Id: 175 Site-Directed Mutagenesis of Residues Surrounding the Thrombin Cleavage Site in Factor Xiii-a Subunit Demonstrates Relevant Sites for Interaction

Abstract

Background and aims: Factor (F)XIII is a protransglutaminase that on activation by thrombin introduces covalent cross-links in fibrin and incorporates inhibitors of fibrinolysis to increase resistance of the clot to proteolysis. Activation of FXIII involves cleavage of R37-G38 in the A-subunit by thrombin. Interaction with thrombin has been investigated for peptides based on FXIII-A sequence, but not for full-length FXIII-A. The only information on full-length FXIII is derived from the naturally occurring V34L variant, which has been shown to increase activation rates. We therefore used site-directed mutagenesis on recombinant FXIII-A to investigate thrombin interaction. Methods: Recombinant full-length FXIII-A was expressed with an N-terminal GST-tag in E coli. The cells expressed fully functional FXIII-A of dimeric form. Site-directed mutagenesis was used to produce mutants of the activation peptide and the region downstream of the thrombin cleavage site. Residues 28-35 and 37-42 were mutated to alanine, V29 also to leucine, and V34 to leucine and methionine, to investigate the role of increase of side-chain mass at these positions. Recombinant proteins were purified to homogeneity using glutathione affinity chromatography. Interaction with thrombin was investigated through generation of cross-linking activity in a biotin-pentylamine incorporation assay. Cleavage of the recombinant proteins by thrombin was confirmed by SDS-PAGE. Results: Mutation of R37 at the thrombin cleavage site to alanine completely abolished activation of FXIII-A by thrombin as expected. Mutation of residues V29, E30, G33 and V34 to alanine significantly reduced activation rates of FXIII-A, indicating a role for these residues of the activation peptide just upstream of the thrombin cleavage site in the interaction between FXIII-A and thrombin. There were no significant effect of mutations of residues G38, V39, N40, L41 or Q42 to alanine on activation rates of FXIII-A, showing that residues immediately downstream of the thrombin cleavage site do not play a role. V34L increased activation rate by thrombin. A similar effect was observed for V29L. Mutation of V34 to different residues showed that an increase in side-chain mass increased activation rates of FXIII-A by thrombin (M>L>V>A34). Conclusion: These data demonstrate that residues R37, V34 and G33 at P1, P4 and P5 positions play a role in full-length FXIII-A for thrombin interaction. They also indicate a further role for residues V29 and E30 in this process. Side-chain mass at residue 34 enhances thrombin interaction, elucidating a mechanism by which V34L increases FXIII activation rates.