Effect of sialidase treatment on complement activation on platelets. (A) Washed human platelets were treated with sialidase or the respective buffer. Removal of sialic acid was confirmed by using flow cytometry to show decreased binding of fluorescently labeled MAL II. (A,C) Representative histograms of an assay performed 4 times. (B) The activation state of untreated and sialidase-treated platelets before and after serum exposure was determined by measuring P-selectin expression. Binding of anti-P-selectin antibody was detected by flow cytometry. (C-G) Platelets were incubated in 33% NHS, and FH19-20 fragments were added to disturb FH-mediated complement regulation. Fluorescently labeled C3 was included in the serum to allow measurement of complement activation as a function of deposition of fluorescent C3 fragments on platelets. (C) Addition of FH19-20 wt to serum caused an increase in C3b-deposition on untreated platelets. Shown is a representative histogram of an assay performed 5 times. (D) C3b-deposition on normal platelets incubated in serum with wt or mutant FH19-20. (E) C3b deposition on sialidase-treated platelets incubated in serum with wt or mutant FH19-20. (F-G) Assays described in (C) and (D) were also performed with control mutants FH19-20. Assays were performed 4 times. Shown are GMFI (± SD). Either (C) Student t tests or (D-G) one-way ANOVA with Tukey’ multiple comparison posttests were performed. **P < .01; ***P < .001.