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

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

“…We therefore consider these regions and lysine residues "soft spots." The effect of the soft-spot lysine residues may be explained by conformational changes; lysine residue 1693 is directly facing the hot-spot lysine at position 1818, and the softspot lysine residue 1967 has been shown to interact with the A2 domain of FVIII (46). As for the soft-spot regions identified by HDX-MS, region 1735-1746 is located within the A3 region and consists of the amino acids FQEFTDGSFTQP.…”

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

“…The chemical footprinting approach has also revealed that the lysine residues in region 1803-1818 are more surface-exposed in dissociated FVIIIa than in intact FVIII (19). This suggests that this region might contribute to the direct interaction with the A2 domain in FVIIIa.…”

“…With this approach, we established that lysine residues 1967 and 1968 are more surface-exposed in dissociated FVIIIa than in intact FVIII. Sitedirected mutagenesis revealed that these residues have an opposite contribution to the stability of FVIIIa (19).…”

Factor VIII C1 Domain Spikes 2092–2093 and 2158–2159 Comprise Regions That Modulate Cofactor Function and Cellular Uptake

“…HEK293 cell lines stably expressing B domaindeleted FVIII and the R2159N variant were produced as described (23) and grown in DMEM-F12 medium supplemented with 10% FCS. Recombinant FVIII-WT and the R2159N variant were purified and analyzed as described using CLB-VK34 IgG1 coupled to CNBr-Sepharose 4B as an affinity matrix (16,24). Recombinant VWF was prepared as described previously (25).…”

“…These contained PS/L-␣-phosphatidylethanolamine (transphosphatidylated)/L-␣-phosphatidylcholine in a molar ratio of 15/20/65 (15% PS) or 5/20/85 (5% PS) and were prepared by sonication in 40 mM Tris-HCl (pH 7.8), 150 mM NaCl. FX was incubated with FIXa and FVIII-WT or FVIII-R2159N (0.3 nM) in the presence of thrombin (1 nM) and phospholipids in a buffer containing 40 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1.5 mM CaCl 2 , and 0.2% (w/v) BSA at 25°C in a final volume of 40 l. After varying time points (1-3 min), the reaction was terminated by adding 50 l of the same buffer containing 16 mM EDTA, and finally 10 l of 1.8 mM S-2765 was added to quantify activated factor X (14,24).…”