Abstract 13986: Nanodiscs as a Platform for Structural Studies of Membrane-bound Factor VIII
Factor VIII (FVIII) is a multidomain blood plasma glycoprotein critical for blood hemostasis. Activated FVIII (FVIIIa) acts as a co-factor within the membrane-bound Tenase complex thus enhancing Factor Xa and consecutive thrombin generation more than 105 times, which is crucial for blood clot formation. Defect or deficiency of FVIII causes Hemophilia A, a severe hereditary bleeding disorder and the most efficient therapy of Hemophilia A is recombinant forms of FVIII concentrates. Despite the human FVIII crystal strcuture has been resolved by X-ray, the functional FVIII membrane-bound structure is not yet known. Defining the FVIII membrane-bound organization for different recombinant FVIII forms alone and within the tenase complex will provide critical information for FVIII physiological function. Resolving the FVIII membrane-bound structure will open new avenues for antithrombotic therapies at the level of the tenase complex.
In this study we propose a novel approach to solve the membrane-bound structure of clinically relevant recombinant FVIII forms, bound to functionalized Nanodiscs (ND) by Cryo-electron microscopy (Cryo-EM) and Single particle analysis (SPA). Cryo-EM is a method, which can give direct structural information at subnanometer resolution for fully hydrated samples and in a membrane environment. We have successfully attached two highly homologous clinically active FVIII forms: human and porcine on ND resembling the activated platelet surface. Nanodiscs (ND) are a unique membrane nanosystem (~10 nm in diameter), consisting of a phospholipid bilayer, stabilized by two copies of an amphipathic helical membrane scaffold protein. These nanolipid bilayers are very stable and homogeneously distributed in solution, therefore a suitable platform for structural studies of membrane-associated proteins and complexes such as FVIII its complex with FIXa by Cryo-EM and SPA.
We have used SPA based 3D reconstruction algorithms to calculate the structure of both human and porcine FVIIII on ND mimicking the activated platelet surface. Our first intermediate resolution (2 nm) 3D reconstructions show that the FVIII-ND complexes are suitable for further high resolution (< 1 nm) structure determination by Cryo-EM.
- © 2013 by American Heart Association, Inc.