Zinc ions bind to and inhibit activated protein C

Tianqing Zhu; Wimal Ubhayasekera; Noëlle Nickolaus; Wei Sun; Susanne Tingsborg; Sherry L. Mowbray; Sophia Schedin-Weiss

doi:10.1160/TH09-12-0862

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2010; 104(03): 544-553
DOI: 10.1160/TH09-12-0862

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Zinc ions bind to and inhibit activated protein C

Tianqing Zhu

¹Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

,

Wimal Ubhayasekera

²Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

⁴Present address: MAX-lab, Lund University, Lund, Sweden and Institute of Medicinal Chemistry, University of Copenhagen, Copenhagen, Denmark

,

Noëlle Nickolaus

¹Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

,

Wei Sun

¹Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

,

Susanne Tingsborg

¹Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

,

Sherry L. Mowbray

²Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

³Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

,

Sophia Schedin-Weiss

¹Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

› Author Affiliations

Abstract

Summary

Zn²⁺ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K _I was 13 ± 2 μM. k _cat was seven fold decreased whereas K _M was unaffected in the presence of 10 μM Zn²⁺. The inhibitory effect of Zn²⁺ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn²⁺ with APC was accompanied by a reversible ~40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K _D was 7.4 ± 1.5 μM and thus correlated well with the apparent K _I. In the presence of physiological Ca²⁺ concentration the K _I and K _D values were three to four fold enhanced, presum-ably due to the Ca²⁺-induced conformational change affecting the conformation of the Zn²⁺-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca²⁺. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn²⁺ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

Keywords

Activated protein C - serine protease - zinc - blood coagulation - enzyme inhibition

Full Text

References

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