Membrane-Induced Dimerization of Coagulation Factor VIII

Abstract

Coagulation Factor VIII (FVIII) is a blood plasma protein expressed as five domains denoted as A1, A2, A3, C1 and C2 that exist as a non-covalently bound heterodimer of a light (A3, C1, C2) and heavy (A1, A2) chain with a combined molecular mass of ∼170 kDa. FVIII is activated by thrombin to FVIIIa, which is the cofactor to the serine protease Factor IXa (FIXa) within the membrane-bound Tenase complex that assembles on the activated platelets’ surface during the propagation phase of coagulation. FVIIIa is a heterotrimer, due to an additional cleavage between the A1 and A2 domains.Although the function of FVIII has been well characterized, little is known about its membrane-bound structure and mechanism of the Tenase complex assembly. To fill this knowledge gap, we have engineered negatively charged lipid nanotubes (LNT) that resemble the activated platelet surface and on which FVIII and FVIIIa can be helically organized. The helically organized FVIII-LNT and FVIIIa-LNT were further subjected to structural analysis by Cryo-electron microscopy (Cryo-EM) to elucidate the structural basis of FVIII activation and function. To achieve this, we carried out helical reconstruction with the iterative real space helical reconstruction algorithm (IHRSR). Our results show that membrane-bound FVIII forms dimers that associate tightly through heavy chain - heavy chain interactions. These interactions involve the A1 and A2 domains surface that don’t overlap with the A2-A3 interface containing the FVIIIa binding sites to FIXa. We therefore propose that membrane-induced dimerization of FVIII is required to stabilize its membrane-bound conformation and facilitate its interaction with FIXa. The resulting model of a binary Tenase complex would therefore consist of two molecules of FIXa and two molecules of FVIIIa, which fits its function in coagulation: increasing of more than a 100,000 fold the FIXa proteolytic activity.