OC 73.2 - Cryo-EM Structure of Coagulation Factor V short

Background: A recent cryogenic electron microscopy (cryo-EM) structure of coagulation factor V (fV) has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved due to intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for binding of tissue factor pathway inhibitor a (TFPIa). Upregulation of fV short expression as seen in the East Texas bleeding disorder increases TFPIa concentration in plasma and decreases thrombin generation.

Aims: Solve the structure of fV short.

Methods: Recombinant fV short (fV ∆756-1458) was expressed in Expi293 cells and purified by ion-exchange and immunoaffinity chromatography. Purified fV short was concentrated and buffered exchanged before being plunge frozen at 0.1 mg/mL on cryo-EM grids for data collection on a Titan Krios 300 KeV microscope.

Results: The cryo-EM structure of fV short was solved at 3.2 Å resolution and reveals the arrangement of the entire A1-A2-B-A3-C1-C2 assembly for the first time. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2 and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a likely binding site for the basic C-terminal end of TFPIa. In fV, these epitopes may bind intramolecularly the basic region of the B domain.

Conclusion(s): The cryo-EM structure advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIa, protein S and fXa.