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.

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).

DAF^−/−/Crry^−/−/C3^−/− platelets are more sensitive to alternative pathway complement attack than DAF^−/−/Crry^−/−/C3^−/− erythrocytes. FACS analysis of C3 deposition on DAF^−/−/Crry^−/−/C3^−/− platelets and erythrocytes after 20 minutes (A-C) or 60 minutes (D-F) of incubation with various concentrations of wild-type mouse plasma in 10 mM EGTA-MgCl₂. MFI indicates mean fluorescence intensity. Bars in panels C and F represent means (± SEM) of triplicate assays.

Expression of DAF, Crry, and CD59 on murine platelets. FACS analysis of DAF (A) and Crry (B) on wild-type mouse platelets. Platelets from DAF^−/−/C3^−/− and Crry^−/−/C3^−/− mice were used as negative controls. (C) FACS analysis of CD59a levels on WT (614 ± 32 molecules/cell) and various knockout mouse platelets. Platelets from DAF^−/−/CD59a^−/− mice were used as negative controls. (D) Comparison of the number of DAF and Crry molecules on WT, DAF^−/−/C3^−/−, and Crry^−/−/C3^−/− mouse platelets. WT and Crry^−/−/C3^−/− mouse platelets expressed 286 (± 98) and 282 (± 57) DAF molecules/platelet, respectively. WT and DAF^−/−/C3^−/− mouse platelets expressed 294 (± 86) and 274 (± 71) Crry molecules/platelet, respectively. Error bars represent SEM.

Rescue of DAF^−/−/Crry^−/−/C3^−/− mouse platelets by retroviral transgenic expression of Crry. (A) Tracking by FACS of the survival of nontransgenic (population X) and Crry-transgenic (population Y) DAF^−/−/Crry^−/−/C3^−/− platelets in WT recipients. Donor platelets were labeled with CFSE and were distinguishable from endogenous WT platelets (population Z). (B) Plot of elimination kinetics of populations X and Y platelets in panel A. The percentage of platelets remaining at each time point was normalized to the percentage of platelets remaining at 10 minutes after transfusion.

Role of macrophage and complement receptors in the elimination of DAF^−/−/Crry^−/−/C3^−/− erythrocytes. (A) Treatment of recipient mice with clodronate-liposomes (■; n = 4) but not PBS-liposomes (□; n = 4, negative control) prevented accelerated elimination of DAF^−/−/Crry^−/−/C3^−/− erythrocytes. C3^−/− mice (●; n = 4) were used as control recipients. (B) Splenectomy (SPX) did not prevent DAF^−/−/Crry^−/−/C3^−/− erythrocytes from accelerated elimination. DAF^−/−/Crry^−/−/C3^−/− erythrocytes transfused into splenectomized WT recipients (■; n = 3) had similar half-life to those transfused into sham-operated WT mice (□; n = 3), and both were rapidly eliminated. WT erythrocytes transfused into splenectomized WT recipients (●; n = 3) remained stable. (C) DAF^−/−/Crry^−/−/C3^−/− mouse erythrocytes were not rescued in CRIg^−/− (□; n = 4) or CR3^−/− (▴; n = 3) mice. WT (■; n = 3) and C3^−/− (●; n = 3) mice were used as control recipients. (D) Neither blockade of CRIg by antibody (16G8.2) in CR3^−/− mice (▴; n = 2) nor blockade of CRIg, CR1/2, CR4 by antibody (16G8.2), (7G6), and (N418) all in CR3^−/− mice (▵; n = 3) did not rescue DAF^−/−/Crry^−/−/C3^−/− erythrocytes. WT (■; n = 2) and C3^−/− (●; n = 2) mice were used as control recipients. All error bars means represent (±SEM).

Factor H is constitutively present on mouse platelets but not erythrocytes. Although FACS analysis detected reactivity to anti–human factor H antibodies on the mouse platelets (A) and erythrocytes (B), Western blot analysis confirmed fH association with platelets but not with erythrocytes (C right). Factor H in human and mouse sera were used as a positive control for antibody reactivity (C left), and GAPDH signal was used as a loading control for platelet and erythrocyte proteins. The positions of molecular weight markers (in kilodaltons) were indicated on the right side of panel C.