Resolving the Structural Basis of Factor VIII Activation

Abstract

Activated Factor VIII (FVIIIa) is the essential co-factor for the serine protease Factor IXa (FIXa) in the membrane-bound Tenase complex. In the blood coagulation process, the interaction between FVIIIa and FIXa in presence of Ca2+ and phospholipids amplifies the proteolytic activity of Factor IXa more than 100.000 times. The Tenase complex catalyzes the activation of Factor X and initiates a cascade of multi-protein complex formation which amplifies the Thrombin production and secures the formation of a blood clot.Here we report for the first time the 3D-structure of the membrane-bound FVIIIa form, which is the most physiologically relevant in relation to the delicate equilibrium of hemostasis and thrombosis.The structure of the membrane bound light chain (domains A3-C1-C2) of human FVIII helically organized onto lipid bilayer nanotubes (LNT) clarified that the domains adopt a different organization than in the crystal structure which represents the molecule in solution. These differences of the membrane-bound FVIII structure have to be confirmed for the full length FVIII protein and the active form. To achieve this, high resolution Cryo-EM data of porcine FVIII and FVIIIa helically organized on LNT at closest to physiological conditions were collected. A newly developed process of iterated rounds of 2D refinement was applied to select only highly organized homogenous helical segments for an effective iterative helical real-space reconstruction (IHRSR). The calculated porcine FVIII and FVIIIa membrane-bound structures at subnanometer resolution were combined with flexible docking to define the changes in the FVIIIa interfaces important for its function.Understanding the structural differences and similarities between the membrane-bound activated FVIII and the non-activated FVIII will make an indispensable contribution to the field of coagulation in determining unique targets for the design of specific therapeutic drugs against Hemophilia A and thrombosis.