SAXS Analysis of the Intrinsic Tenase Complex Bound to Lipid Nanodisc Highlights Intermolecular Contacts between Factors VIIIa/IXa

The intrinsic tenase (Xase) complex is formed by activated clotting factors VIII (fVIIIa) and IX (fIXa) on activated platelet surfaces and catalyzes the activation of factor X to promote blood coagulation. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering a mechanistic understanding of how mutations to fVIIIa or fIXa lead to thrombotic disorders. In the present study, we aimed to structurally characterize the Xase complex bound to a lipid nanodisc using biolayer interferometry and small-angle X-ray scattering. By immobilizing lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results suggest that interactions between the Gla and catalytic domains of fIXa and the C2 and A2 domains of fVIIIa, respectively, mediate Xase complex assembly. Additionally, our computational modelling highlighted a novel interaction between fIXa residue K80 and an acidic patch formed at the fVIIIa A1/A3 domain interface. Our model of the Xase complex sheds light on how hemophilia A/B-related mutations with varying severities disrupt Xase complex assembly. We also speculate on how gain-of-function mutations to fIXa residue R338, such as the Padua (R338L) and Shanghai (R338Q) variants, bind to the fVIIIa A2 domain and stabilize the Xase complex, leading to excessive blood clotting. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.