Factor VIII Region 1811–1818 Is Involved in Factor IXa Binding and Factor VIIIa Stability

Abstract 3357

Previously, we identified a role for the lysine residue couple 1967/1968 in the stability of activated factor VIII (FVIIIa). Using tandem mass tags (TMT 126/127) in combination with mass spectrometry, we identified lysine residues involved in the interaction between the A2 domain and the rest of heterodimer (A1/A3-C1-C2) of FVIIIa (Bloem et al., J Biol Chem 2012;287:5575–83). Upon FVIII activation and A2 domain dissociation, the highest increase in surface exposure occurred for the lysine couple 1967/1968, and functional studies confirmed the role thereof in FVIIIa stability. In addition to lysines 1967/1968 also other lysines displayed an increased surface exposure, including those in positions 1804, 1808, 1813 and 1818. The A3 domain region 1803–1818 has previously been implicated in interactions with ligands such as activated factor IX (FIXa). As such, one might expect increased surface exposure due to FVIII activation. On the other hand, A2 domain dissociation may have rearranged the A3 domain surface in this region. The relation between FIXa assembly and A2 domain retention was therefore explored in the present study.

To unravel the role of region 1803–1818 in FVIIIa stability and FIXa binding, either region 1803–1810 or 1811–1818 was replaced by the corresponding regions of the homologous factor V. Additionally, as Asn1810 is N-linked glycosylated and this glycan is maintained in both chimeras, a variant that lacks this glycan (N1810C) was investigated.

Factor × activation kinetics were used to investigate the apparent FIXa binding affinity of the FVIII variants. FXa generation was assessed on 15% phosphatidyl serine (PS) containing membranes. FIXa titration experiments showed that the affinity for the 1811–1818 variant is reduced (apparent Kd from 1.3 nM to 2.4 nM). Removal of the glycan and substitution of 1803–1810 has little or no effect on the apparent FIXa binding. FVIIIa-FIXa assembly on membranes containing 15% PS was studied using lipid- coated glass beads (lipospheres). Lipospheres were incubated with fluorescein-labeled FIXa and different FVIIIa concentrations. FIXa did only display liposphere binding in the presence of FVIIIa. Therefore, the mean fluorescent intensity on the lipospheres at increasing FVIIIa concentration could be used as a measure for FVIIIa-FIXa assembly on lipids. Results from these experiments showed that the 1811–1818 variant fails to promote FVIIIa-FIXa assembly, whereas the other variants behave like WT. To investigate FVIIIa stability, and thereby the role of the mutations on A2 domain dissociation, the activity of the variants was followed over time. Results showed that the 1811–1818 variant has a decreased half life of 1.5 min, compared to 6.9 min for WT. Also substitution of region 1803–1810 results in a lower half life, although to a lesser extent (2.8 min), whereas the glycan lacking variant behaves like WT (6.8 min). Incubation of FVIIIa variants with FIXa is known to stabilize FVIIIa and leads to an increased half life for all variants. However, the 1803–1810 variant is most efficiently stabilized by FIXa, shown by a 3-fold increase in half life, instead of 1.6-fold as seen for both WT and N1810C. The 1811–1818 variant is stabilized by 2.2-fold and therefore remains significantly less stable than WT.

Together these results show that the 1811–1818 region is not only involved in FIXa binding, but additionally plays a major role in A2 domain retention. Region 1803–1810 also plays a role in FVIIIa stability, although to a lesser extent. Remarkably, the glycan at position Asn1810 does not influence neither FIXa binding nor FVIIIa stability, and apparently serves some other function.