Cryo-EM structure of coagulation factor V short.

Area of Science:

• Biochemistry
• Structural Biology
• Hematology

Background:

• Coagulation factor V (fV) is crucial for blood clotting, acting as the precursor to activated factor V (fVa) in the prothrombinase complex.
• fV also regulates inhibitory pathways, including the tissue factor pathway inhibitor alpha (TFPIα) and protein C pathways.
• Previous cryogenic electron microscopy (cryo-EM) studies revealed fV architecture but not the mechanism of its inactive state due to B domain disorder.

Purpose of the Study:

• To determine the cryo-EM structure of the fV splice variant, fV short, which lacks the B domain and exhibits constitutive activity.
• To elucidate the structural basis for fV inactivation and identify potential binding sites for TFPIα.
• To provide insights into the regulation of the coagulation cascade.

Main Methods:

• Cryogenic electron microscopy (cryo-EM) was used to solve the structure of fV short at 3.2 Å resolution.
• Analysis of the structural arrangement of the fV short A1-A2-B-A3-C1-C2 assembly.
• Identification of potential TFPIα binding sites within the fV structure.

Main Results:

• The cryo-EM structure of fV short revealed the complete A1-A2-B-A3-C1-C2 assembly, with a shortened B domain.
• The B domain in fV short spans the protein width, contacting A1, A2, and A3 domains, while being suspended over C1 and C2.
• Specific hydrophobic clusters and acidic residues in the B domain suggest a binding site for the C-terminal end of TFPIα.

Conclusions:

• The structure provides a mechanistic understanding of how fV remains inactive, potentially through intramolecular binding of TFPIα to the B domain.
• The findings identify new targets for mutagenesis studies to probe fV function.
• This structural information facilitates future investigations of fV short in complex with TFPIα, protein S, and factor Xa (fXa).