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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
› Author Affiliations Funding This research was supported by a grant from the National Institutes of Health [R15 HL120068 to MCM].
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Abstract

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.

Keywords

fibrinogen - transglutaminase - factor XIII - substrate specificity - activation peptide

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



Publication History

Received: 14 August 2023

Accepted: 30 November 2023

Accepted Manuscript online:
01 December 2023

Article published online:
09 January 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

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