Complement susceptibility of WT and mutant mouse platelets. (A) Normal survival of transfused WT (■; n = 4), Crry^−/−/C3^−/− (○; n = 5), and DAF^−/−/C3^−/− (●; n = 4) mouse platelets in WT recipients; n refers to the number of recipient mice. Platelets were pooled from 2 to 3 donor mice. (B) Accelerated elimination of DAF^−/−/Crry^−/−/C3^−/− (□; n = 4) but not DAF^−/−/CD59a^−/− (♦; n = 4) mouse platelets compared with WT (■; n = 3) mouse platelets. In panels A and B, platelets were pooled from 2 to 3 donor mice. Blood samples were taken at 10 minutes after labeled platelets were intravenously infused, and the percentage of labeled platelets at this time point was taken as 100% for later reference. (C) FACS analysis of C3 deposition on donor platelets from panel A (WT, Crry^−/−/C3^−/−, DAF^−/−/C3^−/−) and panel B (DAF^−/−/Crry^−/−/C3^−/−). MFI indicates mean fluorescence intensity. C3 deposition on DAF^−/−/Crry^−/−/C3^−/− platelets at 12 hours and 24 hours were significantly higher than C3 deposition on any other platelets (P < .05, Student t test). (D) FACS analysis of C3 deposition on WT, DAF^−/−/C3^−/−, Crry^−/−/C3^−/−, and DAF^−/−/Crry^−/−/C3^−/− mouse platelets in vitro. Washed platelets were incubated with mouse serum (20% diluted in Tyrode buffer) at 37°C for 30 minutes. Similar results were obtained with 40% serum. Three mice were used in each group. Triplicate assays for each mouse were performed. (E) FACS analysis of activated αIIbβ3 integrin on CFSE-labeled DAF^−/−/Crry^−/−/C3^−/− platelets 2 hours after transfer into WT recipients. (F) FACS analysis of activated αIIbβ3 integrin on WT and DAF^−/−/Crry^−/−/C3^−/− mouse platelets after in vitro incubation (30 minutes, 37°C) with 10% or 40% WT mouse plasma (in 10 mM EGTA-MgCl₂). Numbers on plots are percentages of cells in gates. (G) ELISA analysis of C3a/C3a-desArg concentration in mouse plasma after in vitro incubation with WT or DAF^−/−/Crry^−/−/C3^−/− mouse platelets. Intact plasma (not incubated) and plasma incubated in the absence of added platelets were used as controls. All plasma samples, including controls, were diluted to 40% in 10 mM EGTA-MgCl₂ before assay_. (H) ELISA analysis of C5a/C5a-desArg concentration in mouse plasma after in vitro incubation. Experimental conditions were the same as panel G. Error bars represent mean (± SEM) of triplicate assays. *P < .001, **P < .05, Student t test.