Related Experiment Videos
The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be
1Howard Hughes Medical Institute, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Insights
Calcium ions (Ca2+) play a dual role in protein C activation. Aspartic acid residues are crucial for Ca2+-dependent inhibition by thrombin alone, but not for activation by the thrombin-thrombomodulin complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Protein C activation is a critical anticoagulant pathway.
- This process is regulated by thrombin and thrombomodulin on the endothelium.
- Calcium ions (Ca2+) influence protein C activation, stimulating it with thrombomodulin but inhibiting it without.
Purpose of the Study:
- To investigate the role of Asp residues at P3 and P3' positions in protein C.
- To determine their contribution to calcium's effect on protein C activation.
- To elucidate the mechanism of Ca2+ dependent inhibition and stimulation.
Main Methods:
- Expression of human Gla-domainless protein C and its Asp mutants (Asp-->Gly substitutions).
- Monitoring Ca2+ interaction using intrinsic fluorescence.
- Assessing Ca2+ dependence of activation by thrombin and thrombin-thrombomodulin complex.
Main Results:
- Mutations reduced Ca2+ affinity 3-6 fold, altering Ca2+ concentration for half-maximal activation.
- Ca2+ no longer effectively inhibited activation of mutants by thrombin alone.
- Asp residues are critical for Ca2+-dependent inhibition by thrombin, but not essential for thrombin-thrombomodulin-mediated activation.
Conclusions:
- Asp residues at P3 and P3' sites are key to Ca2+-dependent inhibition of protein C by thrombin.
- These mutations affect Ca2+ binding affinity to the high-affinity site.
- Asp residues do not positively contribute to rapid activation by the thrombin-thrombomodulin complex.
Abstract:
Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.