Background: Extracellular histone H3 can induce pathological effects in vivo including inflammation and cell death. The neutralization of histone H3 is therefore beneficial in various diseases, such as sepsis. Activated protein C (APC) can cleave histone H3, which reduces its cytotoxicity. However, due to the anticoagulant properties of APC, use of wildtype APC is not optimized for the treatment of histone-mediated cytotoxicity, because it may cause bleeding.
Aims: We aimed to investigate the molecular interaction between APC and histone H3 and subsequently generated rationally designed APC variants. These variants are predicted to have reduced anticoagulant activity, whilst also possessing an enhanced ability to bind and cleave histones, as compared to wildtype APC.
Methods: We used computational design to identify key residues that mediate the interaction between APC and histone H3. By rational design we introduced in silico mutations in APC, to obtain variants with optimized functional characteristics. Next, ability of the variants to function as an anticoagulant, to bind to histones and to cleave histones was tested, and correlated to the cytoprotective properties.
Results: Analysis of APC-mediated histone H3 proteolysis and structural modelling identified the cleavage sites in histone H3 and APC residues that are important for the interaction with histone H3. APC variants were engineered and generated by mutation of these identified key cationic residues to neutral or anionic residues, resulting in the novel 5D-APC and 5D2A-APC variants.
Compared to wildtype APC, both the 5D-APC and 5D2A-APC variant showed a significant decreased anticoagulant activity and increased binding to histone H3. In addition, the APC variants showed a similar ability to proteolyze histone H3 as wildtype APC.
Conclusions: It is possible to rationally design novel optimized APC variants that can be further developed and utilized as a therapy for histone-mediated disease, to reduce their associated cytotoxic properties, whilst not inducing an increased bleeding risk.
To cite this abstract in AMA style:Huckriede J, Beurskens D, Wichapong K, Wildhagen K, Hackeng T, Reutelingsperger C, Nicoleas G. Design and Characterization of Novel Activated Protein C (APC) Variants for the Proteolysis of Cytotoxic Extracellular Histone H3 [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/design-and-characterization-of-novel-activated-protein-c-apc-variants-for-the-proteolysis-of-cytotoxic-extracellular-histone-h3/. Accessed November 26, 2020.
« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/design-and-characterization-of-novel-activated-protein-c-apc-variants-for-the-proteolysis-of-cytotoxic-extracellular-histone-h3/