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Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway
M Baroni1, G Pavani2, M Pinotti1
1Department of Life Sciences and Biotechnology, University of Ferrara, Italy.
Arginine 386 in coagulation factor X (FX) is crucial for activation by the intrinsic pathway. Mutations here reduce thrombin generation, impacting coagulation and potentially explaining bleeding tendencies.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Coagulation factor X (FX) activation is essential for hemostasis, mediated by extrinsic (FVIIa/TF) and intrinsic (FIXa/FVIIIa) pathways.
- Dysregulation of FX activation can lead to bleeding or clotting disorders.
- Molecular mechanisms of FX substrate recognition by activation complexes are not fully understood.
Purpose of the Study:
- To investigate the role of arginine 386 (Arg386) in the catalytic domain of coagulation factor X (FX).
- To characterize the functional impact of FX mutations at position 386, specifically FX386Cys and FX386Ala.
- To elucidate the contribution of Arg386 to FX activation via different pathways.
Main Methods:
- Recombinant wild-type FX (rFXwt), FX386Cys, and FX386Ala were produced and characterized.
- FX activation kinetics by FVIIa/TF and FIXa/FVIIIa complexes were determined.
- Amidolytic activity and plasma-based thrombin generation assays were performed.
- Enzyme kinetic parameters (kcat, Km) were analyzed.
Main Results:
- Recombinant FX386Cys exhibited normal secretion, clearance, FVIIa/TF activation, and amidolytic activity.
- FX386Cys activation by the FIXa/FVIIIa complex was significantly reduced (4.5-fold decrease in kcat) with unaltered Km.
- FX386Cys resulted in reduced thrombin generation and coagulation efficiency in plasma.
- FX386Ala displayed similar biochemical properties, indicating Arg386 is key for intrinsic pathway activation.
Conclusions:
- Coagulation factor X Arg386 plays a critical role in activation mediated by the intrinsic FIXa/FVIIIa complex.
- Alterations at FX position 386 impact FX substrate recognition and activation efficiency.
- These findings help explain bleeding tendencies in FX deficiency cases with FX386Cys and suggest potential for targeted therapies.
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