title: Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions.
abstract: Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H -fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H -fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for  γ D364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than  for γD364H -fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C -fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.
authors.affiliation: Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Soya
authors.forename: Fumiko
authors.initials: F
authors.lastname: Terasawa
authors.forename: Nobuo
authors.initials: N
authors.lastname: Okumura
mesh_terms: D012103:Batroxobin / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005337:Fibrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015241:Fibrinogens, Abnormal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D005344:Fibrinopeptide A / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D058105:Polymerization; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: C115499:BBeta fibrinogen; D015241:Fibrinogens, Abnormal; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; C464794:fibrinogen Aalpha; D005344:Fibrinopeptide A; D005337:Fibrin; D005340:Fibrinogen; D012103:Batroxobin; D013917:Thrombin