Zinc ions bind to and inhibit activated protein C

 

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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.

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