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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
1   Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
,
Wimal Ubhayasekera
2   Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
4   Present address: MAX-lab, Lund University, Lund, Sweden and Institute of Medicinal Chemistry, University of Copenhagen, Copenhagen, Denmark
,
Noëlle Nickolaus
1   Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
,
Wei Sun
1   Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
,
Susanne Tingsborg
1   Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
,
Sherry L. Mowbray
2   Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
3   Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
,
Sophia Schedin-Weiss
1   Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
› Author Affiliations Financial support:This work was supported by Swedish Research Council grants 2003–6107, 2005–6412 and 2007–4444 and by the Magnus Bergvall foundation (to S.S.W.).
Further Information

Publication History

Received: 23 December 2009

Accepted after major revision: 22 May 2010

Publication Date:
23 November 2017 (online)

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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
 

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