Active FSAP proteolyzes all histone subtypes and provides protection against histone-induced cytotoxicity in HEK293 cells. (A-B) Calf thymus histones (100 µg/mL or ∼7 µM) were incubated with different concentrations of plasma-purified active FSAP (12.5, 25, 50, or 100 nM) and the same concentrations of active rFSAP (A) or APC (B) at 37°C for 30 minutes to determine proteolysis of the different histone subtypes. Cleavage products were visualized by SDS-PAGE and subsequent silver staining. Normal plasma concentration of FSAP: ∼185 nM. Arrows indicate the different histone subtypes. (C-D) Histones (100 µg/mL) were incubated with 20 nM of plasma-purified active FSAP, active rFSAP, or inactive rFSAP for 30 minutes before their addition to HEK293 cells and overnight incubation. To determine cytotoxicity, lactate dehydrogenase levels were quantified in the supernatant (C), whereas the viability of the cells was assessed by conversion of thiazolyl blue tetrazolium bromide (D). Cytotoxicity is expressed as a percentage of cytotoxicity induced by 0.1% saponin, and cell viability is expressed as a percentage of untreated cells. Data are expressed as mean ± standard error of the mean obtained from 3 independent experiments, each performed in triplicate. *P < .05, **P < .01, ***P < .001 (calculated using an unpaired 2-tailed Student t test). inact, inactive.