Role of complement, macrophage, and complement receptors in the elimination of DAF/Crry-deficient platelets. (A) DAF^−/−/Crry^−/−/C3^−/− platelets had accelerated elimination in C4^−/−, C5^−/−, JH^−/−, and FcRγ^−/− mice but not in fB^−/− and C3^−/− mice (n = 3 for each type of recipients). (B) FACS analysis of C3 deposition on DAF^−/−/Crry^−/−/C3^−/− mouse platelets (n = 3) after in vitro incubation with 20% WT, C4^−/−, fB^−/−, or C3^−/− mouse serum in Tyrode buffer, 5 mM MgCl₂-EGTA, or 10 mM EDTA. Similar results were obtained with 40% serum. Sera were pooled from 3 to 4 mice of each genotype. Error bars represent means ± SEM of triplicate assays. (C) Treatment of recipient mice with clodronate-liposomes (■; n = 4) but not PBS-liposomes (□; n = 4, negative control) prevented accelerated elimination of DAF^−/−/Crry^−/−/C3^−/− platelets. C3^−/− mice (●; n = 4) were used as control recipients for the rescue experiment. (D) Splenectomy (SPX) did not prevent DAF^−/−/Crry^−/−/C3^−/− platelets from accelerated elimination. DAF^−/−/Crry^−/−/C3^−/− platelets transfused into splenectomized WT recipients (■; n = 3) had similar half-life to those transfused into sham-operated WT mice (□; n = 3), and both had accelerated elimination compared with WT platelets transfused into splenectomized WT recipients (●; n = 3). (E) Blockade of CR3 in WT mice by anti-CR3 (M1/70) (■; n = 3) or CR3 gene knockout (▴; n = 3) did not rescue DAF^−/−/Crry^−/−/C3^−/− platelets from accelerated elimination. Blockade of CR4 in CR3^−/− mice by anti-CR4 (N418) (○; n = 2) and blockade of CR1/2 in WT mice by anti–CR1/2 (7G6) (▾; n = 3) also did not prevent the elimination. WT (□; n = 3) and C3^−/− (●; n = 3) were used as control recipients. (F) Blockade of CRIg in WT mice by anti-CRIg (14G8.2) (●; n = 3) or CRIg gene knockout (■; n = 3) prevented DAF^−/−/Crry^−/−/C3^−/− platelets from accelerated elimination. WT (□; n = 3) and C3^−/− (○; n = 3) were used as control recipients. All error bars represent mean (± SEM).