A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function

Summary

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95–98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

Keywords

Fibrinogen - platelet aggregation - RGD sequence - thrombosis - pregnancy loss

• References

• 1 Weisel JW. Fibrinogen and fibrin. Adv Prot Chem 2005; 70: 247-299.
• 2 Hill M, Dolan G. Diagnosis, clinical features and molecular assessment of the dysfibrinogeneamias. Haemophilia 2008; 14: 889-897.
• 3 Asselta R, Duga S, Tenchini ML. The molecular basis of quantitative fibrinogen disorders. J Thromb Haemost 2006; 4: 2115-2129.
• 4 Okumura N, Terasawa F, Hirota-Kawadobora M. et al. A novel variant fibrinogen, deletion of Bβ111Ser in coiled-coil region, affecting fibrin lateral aggregation. Clin Chim Acta 2006; 365: 160-167.
• 5 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Hae-most 2005; 3: 1894-1904.
• 6 Kotlín R, Reicheltová Z, Suttnar J. et al. Two novel fibrinogen variants in the C-terminus of the Bβ-chain: fibrinogen Rokycany and fibrinogen Znojmo. J Thromb Thrombolysis 2010; 30: 311-318.
• 7 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val --> Asp). J Clin Invest 1995; 96: 2854-2858.
• 8 Suttnar J, Dyr JE, Foftová H. et al. Determination of fibrinopeptides by high performance liquid chromatography. Biochem Clin Bohemoslov 1989; 18: 17-25.
• 9 Kotlín R, Sobotková A, Suttnar J. et al. A novel fibrinogen variant - Liberec: dys-fibrinogenaemia associated with γ Tyr262Cys substitution. Eur J Haematol 2008; 81: 123-129.
• 10 Miller SA, Dykes PD, Polensky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acid Res 1988; 16: 1215.
• 11 Riedel T, Brynda E, Dyr JE. et al. Controlled preparation of thin fibrin films immobilized at solid surfaces. J Biomed Mater Res A 2009; 88A: 437-447.
• 12 Reicheltova Z, Majek P, Riedel T. et al. Simplified platelet sample preparation for SDS-PAGE-based proteomic studies. Proteomics Clin Appl 2012; 6: 374-381.
• 13 Majek P, Reicheltova Z, Stikarova J. et al. Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin. Proteome Sci 2010; 8: 56.
• 14 Kollman JM, Pandi L, Sawaya MR. et al. Crystal structure of human fibrinogen. Biochemistry 2009; 48: 3977-3986.
• 15 Dear A, Brennan SO, Dempfle CE. et al. Hypofibrinogenaemia associated with a novel heterozygous gamma289 Ala -->Val substitution (fibrinogen Dorfen). Thromb Haemost 2004; 92: 1291-1295.
• 16 Undas A, Zdziarska J, Iwaniec T. et al. Fibrinogen Krakow: a novel hypo/dysfi-brinogenemia mutation in fibrinogen gamma chain (Asn325Ile) affecting fibrin clot structure and function. Thromb Haemost 2009; 101: 975-976.
• 17 Takagi T, Doolittle RF. Amino acid sequence studies on plasmin-derived fragments of human fibrinogen: N-terminal sequence of intermediate and terminal fragments. Biochemistry 1975; 14: 940.
• 18 Lugovskoy EV, Gritsenko PG, Kolesnikova IN. et al. A neoantigenic determinant in coiled coil region of human fibrin β-chain. Thromb Res 2009; 123: 765-770.
• 19 Sugo T, Nakamikawa C, Takano H. et al. Fibrinogen Niigata with impaired fibrin assembly: an inherited dysfibrinogen with a Bbeta Asn-160 to Ser substitution associated with extra glycosylation at Bbeta Asn-158. Blood 1999; 94: 3806-3813.
• 20 Lounes KC, Lefkowitz JB, Henschen-Edman AH. et al. The impaired polymerization of fibrinogen Longmont (Bbeta 166 Arg->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cystein-linked monomers. Blood 2001; 98: 661-667.
• 21 Soe G, Kohno I, Inuzuka K. et al. A monoclonal antibody that recognizes a neo-antigen exposed in the E domain of fibrin monomer complexed with fibrinogen or its derivatives: its application to the measurement of soluble fibrin in plasma. Blood 1996; 88: 2109-2117.
• 22 Morris TA, Marsh JJ, Chiles PG. et al. High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension. Blood 2009; 114: 1929-1936.
• 23 Doolittle RF. Determining the crystal structure of fibrinogen. J Thromb Hae- most 2004; 2: 683-689.
• 24 Hunter CA, Singh J, Thornton JM. π-π interactions: the geometry and energetics of phenylalanine-phenylalanine interactions in proteins. J Mol Biol 1991; 218: 837-846.
• 25 Marchi R, Arocha-Piñango CL, Nagy H. et al. The effects of additional carbohydrate in the coiled-coil region of fibrinogen on polymerization and clot structure and properties: characterization of the homozygous and heterozygous forms of fibrinogen Lima (Aa Arg141->Ser with extra glycosylation). J Thromb Haemost 2004; 2: 940-948.
• 26 Cheresh DA, Berliner SA, Vicente V. et al. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 945-953.
• 27 Farrell DH, Thiagarajan P, Chung DW. et al. Role of fibrinogen alpha and gamma chain sites in platelet aggregation. Proc Natl Acad Sci USA 1992; 89: 10729-10732.
• 28 Kotlín R, Reicheltová Z, Maly M. et al. Two cases of congenital dysfibrinogen-emia associated with thrombosis - Fibrinogen Praha III a Fibrinogen Plzeñ. Thromb Haemost 2009; 102: 479-486.
• 29 Collet JP, Allali Y, Lesty C. et al. Altered fibrin architecture is associated with hy-pofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol 2006; 26: 2567-2573.
• 30 Krabbendam I, Franx A, Bots ML. et al. Thrombophilias and recurrent pregnancy loss: a critical appraisal of the literature. Eur J Obstet Gynecol Reprod Biol 2005; 118: 143-153.
• 31 Haverkate F, Samama M. Familial dysfibrinogenemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen. Thromb Haemost 1995; 73: 151-161.