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Thromb Haemost 2014; 112(03): 495-502
DOI: 10.1160/TH14-01-0086
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Enhanced factor VIIIa stability of A2 domain interface variants results from an increased apparent affinity for the A2 subunit

Morgan Monaghan
1   Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA
,
Hironao Wakabayashi
1   Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA
,
Amy Griffiths
1   Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA
,
Jennifer Wintermute
1   Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA
,
Philip J. Fay   †
1   Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York, USA
› Author Affiliations Financial support: This work was supported by NIH Grant HL38199 (to P. J. F.) and NIH predoctoral trainee-ship GM068411 from the Institutional Ruth L. Kirschstein National Research Award (to M. M.).
Further Information

Publication History

Received: 27 January 2014

Accepted after major revision: 10 April 2014

Publication Date:
20 November 2017 (online)

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Summary

Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.

Keywords

Factor VIII - factor VIIIa - inter-subunit binding affinity - protein stability

† Dr. Philip Fay has recently passed away.


 

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