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Polymerization site a function dependence on structural integrity of its nearby calcium binding site
K C Lounes1, N Okumura, K A Hogan
1Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA.
Annals of the New York Academy of Sciences
|July 20, 2001
Summary
The calcium binding site is crucial for the function of the polymerization site in fibrinogen. Disrupting the calcium site impairs polymerization, but altering the polymerization site does not affect calcium binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Structure-Function
Background:
- Fibrinogen is a key protein in blood coagulation, forming a fibrin clot.
- The polymerization site (a) and a high-affinity calcium (Ca2+) binding site are critical for fibrin formation.
- The functional relationship between these sites is not fully understood.
Purpose of the Study:
- To investigate the functional interplay between the fibrinogen polymerization site (a) and the adjacent high-affinity calcium binding site.
- To determine how alterations in these sites affect fibrinogen's interaction with thrombin and plasmin.
Main Methods:
- Analysis of four variant fibrinogens with specific amino acid substitutions at the polymerization (gamma D364A) and calcium binding sites (gamma D318A, gamma D320A, gamma D318 + gamma D320A).
- Assessment of fibrinopeptide A release and thrombin-catalyzed polymerization.
- Plasmin protection assays in the presence of Ca2+ or the GPRP peptide (a site ligand).
- SDS-PAGE analysis of plasmin cleavage products.
Main Results:
- All variant fibrinogens showed normal fibrinopeptide A release but impaired thrombin-catalyzed polymerization.
- Gamma D364A fibrinogen was protected from plasmin by Ca2+, but not by the GPRP peptide.
- Gamma D318A, gamma D320A, and gamma D318 + gamma D320A fibrinogens were not protected from plasmin by either Ca2+ or the GPRP peptide.
Conclusions:
- The structural integrity of the calcium binding site is essential for the proper function of the fibrinogen polymerization site.
- The polymerization site's integrity does not influence Ca2+ binding to its high-affinity site.
- These findings elucidate a critical structural and functional link between calcium binding and fibrin polymerization.
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