Rigid RBCs and CD47-blocked RBCs both promote opsonization-driven phagocytosis unless myosin-II is directly inhibited. (A) Phagocytic uptake shows an increasing and saturating response to antiserum concentration. High antiserum is defined as treatment with antiserum concentration greater than that required to result in half of saturating levels of engulfment, as highlighted by the arrows and gray box. (B) High antiserum opsonized RBCs were incubated with THP-1 macrophages for 45 minutes at 37°C. A microscopy-based phagocytosis assay indicated that native RBCs were engulfed more so when they were CD47 blocked, unless macrophages were pretreated with blebbistatin (20 μM). Rigidified GA discocyte engulfment proved independent of CD47 blocking (*P < .05). However, engulfment of GA discocytes, like native RBCs, was inhibited by pretreating macrophages with blebbistatin. The gray bar thus highlights a baseline level of myosin-II-independent phagocytosis. Phagocytosis is significantly higher than baseline when RBC targets are either CD47 blocked or GA rigidified. Blebbistatin pretreatment of both native-blocked and rigid cells keep uptake at the baseline. (C) High-throughput flow cytometry assay for phagocytic uptake with opsonization per supplemental Table 2 shows that for high antiserum opsonization, phagocytosis of GA discocytes is unaffected by blocking of CD47 (not significant) (i), whereas at low antiserum opsonization, blocking of CD47 on GA discocytes does increase phagocytosis) (ii). For high antiserum opsonized deformable native RBCs, phagocytosis increases with blocking of CD47 (i), whereas, at low antiserum opsonization, phagocytosis of native cells is insignificant regardless of blocking CD47 (not significant) (ii). Hyperbolic fits for i and ii give K = 90 nM and an ∼2.5-fold increase from baseline to saturation. Clearly, the anti-CD47 blocking treatment alone is not sufficient to drive engulfment of native RBCs. In the absence of antiserum, or with zero antiserum treatment, blocking CD47 on GA discocytes reduces phagocytosis (iii). This suggests the functional CD47-SIRPA interaction on rigidified cells indicated by the SIRPA binding studies here (Figure 1B). For all experiments, *P < .05; n ≥ 3 ± standard error of the mean). (D) At intermediate antiserum opsonization (supplemental Table 2), CD47 signaling can be blocked. Phagocytosis of native RBCs and rigid GA discocytes significantly increases on blocking with anti-CD47 (90 nM). An anti-SIRPA antibody (90 nM) and an SHP-1 inhibitor (NSC87877, 60 nM) enhance uptake of GA discocytes. (E) NSG splenic macrophages take up stiff, highly opsonized hRBCs in vivo, independent of CD47. High antiserum opsonized GA rigidified (17 mM) RBCs were anti-CD47 (CD47 block) treated or not (CD47⁺), mixed 1:1, and injected into NSG mice via the tail vein (n = 4). Rigid discs were prelabeled with 2 lipophilic dyes, either DiR or PKH26, to be distinguished after mixing. Spleens were isolated 15 minutes after injection, dissociated, and analyzed by flow cytometry. Splenic macrophages were distinguished from splenocytes by Cd11b expression and quantified for the percentage positive RBC uptake. Although 1% of splenic macrophages were positive for CD47 blocked and CD47⁺ hRBCs, double-positive macrophages were rare, as were positive nonmacrophage cells (CD11b⁻).