A 3.3-Å cryo-EM structure of an engineered high-affinity human prothrombinase complex
Fatma Işık Üstok1, Alexandre Faille1, James A Huntington1
1Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
Blood
|October 27, 2025
Summary
Researchers engineered a high-affinity human prothrombinase complex, M17, that functions independently of phospholipid membranes. This breakthrough enabled cryo-electron microscopy (cryo-EM) structural analysis, revealing key interactions within the prothrombinase complex.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Medicine
Background:
- Thrombin generation from prothrombin is catalyzed by prothrombinase (factor Xa and factor Va).
- Native prothrombinase requires phospholipid membranes for assembly and function due to low factor Xa-factor Va affinity.
- Venom factor Xa variants exhibit high affinity for factor Va, enabling prothrombin activation independent of phospholipids.
Purpose of the Study:
- To engineer a high-affinity, phospholipid-independent human prothrombinase complex.
- To determine the high-resolution structure of the engineered prothrombinase complex using cryo-electron microscopy (cryo-EM).
- To elucidate the molecular interactions governing prothrombinase assembly and function.
Main Methods:
- Site-directed mutagenesis of human factor Xa to create a high-affinity variant (M17).
- Cryogenic electron microscopy (cryo-EM) for structural determination of the M17-prothrombinase complex at 3.3 Å resolution.
- High-resolution crystal structure analysis of factor Xa complexed with a synthetic a2-peptide to resolve loop interactions.
Main Results:
- A novel M17-prothrombinase complex with significantly increased affinity and phospholipid independence was successfully engineered.
- Cryo-EM structure revealed detailed interactions between factor Xa (serine protease and EGF2 domains) and factor Va (A2 and A3 domains), burying 4,900 Ų of surface area.
- Structural analysis identified key side-chain interactions, including the interaction of the acidic a2-loop with the basic heparin-binding site of factor Xa.
Conclusions:
- The engineered M17-prothrombinase provides a structural basis for understanding high-affinity complex formation.
- The structure is consistent with existing biochemical and mutagenesis data, offering new insights into prothrombinase assembly.
- This work advances the understanding of blood coagulation factor interactions and potential therapeutic targets.
