Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions

Summary

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 γD364H-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.

Keywords

Fibrin polymerisation - B:b interaction - fibrinopeptide A release - knob ‘A’, hole ‘a’

• References

• 1 Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70: 247-299.
• 2 Everse SJ, Spraggon G, Veerapandian L. et al. Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide. Biochemistry 1999; 38: 2941-2946.
• 3 Gorkun OV, Veklich YI, Medved LV. et al. Role of the alpha C domains of fibrin in clot formation. Biochemistry 1994; 33: 6986-6997.
• 4 Yang ZI, Mochalkin I, Doolittle RF. A model of fibrin formation based on crystal structures of fibrinogen and fibrin fragments complexed with synthetic peptides. Proc Natl Acad Sci USA 2000; 97: 14156-14161.
• 5 Weisel JW, Veklich Y, Gorkun O. The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots. J Mol Biol 1993; 232: 285-297.
• 6 Weisel JW. Fibrin Assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides. Biophys J 1986; 50: 1079-1093.
• 7 Doolittle RF, Pandi L. Binding of synthetic B-knobs to fibrinogen changes the character of fibrin and inhibits its ability to activate tissue plasminogen activator and its destruction by plasmin. Biochemistry 2006; 45: 2657-2667.
• 8 Doolittle RF, Pandi L. Probing the β-chain hole of fibrinogen with synthetic peptides that differ at their amino termini. Biochemistry 2007; 46: 10033-10038.
• 9 Okumura N, Terasawa F, Haneishi A. et al. B:b interactions are essential for polymerisation of variant fibrinogens with impaired holes ’a’. J Thromb Haemost 2007; 05: 2352-2359.
• 10 Okumura N, Gorkun OV, Lord ST. Severely impaired polymerisation of recombinant fibrinogen γ-364 Asp-> His, the substitution discovered in a heterozygous individual. J Biol Chem 1997; 272: 29596-29601.
• 11 Okumura N, Terasawa F, Takezawa Y. et al. Heterozygous Bβ-chain C-terminal 12 amino acid elongation variant, BβX462W (Kyoto VI), showed dysfibrinogenemia. Blood Coagul Fibrinolysis 2012; 23: 87-90.
• 12 Lewis SD, Shields PP, Shafer JA. Characterisation of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin. J Biol Chem 1985; 260: 10192-10199.
• 13 Bowley SR, Merenbloom BK, Okumura N. et al. Polymerisation-defective fibrinogen variant γD364A binds knob “A” peptide mimic. Biochemistry 2008; 47: 8607-8613.
• 14 Laudano A, Doolittle RF. Synthetic peptide derivatives that bind to fibrinogen and prevent the polymerisation of fibrin monomers. Proc Natl Acad Sci USA 1978; 75: 3085-3089.
• 15 Litvinov RI, Gorkun OV, Owen SF. et al. Polymerisation of fibrin: specificity strength, and stability of knob-hole interactions studied at the single-molecular level. Blood 2005; 106: 2944-2951.
• 16 Wada Y, Niwa M, Maekawa H. et al. A new type of congenital dysfibrinogen, fibrinogen Bremen, with an AαGly-17 to Val substitution associated with hemorrhagic diathesis and delayed wound healing. Thromb Haemost 1993; 70: 397-403.
• 17 Weisel JW, Nagaswami C. Computer modeling of fibrin polymerisation kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophys J 1992; 63: 111-128.
• 18 Laudano A, Doolittle RF. Influence of calcium ion on the binding of fibrin amino terminal peptides to fibrinogen. Science 1981; 212: 457-459.
• 19 Soria J, Soria C, Samama M. et al. Fibrinogen Troyes - Fibrinogen Metz. Two new cases of congenital dysfibrinogenemia. Thromb Diath Haemorrh 1972; 27: 619-633.
• 20 Galanakis D, Spitzer S, Scharrer I. Unusual Aα16Arg→Cys dysfibrinogenaemic family: absence of normal Aα-chains in fibrinogen from two of four heterozygous siblings. Blood Coagul Fibrinolysis 1993; 04: 67-71.
• 21 Galanakis DK, Neerman-Arbez M, Scheiner T. et al. Homophenotypic AαR16H fibrinogen (Kingsport): uniquely altered polymerization associated with slower fibrinopeptide A than fibrinopeptide B releas. Blood Coagul Fibrinolysis 2007; 18: 731-737.