Factor VIII organisation on nanodiscs with different lipid composition.

Area of Science:

• Structural biology
• Biochemistry
• Membrane protein studies

Background:

• Nanodiscs (NDs) are nanoscale lipid bilayers stabilized by scaffold proteins, ideal for membrane protein research.
• Blood coagulation relies on the assembly of complexes like Factor VIII (FVIII) on activated platelet membranes.
• Understanding these interactions is critical for hemostasis and treating bleeding disorders.

Purpose of the Study:

• To optimize nanodiscs (NDs) for binding and structural analysis of membrane-bound Factor VIII (FVIII).
• To investigate the influence of phosphatidylserine (PS) content and scaffold protein (MSP1D1) to lipid ratios on FVIII binding.
• To enable structural studies of the FVIII and intrinsic tenase complex using electron microscopy (EM).

Main Methods:

• Preparation of nanodiscs (NDs) with varying phosphatidylserine (PS) compositions and MSP1D1 to lipid ratios.
• Binding studies of Factor VIII (FVIII) to optimized NDs.
• Analysis using electron microscopy (EM) and single particle analysis for 3D reconstruction.

Main Results:

• Nanodiscs (NDs) with 80% phosphatidylserine (PS) content and a 1:47 MSP1D1 to lipid ratio showed optimal FVIII binding.
• Preliminary 3D reconstruction of membrane-bound FVIII on PS-containing NDs confirmed suitability for EM studies.
• Optimized NDs provide a platform for studying FVIII and the intrinsic tenase complex assembly.

Conclusions:

• Optimized nanodiscs (NDs) are effective platforms for the structural determination of membrane-bound Factor VIII (FVIII) using single particle EM.
• These findings advance the structural understanding of blood coagulation factors and their interactions.
• Future work will focus on the FVIIIa-FIXa complex to elucidate critical protein-protein and protein-membrane interfaces.