, a homozygous Fibrinogen Variant with truncated A alpha-chains (A alpha 452 Gly-Pro-Asp-->Trp-Ser-Stop). These results confirm that the C-terminal parts of the A alpha-chains beyond residue 451 Ile are not involved in calcium binding. The thrombin time was severely prolonged and the final clot turbidity was strongly reduced in Fibrinogen Milano III. Moreover, calcium ions did not significantly improve the abnormal clotting behavior of this dysFibrinogen. The polymerisation defect in Fibrinogen Milano III appears to be due to truncated A alpha-chains as well as to the disulphide-linked albumin.
Oleg V. Gorkun - One of the best experts on this subject based on the ideXlab platform.
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An engineered Fibrinogen Variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links.
Thrombosis and haemostasis, 2012Co-Authors: Rojin Park, Jong Rak Choi, Oleg V. Gorkun, Lifang Ping, Jaewoo Song, Joo-young Seo, Tae-youn Choi, Susan T. LordAbstract:A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular e-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 α chain can support FXIIIa-catalysed fibrin cross-linking.
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Polymerization-Defective Fibrinogen Variant gammaD364A Binds Knob “A” Peptide Mimic
Biochemistry, 2008Co-Authors: Sheryl R. Bowley, Nobuo Okumura, Betsy K. Merenbloom, Laurie Betts, Annie Heroux, Oleg V. Gorkun, Susan T. LordAbstract:Fibrin polymerization is supported in part by interactions called 'A:a'. Crystallographic studies revealed ?364Asp is part of hole 'a' that interacts with knob 'A' peptide mimic, GPRP. Biochemical studies have shown ?364Asp is critical to polymerization, as polymerization of Variants ?D364A, ?D364H, and ?D364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from ?D364A. Surprisingly, the structure (rfD-?D364A+GP) showed near normal 'A:a' interactions with GPRP bound to hole 'a' and no change in the overall structure of ?D364A. Of note, inspection of the structure showed negative electrostatic potential inside hole 'a' was diminished by this substitution. We examined GPRP binding to the ?364Asp Variants in solution by plasmin protection assay. We found no protection of either ?D364H or ?D364V but partial protection of ?D364A, indicating the peptide does not bind to either ?D364H or ?D364V and binds more weakly than normal to ?D364A. We also examined protection by calcium and found all Variants were indistinguishable from normal, suggesting the global structures of the Variants are not markedly different from normal. Our data imply that ?364Asp per se is not required for knob 'A' binding to hole 'a'; rather,more » this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.« less
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polymerization defective Fibrinogen Variant gammad364a binds knob a peptide mimic
Biochemistry, 2008Co-Authors: Sheryl R. Bowley, Nobuo Okumura, Betsy K. Merenbloom, Laurie Betts, Annie Heroux, Oleg V. Gorkun, Susan T. LordAbstract:Fibrin polymerization is supported in part by interactions called 'A:a'. Crystallographic studies revealed ?364Asp is part of hole 'a' that interacts with knob 'A' peptide mimic, GPRP. Biochemical studies have shown ?364Asp is critical to polymerization, as polymerization of Variants ?D364A, ?D364H, and ?D364V is exceptionally impaired. To understand the molecular basis for the aberrant function, we solved the crystal structure of fragment D from ?D364A. Surprisingly, the structure (rfD-?D364A+GP) showed near normal 'A:a' interactions with GPRP bound to hole 'a' and no change in the overall structure of ?D364A. Of note, inspection of the structure showed negative electrostatic potential inside hole 'a' was diminished by this substitution. We examined GPRP binding to the ?364Asp Variants in solution by plasmin protection assay. We found no protection of either ?D364H or ?D364V but partial protection of ?D364A, indicating the peptide does not bind to either ?D364H or ?D364V and binds more weakly than normal to ?D364A. We also examined protection by calcium and found all Variants were indistinguishable from normal, suggesting the global structures of the Variants are not markedly different from normal. Our data imply that ?364Asp per se is not required for knob 'A' binding to hole 'a'; rather,more » this residue's negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.« less
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Probing the gamma2 calcium-binding site: studies with gammaD298,301A Fibrinogen reveal changes in the gamma294-301 loop that alter the integrity of the "a" polymerization site.
Biochemistry, 2007Co-Authors: Michael S. Kostelansky, Oleg V. Gorkun, Lifang Ping, Karim C. Lounes, Sarah K Dickerson, Susan T. LordAbstract:To determine the significance of the gamma2 calcium-binding site in fibrin polymerization, we synthesized the Fibrinogen Variant, gammaD298,301A. We expected these two alanine substitutions to prevent calcium binding in the gamma2 site. We examined the influence of calcium on the polymerization of gammaD298,301A Fibrinogen, evaluated its plasmin susceptibility, and solved 2.7 and 2.4 A crystal structures of the Variant with the peptide ligands Gly-Pro-Arg-Pro-amide (GPRP) and Gly-His-Arg-Pro-amide (GHRP), respectively. We found that thrombin-catalyzed polymerization of gammaD298,301A Fibrinogen was modestly impaired, whereas batroxobin-catalyzed polymerization was significantly impaired relative to normal Fibrinogen. Notably, the influence of calcium on polymerization was the same for the Variant and for normal Fibrinogen. Fibrinogen gammaD298,301A was more susceptible to plasmin proteolysis in the presence of GPRP. This finding suggests structural changes in the near-by "a" polymerization site. Comparisons of the structures revealed minor conformational changes in the gamma294-301 loop that are likely responsible for the weakened "a" site. When considered altogether, the data suggest that the gamma2 calcium-binding site does not significantly modulate polymerization. We cannot, however, rule out the possibility that the weakened "a" polymerization site masks an important role for the gamma2 calcium-binding site in normal polymerization. Somewhat unexpectedly, the structure data showed that GPRP bound to the "b" site and induced the same local conformational changes as GHRP to this site. This structure shows that "A:b" interactions can occur and suggests that these may participate in normal polymerization.