Partial deletion of the αC-domain in the Fibrinogen Perth variant is associated with thrombosis, increased clot strength and delayed fibrinolysis

Summary

Genetic fibrinogen (FGN) variants that are associated with bleeding or thrombosis may be informative about fibrin polymerisation, structure and fibrinolysis. We report a four generation family with thrombosis and heritable dysfibrinogenaemia segregating with a c.[1541delC];[=] variation in FGA (FGN-Perth). This deletion predicts a truncated FGN αC-domain with an unpaired terminal Cys at residue 517 of FGN-Aα. In keeping with this, SDS-PAGE of purified FGN-Perth identified a truncated FGN-Aα chain with increased co-purification of albumin, consistent with disulphide bonding to the terminal Cys of the variant FGN-Aα. Clot visco-elastic strength in whole blood containing FGN-Perth was greater than controls and tPA-mediated fibrinolysis was delayed. In FGN-Perth plasma and in purified FGN-Perth, there was markedly reduced final turbidity after thrombin-mediated clot generation. Consistent with this, FGN-Perth formed tighter, thinner fibrin fibres than controls indicating defective lateral aggregation of protofibrils. Clots generated with thrombin in FGN-Perth plasma were resistant to tPA-mediated fibrinolysis. FGN-Perth clot also displayed impaired tPA-mediated plasmin generation but incorporated α2-anti-plasmin at a similar rate to control. Impaired fibrinolysis because of defective plasmin generation potentially explains the FGN-Perth clinical phenotype. These findings highlight the importance of the FGN αC-domain in the regulation of clot formation and fibrinolysis.

Keywords

Dysfibrinogenaemia - thrombosis - fibrin polymerisation - fibrinolysis

• References

• 1 Hill M, Dolan G. Diagnosis, clinical features and molecular assessment of the dysfibrinogenaemias. Haemophilia 2008; 14: 889-897.
• 2 Shapiro SE, Phillips E, Manning RA. et al. Clinical phenotype, laboratory features and genotype of 35 patients with heritable dysfibrinogenaemia. Br J Haematol 2012; 160: 220-227.
• 3 Cunningham MT, Brandt JT, Laposata M. et al. Laboratory diagnosis of dysfibrinogenemia. Arch Pathol Lab Med 2002; 126: 499-505.
• 4 Smith KA, Adamson PJ, Pease RJ. et al. Interactions between factor XIII and the alphaC region of fibrinogen. Blood 2011; 117: 3460-3468.
• 5 Tsurupa G, Medved L. Identification and characterisation of novel tPA- and plasminogen-binding sites within fibrin(ogen) alpha C domains. Biochemistry 2001; 40: 801-808.
• 6 Tsurupa G, Yakovlev S, McKee P. et al. Noncovalent interaction of alpha(2)-anti-plasmin with fibrin(ogen). localisation of alpha(2)-antiplasmin-binding sites. Biochemistry 2010; 49: 7643-7651.
• 7 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005; 3: 1894-1904.
• 8 Homer VM, Mullin JL, Brennan SO. et al. Novel Aalpha chain truncation (fibrinogen Perth) resulting in low expression and impaired fibrinogen polymerisation. J Thromb Haemost 2003; 1: 1245-1250.
• 9 Jayo A, Arnold E, Gonzalez-Manchon C. et al. Hypodysfibrinogenemia causing mild bleeding and thrombotic complications in a compound heterozygote of AalphaIVS4+1G>T mutation and Aalpha4841delC truncation (Aalpha(Perth)). Thromb Haemost 2009; 101: 770-772.
• 10 Furlan M, Steinmann C, Jungo M. et al. A frameshift mutation in Exon V of the A alpha-chain gene leading to truncated A alpha-chains in the homozygous dysfibrinogen Milano III. J Biol Chem 1994; 269: 33129-33134.
• 11 Koopman J, Haverkate F, Grimbergen J. et al. Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA). Blood 1992; 80: 1972-1979.
• 12 Scott DJ, Prasad P, Philippou H. et al. Clot architecture is altered in abdominal aortic aneurysms and correlates with aneurysm size. Arteriosclerosis, thrombosis, and vascular biology 2011; 31: 3004-3010.
• 13 Allan P, Uitte de Willige S, Abou-Saleh RH. et al. Evidence that fibrinogen gamma' directly interferes with protofibril growth: implications for fibrin structure and clot stiffness. J Thromb Haemost 2012; 10: 1072-1080.
• 14 Schroder V, Kohler HP. Effect of factor XIII Val34Leu on alpha2-antiplasmin incorporation into fibrin. Thromb Haemost 2000; 84: 1128-1130.
• 15 Bobbink IW, Tekelenburg WL, Sixma JJ. et al. Glycated proteins modulate tissue-plasminogen activator-catalysed plasminogen activation. Biochemical and biophysical research communications 1997; 240: 595-601.
• 16 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.
• 17 Wada Y, Lord ST. A correlation between thrombotic disease and a specific fibrinogen abnormality (A alpha 554 Arg-->Cys) in two unrelated kindred, Dusart and Chapel Hill III. Blood 1994; 84: 3709-3714.
• 18 Hanss M, Vergnes C, Rugeri L. et al. A new electrophoretic variant of fibrinogen associated with venous thromboembolism, fibrinogen Bordeaux Aalpha Arg439-->Cys. J Thromb Haemost 2008; 6: 1422-1424.
• 19 Brennan SO, Mosesson MW, Lowen R. et al. Dysfibrinogenemia (fibrinogen Wilmington) due to a novel Aalpha chain truncation causing decreased plasma expression and impaired fibrin polymerisation. Thromb Haemost 2006; 96: 88-89.
• 20 Ridgway HJ, Brennan SO, Gibbons S. et al. Fibrinogen Lincoln: a new truncated alpha chain variant with delayed clotting. Br J Haematol 1996; 93: 177-184.
• 21 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.
• 22 Litvinov RI, Yakovlev S, Tsurupa G. et al. Direct evidence for specific interactions of the fibrinogen alphaC domains with the central E region and with each other. Biochemistry 2007; 46: 9133-9142.
• 23 Tsurupa G, Mahid A, Veklich Y. et al. Structure, stability, and interaction of fibrin alphaC domain polymers. Biochemistry 2011; 50: 8028-8037.
• 24 Gorkun OV, Veklich YI, Medved LV. et al. Role of the alpha C domains of fibrin in clot formation. Biochemistry 1994; 33: 6986-6997.
• 25 Gorkun OV, Henschen-Edman AH, Ping LF. et al. Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerisation, albeit more subtle than anticipated from the analogous proteolytic fragment X. Biochemistry 1998; 37: 15434-15441.
• 26 Baradet TC, Haselgrove JC, Weisel JW. Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching. Biophys J 1995; 68: 1551-1560.
• 27 Matsuka YV, Medved LV, Migliorini MM. et al. Factor XIIIa-catalysed cross-linking of recombinant alpha C fragments of human fibrinogen. Biochemistry 1996; 35: 5810-5816.
• 28 Carlisle CR, Sparks EA, Der Loughian C. et al. Strength and failure of fibrin fiber branchpoints. J Thromb Haemost 2010; 8: 1135-1138.
• 29 Collet JP, Woodhead JL, Soria J. et al. Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots. Biophys J 1996; 70: 500-510.
• 30 Gabriel DA, Muga K, Boothroyd EM. The effect of fibrin structure on fibrinolysis. J Biol Chem 1992; 267: 24259-24263.