Thromb Haemost 2024; 124(05): 408-422
DOI: 10.1055/a-2220-7544
Coagulation and Fibrinolysis

Identification of Factor XIII β-Sandwich Residues Mediating Glutamine Substrate Binding and Activation Peptide Cleavage

Rameesa D. Syed Mohammed
1   Department of Chemistry, University of Louisville, Louisville, Kentucky, United States
Kellianne M. Piell
2   Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, Kentucky, United States
Muriel C. Maurer
1   Department of Chemistry, University of Louisville, Louisville, Kentucky, United States
› Institutsangaben
Funding This research was supported by a grant from the National Institutes of Health [R15 HL120068 to MCM].


Background Factor XIII (FXIII) forms covalent crosslinks across plasma and cellular substrates and has roles in hemostasis, wound healing, and bone metabolism. FXIII activity is implicated in venous thromboembolism (VTE) and is a target for developing pharmaceuticals, which requires understanding FXIII – substrate interactions. Previous studies proposed the β-sandwich domain of the FXIII A subunit (FXIII-A) exhibits substrate recognition sites.

Material and Methods Recombinant FXIII-A proteins (WT, K156E, F157L, R158Q/E, R171Q, and R174E) were generated to identify FXIII-A residues mediating substrate recognition. Proteolytic (FXIII-A*) and non-proteolytic (FXIII-A°) forms were analyzed for activation and crosslinking activities toward physiological substrates using SDS-PAGE and MALDI-TOF MS.

Results All FXIII-A* variants displayed reduced crosslinking abilities compared to WT for Fbg αC (233 – 425), fibrin, and actin. FXIII-A* WT activity was greater than A°, suggesting the binding site is more exposed in FXIII-A*. With Fbg αC (233 – 425), FXIII-A* variants R158Q/E, R171Q, and R174E exhibited decreased activities approaching those of FXIII-A°. However, with a peptide substrate, FXIII-A* WT and variants showed similar crosslinking suggesting the recognition site is distant from the catalytic site. Surprisingly, FXIII-A R158E and R171Q displayed slower thrombin activation than WT, potentially due to loss of crucial H-bonding with neighboring activation peptide (AP) residues.

Conclusion In conclusion, FXIII-A residues K156, F157, R158, R171, and R174 are part of a binding site for physiological substrates [fibrin (α and γ) and actin]. Moreover, R158 and R171 control AP cleavage during thrombin activation. These investigations provide new molecular details on FXIII – substrate interactions that control crosslinking abilities.

Authors' Contribution

R.D.S.M. and M.C.M. designed the research. R.D.S.M and K.M.P performed the experiments. M.C.M. and R.D.S.M. interpreted the data and wrote the manuscript. All authors reviewed the manuscript and approved its final version.

Supplementary Material


Eingereicht: 14. August 2023

Angenommen: 30. November 2023

Accepted Manuscript online:
01. Dezember 2023

Artikel online veröffentlicht:
09. Januar 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

