Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

• Published in: The Journal of biological chemistry 2012-02, Vol.287 (8), p.5775-5783
• Main Authors: Bloem, Esther, Meems, Henriet, van den Biggelaar, Maartje, van der Zwaan, Carmen, Mertens, Koen, Meijer, Alexander B.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Enzyme Kinetics; Factor VIII; Factor VIIIa - chemistry; Factor VIIIa - metabolism; Lysine - metabolism; Mass Spectrometry; Mass Spectrometry (MS); Molecular Sequence Data; Protein Stability; Protein Structure; Protein Structure and Folding; Surface Plasmon Resonance
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.308627

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys1967 and Lys1968 of region Thr1964-Tyr1971 are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys1967-Lys1968 within region Thr1964-Tyr1971 has an opposite contribution to the stability of FVIIIa. Activated FVIII (FVIIIa) stability is critical for cofactor function. Mass spectrometry analysis reveals that Lys1967 and/or Lys1968 are buried in factor VIII and surface-exposed in dissociated FVIIIa. Lys1967/Lys1968 variants differentially affect FVIIIa activity over time. Lys1967 and Lys1968 have an opposite contribution to FVIIIa stability. Insight is increased about how FVIIIa activity is controlled to prevent thrombosis or bleeding.