SIRPA binds CD47 on both rigid and native RBCs. (A) Downstream of FcγR binding of IgG, kinases phosphorylate multiple cytoskeletal proteins, including myosin-II, which drive assembly of the phagocytic cup and promote uptake. CD47-SIRPA signaling leads to activation of SHP-1 phosphatase that can deactivate myosin-II. Because substrate rigidity initiates assembly and polarization of myosin-II in many cell types, phagocytic target rigidity is expected to counterbalance CD47-mediated inhibition of the motor. Our working hypothesis is that with flexible self-cells (left), CD47 initiated inhibition can overcome myosin-II activation, whereas with rigid self-cells (right), the myosin-II driven cytoskeleton is not diminished by CD47-SIRPA self signals. (B) Flow cytometry histograms show SIRPA-GST binds to GA- and MDA-rigidified RBCs unless partially blocked by pretreating RBCs with anti-CD47. Cell aggregation by this antibody prevents a demonstration of complete inhibition. (C) SIRPA-GST-Fluor is covalently labeled with fluorophore and binds both native and aldehyde treated RBCs (scale bar, 5 μm). SIRPA-GST-Fluor was used to bind the MDA-RBCs in B, whereas anti-GST was used to detect binding on native and GA-RBCs. Supplemental Figure 1 further illustrates the saturable and specific binding, as well as CD47 blocking. (D) Aspiration of RBCs into micropipettes with diameters similar to phagocytic cups and in vivo capillaries shows GA treatment rigidifies cells, as does storage at ambient conditions. The maximal RBC length and width under aspiration were quantified by image analysis and normalized by pressure and pipette cross section (native, n = 9; 17 mM GA discocyte, n = 23; 50 mM GA discocyte, n = 2; error bar = standard deviation [SD]). *P ≤ .05 compared with native; trend line R² = 0.99.