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Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor
C R Kelly1, C D Dickinson, W Ruf
1Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
The Journal of Biological Chemistry
|July 11, 1997
Summary
Calcium binding to the epidermal growth factor-like domain in factor VIIa is crucial for stabilizing its structure and maintaining tissue factor binding and proteolytic function in coagulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Coagulation factors VII, IX, and X possess epidermal growth factor-like (EGF) domains with calcium (Ca2+) binding sites.
- These sites stabilize the structural orientation of the gamma-carboxyglutamic acid-rich (Gla) domain relative to the EGF-1 domain.
- Factor VIIa (VIIa), in complex with tissue factor (TF), initiates the coagulation cascade.
Purpose of the Study:
- To investigate the functional significance of Ca2+ binding to the EGF-1 domain in factor VIIa.
- To analyze how mutations affecting Ca2+ coordination impact TF binding and proteolytic activity.
Main Methods:
- Site-directed mutagenesis was used to create mutants in the EGF-1 domain of factor VIIa.
- Mutants were analyzed for their affinity to tissue factor (TF).
- Proteolytic function of selected mutants was assessed.
Main Results:
- Mutations replacing Asp63 or Gln49 with Ala reduced TF affinity, correlating with lost Ca2+-coordinating oxygen atoms.
- Ca2+ binding to EGF-1 did not directly alter contacts with TF residue Gln110 or specific VIIa residues, suggesting an indirect effect on docking via Gla-EGF-1 orientation.
- Mutations at Asp46 increased Gla domain flexibility, leading to significant loss of function.
- Some EGF-1 Ca2+ site mutants exhibited reduced proteolytic activity.
Conclusions:
- Ca2+ binding to the EGF-1 domain is essential for maintaining the correct structural orientation of the Gla domain, which is critical for factor VIIa function.
- The stabilized Gla-EGF-1 orientation influences the overall docking of factor VIIa with TF.
- Disrupting Ca2+ coordination in the EGF-1 site can impair both TF binding and proteolytic activity, highlighting the importance of this high-affinity Ca2+ binding site for coagulation initiation.