TY - JOUR
T1 - The activation peptide cleft exposed by thrombin cleavage of FXIII-A2 contains a recognition site for the fibrinogen α chain
AU - Smith, Kerrie A.
AU - Pease, Richard J.
AU - Avery, Craig A.
AU - Brown, Jane M.
AU - Adamson, Penelope J.
AU - Cooke, Esther J.
AU - Neergaard-Petersen, Søs
AU - Cordell, Paul A.
AU - Ariëns, Robert A.S.
AU - Fishwick, Colin W.G.
AU - Philippou, Helen
AU - Grant, Peter J.
N1 - Funding Information:
The authors thank Prof Susan Lord for the generous provision of CHO cells expressing fibrinogen; Boothby for technical assistance; and Dr Adam Dowle for expertise in mass spectrometry (Bioscience Technology Facility, University of York, UK). This work was supported by the British Heart Foundation (program grant RG/08/004/25 292).
Publisher Copyright:
© 2013 by The American Society of Hematology.
PY - 2013/3/14
Y1 - 2013/3/14
N2 - 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-A2 (FXIII-A2*); however, the functional role of this interaction and binding site(s) on FXIII-A2* 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-A2* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A2*; and (3) carried out molecular modeling of the FXIII-A2*/peptide complex to identify contact site (s) involved. Results demonstrated that inhibition of the FXIII-A2*/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-A2* localizing αC cross-linking Q366 to the FXIII-A2* 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-A2* as a potential site involved in glutamine-donor substrate recognition.
AB - 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-A2 (FXIII-A2*); however, the functional role of this interaction and binding site(s) on FXIII-A2* 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-A2* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A2*; and (3) carried out molecular modeling of the FXIII-A2*/peptide complex to identify contact site (s) involved. Results demonstrated that inhibition of the FXIII-A2*/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-A2* localizing αC cross-linking Q366 to the FXIII-A2* 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-A2* as a potential site involved in glutamine-donor substrate recognition.
UR - http://www.scopus.com/inward/record.url?scp=84877625984&partnerID=8YFLogxK
U2 - 10.1182/blood-2012-07-446393
DO - 10.1182/blood-2012-07-446393
M3 - Article
C2 - 23303819
AN - SCOPUS:84877625984
SN - 0006-4971
VL - 121
SP - 2117
EP - 2126
JO - Blood
JF - Blood
IS - 11
ER -