  • References

  • 1 Richardson VR, Cordell P, Standeven KF, Carter AM. Substrates of factor XIII-A: roles in thrombosis and wound healing. Clin Sci (Lond) 2013; 124 (03) 123-137
  • 2 Pieters M, Wolberg AS. Fibrinogen and fibrin: an illustrated review. Res Pract Thromb Haemost 2019; 3 (02) 161-172
  • 3 Helms CC, Ariëns RA, Uitte de Willige S, Standeven KF, Guthold M. α-α Cross-links increase fibrin fiber elasticity and stiffness. Biophys J 2012; 102 (01) 168-175
  • 4 Duval C, Allan P, Connell SD, Ridger VC, Philippou H, Ariëns RA. Roles of fibrin α- and γ-chain specific cross-linking by FXIIIa in fibrin structure and function. Thromb Haemost 2014; 111 (05) 842-850
  • 5 Byrnes JR, Duval C, Wang Y. et al. Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking. Blood 2015; 126 (16) 1940-1948
  • 6 Wolberg AS, Sang Y. Fibrinogen and factor XIII in venous thrombosis and thrombus stability. Arterioscler Thromb Vasc Biol 2022; 42 (08) 931-941
  • 7 Schroeder V, Kohler HP. Factor XIII: structure and function. Semin Thromb Hemost 2016; 42 (04) 422-428
  • 8 Nikolajsen CL, Dyrlund TF, Poulsen ET, Enghild JJ, Scavenius C. Coagulation factor XIIIa substrates in human plasma: identification and incorporation into the clot. J Biol Chem 2014; 289 (10) 6526-6534
  • 9 Inbal A, Lubetsky A, Krapp T. et al. Impaired wound healing in factor XIII deficient mice. Thromb Haemost 2005; 94 (02) 432-437
  • 10 Bagoly Z, Katona E, Muszbek L. Factor XIII and inflammatory cells. Thromb Res 2012; 129 (Suppl. 02) S77-S81
  • 11 Myneni VD, Hitomi K, Kaartinen MT. Factor XIII-A transglutaminase acts as a switch between preadipocyte proliferation and differentiation. Blood 2014; 124 (08) 1344-1353
  • 12 Cui C, Wang S, Myneni VD, Hitomi K, Kaartinen MT. Transglutaminase activity arising from factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures. Bone 2014; 59: 127-138
  • 13 Cui C, Kaartinen MT. Serotonin (5-HT) inhibits factor XIII-A-mediated plasma fibronectin matrix assembly and crosslinking in osteoblast cultures via direct competition with transamidation. Bone 2015; 72: 43-52
  • 14 Javed H, Singh S, Ramaraje Urs SU, Oldenburg J, Biswas A. Genetic landscape in coagulation factor XIII associated defects - advances in coagulation and beyond. Blood Rev 2023; 59: 101032
  • 15 Schmitz T, Bäuml CA, Imhof D. Inhibitors of blood coagulation factor XIII. Anal Biochem 2020; 605: 113708
  • 16 Katona E, Pénzes K, Csapó A. et al. Interaction of factor XIII subunits. Blood 2014; 123 (11) 1757-1763
  • 17 Byrnes JR, Wilson C, Boutelle AM. et al. The interaction between fibrinogen and zymogen FXIII-A2B2 is mediated by fibrinogen residues γ390–396 and the FXIII-B subunits. Blood 2016; 128 (15) 1969-1978
  • 18 Alshehri FSM, Whyte CS, Mutch NJ. Factor XIII-A: an indispensable “factor” in haemostasis and wound healing. Int J Mol Sci 2021; 22 (06) 3055
  • 19 Weiss MS, Metzner HJ, Hilgenfeld R. Two non-proline cis peptide bonds may be important for factor XIII function. FEBS Lett 1998; 423 (03) 291-296
  • 20 Muszbek L, Bereczky Z, Bagoly Z, Komáromi I, Katona É. Factor XIII: a coagulation factor with multiple plasmatic and cellular functions. Physiol Rev 2011; 91 (03) 931-972
  • 21 Muszbek L, Polgár J, Boda Z. Platelet factor XIII becomes active without the release of activation peptide during platelet activation. Thromb Haemost 1993; 69 (03) 282-285
  • 22 Somodi L, Beke Debreceni I, Kis G. et al. Activation mechanism dependent surface exposure of cellular factor XIII on activated platelets and platelet microparticles. J Thromb Haemost 2022; 20 (05) 1223-1235
  • 23 Cleary DB, Maurer MC. Characterizing the specificity of activated factor XIII for glutamine-containing substrate peptides. Biochim Biophys Acta 2006; 1764 (07) 1207-1217
  • 24 Doiphode PG, Malovichko MV, Mouapi KN, Maurer MC. Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay. Anal Biochem 2014; 457: 74-84
  • 25 Sugimura Y, Hosono M, Wada F, Yoshimura T, Maki M, Hitomi K. Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA. J Biol Chem 2006; 281 (26) 17699-17706
  • 26 Pénzes K, Kövér KE, Fazakas F, Haramura G, Muszbek L. Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate. J Thromb Haemost 2009; 7 (04) 627-633
