title: The activation peptide cleft exposed by thrombin cleavage of FXIII-A(2) contains a recognition site for the fibrinogen α chain.
abstract: Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the β-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.
authors.affiliation: Division of Cardiovascular and Diabetes Research, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom.
authors.forename: Kerrie A
authors.initials: KA
authors.lastname: Smith
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Pease
authors.forename: Craig A
authors.initials: CA
authors.lastname: Avery
authors.forename: Jane M
authors.initials: JM
authors.lastname: Brown
authors.forename: Penelope J
authors.initials: PJ
authors.lastname: Adamson
authors.forename: Esther J
authors.initials: EJ
authors.lastname: Cooke
authors.forename: Søs
authors.initials: S
authors.lastname: Neergaard-Petersen
authors.forename: Paul A
authors.initials: PA
authors.lastname: Cordell
authors.forename: Robert A S
authors.initials: RA
authors.lastname: Ariëns
authors.forename: Colin W G
authors.initials: CW
authors.lastname: Fishwick
authors.forename: Helen
authors.initials: H
authors.lastname: Philippou
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Grant
mesh_terms: D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D005176:Factor XIII / Q000737:chemistry* / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D013917:Thrombin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D020033:Protein Isoforms; C098000:fibrinogen alphaC; C064015:factor XIII subunit A; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin