DNA and factor VII–activating protease protect against the cytotoxicity of histones

Abstract

Key Points• Free histones, not nucleosomes, are cytotoxic and are degraded by FSAP in serum to protect against cytotoxicity.• Free histone H3 was not detectable in sera of septic baboons and patients with meningococcal sepsis.Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes;however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize histones are present in plasma. Importantly, factor VII-activating protease (FSAP) is activated upon contact with histones and subsequently proteolyzes histones. We aimed to determine the effect of FSAP on the cytotoxicity of both histones and nucleosomes. Indeed, FSAP protected against histone-induced cytotoxicity of cultured cells in vitro. Upon incubation of serum with histones, endogenous FSAP was activated and degraded histones, which also prevented cytotoxicity. Notably, histones as part of nucleosome complexes were not cytotoxic, whereas DNA digestion restored cytotoxicity. Histones in nucleosomes were inefficiently cleaved by FSAP, which resulted in limited cleavage of histone H3 and removal of the N-terminal tail. The specific isolation of either circulating nucleosomes or free histones from sera of Escherichia coli challenged baboons or patients with meningococcal sepsis revealed that histone H3 was present in the form of nucleosomes, whereas free histone H3 was not detected. All samples showed signs of FSAP activation. Markedly, we observed that all histone H3 in nucleosomes from the patients with sepsis, and most histone H3 from the baboons, was N-terminally truncated, giving rise to a similarly sized protein fragment as through cleavage by FSAP.Taken together, our results suggest that DNA and FSAP jointly limit histone cytotoxicity and that free histone H3 does not circulate in appreciable concentrations in sepsis.

Extracellular histones trigger disseminated intravascular coagulation by lytic cell death

“…To test whether histones disrupt cell integrity via its positive charges, histones were mixed with negatively charged DNA or heparin and then co-incubated with macrophages. Preincuation of histones with DNA or heparin protected against death of macrophages (Figure 3D), which is consistent with previous studies showing that histones as part of the nucleosome complex are not cytotoxic 24 and that heparin prevents histone-induced toxicity in vivo. 13 We further observed that histone disrupted membranes of liposomes pre-packaged with Tb3 + , as indicated by the time-dependent increase in Tb3 + release (Figure 3E).…”

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…Indeed a study injecting similar doses of nucleosomes in mice makes no mention of toxicity,15 and others have demonstrated cofactors such as HMGB1 responsible for the immune‐stimulatory effects of nucleosomes 16. Only through the interplay of plasma proteases and nucleases including DNAse1 and factor VII activating protease does nucleosome decondensation occur17; however, this also degrades the histone component and limits cytotoxicity 18. These effects may have significant implications for in vitro signalling studies using recombinant proteins, as effects of isolated nucleosome components may not become apparent in this setup.…”

Genome‐wide analysis of genetic determinants of circulating factor VII‐activating protease (FSAP) activity

“…In line with this, impaired FSAP modulation of TFPI levels was suggested as an explanation for the defective thrombus formation observed in mice made deficient for FSAP (FSAP −/− ) . Other studies have shown that FSAP has a role not only in vascular compartments , but also in liver fibrosis , inflammation , and cancer . In vivo experiments in wild‐type and FSAP −/− mice support a role for FSAP in vascular remodeling, liver fibrosis, neointima formation, and arteriogenesis .…”