  • 27 Smith KA, Adamson PJ, Pease RJ. et al. Interactions between factor XIII and the alphaC region of fibrinogen. Blood 2011; 117 (12) 3460-3468
  • 28 Procyk R, Bishop PD, Kudryk B. Fibrin–recombinant human factor XIII a-subunit association. Thromb Res 1993; 71 (02) 127-138
  • 29 Ablan FDO, Maurer MC. Fbg αC 389–402 enhances factor XIII cross-linking in the fibrinogen αC region via electrostatic and hydrophobic interactions. Biochemistry 2023; 62 (14) 2170-2181
  • 30 Fukue H, Anderson K, McPhedran P, Clyne L, McDonagh J. A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA. Blood 1992; 79 (01) 65-74
  • 31 Hornyak TJ, Shafer JA. Interactions of factor XIII with fibrin as substrate and cofactor. Biochemistry 1992; 31 (02) 423-429
  • 32 Mohammed RDS, Ablan FDO, McCann NM, Hindi MM, Maurer MC. Transglutaminase activities of blood coagulant factor XIII are dependent on the activation pathways and on the substrates. Thromb Haemost 2023; 123 (04) 380-392
  • 33 Smith KA, Pease RJ, Avery CA. et al. The activation peptide cleft exposed by thrombin cleavage of FXIII-A(2) contains a recognition site for the fibrinogen α chain. Blood 2013; 121 (11) 2117-2126
  • 34 Schroeder V, Meili E, Cung T, Schmutz P, Kohler HP. Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder. Thromb Haemost 2006; 95 (01) 77-84
  • 35 Zheng WD, Liu YH, He QY, Chen ZH, Fan XB, Liu HF. Identification of Arg77Cys and Arg174stop double heterozygous mutation in a Chinese family with inherited FXIII deficiency [in Chinese]. Zhonghua Xue Ye Xue Za Zhi 2009; 30 (03) 158-161
  • 36 Biswas A, Ivaskevicius V, Thomas A. et al. Eight novel F13A1 gene missense mutations in patients with mild FXIII deficiency: in silico analysis suggests changes in FXIII-A subunit structure/function. Ann Hematol 2014; 93 (10) 1665-1676
  • 37 Jadhav MA, Goldsberry WN, Zink SE. et al. Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: a strategy for controlling fibrin architecture. Biochim Biophys Acta Proteins Proteomics 2017; 1865 (10) 1246-1254
  • 38 Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 2005; 41 (01) 207-234
  • 39 Anokhin BA, Dean WL, Smith KA. et al. Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A. FEBS J 2020; 287 (03) 452-464
  • 40 Mouapi KN, Bell JD, Smith KA, Ariëns RA, Philippou H, Maurer MC. Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII. Blood 2016; 127 (18) 2241-2248
  • 41 Roy I, Smith O, Clouthier CM, Keillor JW. Expression, purification and kinetic characterisation of human tissue transglutaminase. Protein Expr Purif 2013; 87 (01) 41-46
  • 42 Thomas A, Biswas A, Dodt J. et al. Coagulation factor XIIIA subunit missense mutations affect structure and function at the various steps of factor XIII action. Hum Mutat 2016; 37 (10) 1030-1041
  • 43 Serrano K, Devine DV. Intracellular factor XIII crosslinks platelet cytoskeletal elements upon platelet activation. Thromb Haemost 2002; 88 (02) 315-320
  • 44 Guharoy M, Chakrabarti P. Conserved residue clusters at protein-protein interfaces and their use in binding site identification. BMC Bioinformatics 2010; 11: 286
  • 45 Li B, Kohler HP, Schroeder V. Identification of amino acid residues that are crucial for FXIII-A intersubunit interactions and stability. Blood 2020; 135 (02) 145-152
  • 46 Handrkova H, Schroeder V, Kohler HP. The activation peptide of coagulation factor XIII is vital for its expression and stability. J Thromb Haemost 2015; 13 (08) 1449-1458
  • 47 Stieler M, Weber J, Hils M. et al. Structure of active coagulation factor XIII triggered by calcium binding: basis for the design of next-generation anticoagulants. Angew Chem Int Ed Engl 2013; 52 (45) 11930-11934
  • 48 Duval C, Ali M, Chaudhry WW, Ridger VC, Ariëns RA, Philippou H. Factor XIII A-subunit V34L variant affects thrombus cross-linking in a murine model of thrombosis. Arterioscler Thromb Vasc Biol 2016; 36 (02) 308-316
  • 49 Hamedani NS, Biswas A, Rudan O. et al. Functional and structural characterization of nucleic acid ligands that bind to activated coagulation factor XIII. J Clin Med 2021; 10 (04) 677
  • 50 Achyuthan KE, Slaughter TF, Santiago MA, Enghild JJ, Greenberg CS. Factor XIIIa-derived peptides inhibit transglutaminase activity. Localization of substrate recognition sites. J Biol Chem 1993; 268 (28) 21284-21292