Functional regions in coagulation factor VIII explored by mass spectrometry

Abstract

The molecular mechanisms behind the function of factor VIII (FVIII) have remained poorly understood. FVIII acts in the blood coagulation cascade as cofactor for activated factor IX (FIXa) in the membrane bound activated factor X generating (FXase) complex. A functional absence in FVIII leads to the bleeding disorder haemophilia A, which can be treated by frequent intravenous infusions with purified FVIII to reduce the risk for bleedings. Within its life cycle, FVIII binds to several ligands, including von Willebrand factor, which forms a tight protective complex with FVIII in the circulation, the constituents of the FXase complex, and its clearance receptors, LDL receptor and LDL receptor-related protein (LRP). The binding sites are confined within the light chain (domains: A3-C1-C1) and/or heavy chain of FVIII (domains: A1-A2). However, the identity of the functional regions on FVIII that contribute to the binding of these binding partners has remained a source of confusion.

In the present study, chemical footprinting in absence and presence of binding partners is employed to assess functional regions in proteins. We have developed a novel mass spectrometry approach to specifically identify differential exposure of lysine residues. To validate this approach we explore the exposure of lysine residues involved in binding in the complex between receptor associated protein (RAP) and LRP. Using our method, we successfully identify all established lysine residues as well as novel lysine residues that contribute to complex formation.

FVIII requires activation to perform its role as a cofactor. This activated FVIII (FVIIIa) rapidly looses its activity due to spontaneous A2-domain dissociation. Using our novel approach we investigated differences in surface exposure of lysine residues upon FVIII activation. The results show that Lys1967 and Lys1968 at the interface of the A1-A2-A3 domains exhibit an increased exposure to the solvent in dissociated FVIIIa. Site-directed mutagenesis of these residues reveal that both residues have an opposite contribution to A2-domain retention. In addition, lysine residues within region 1803-1818 also show increased surface exposure in dissociated FVIIIa. As this region was previously suggested to play a role in FIXa binding, site-directed mutagenesis is employed to investigate its involvement in FIXa and/or A2-domain binding. Results reveal a role for this complete region in A2-domain retention. In addition, the results exclusively confirm region 1811-1818 to be a FIXa binding region. Finally, using hydrogen-deuterium exchange (HDX) mass spectrometry we have identified a functional hot-spot on FVIII, i.e. region 2092-2093 and 2158-2159. Introduction of a glycan in region 2158-2159 reveals that this region, like the previously investigated region 2092-2093, contributes to phospholipid binding, and to cellular uptake by LRP expressing cells. In addition, these regions are involved in the uptake of FVIII by the antigen presenting dendritic cells which mediate the initiation of the immune response against FVIII, which can occur during haemophilia A treatment.

In conclusion, in this thesis we employ the latest mass spectrometry approaches to gain novel insight into the molecular mechanisms behind the biology of FVIII. Using these techniques, important functional roles for several regions are identified