Heme-MP association in SCD plasma. Circulating PS+ MPs (A) were quantified in platelet-free plasma (PFP) from steady-state SCD patients (n = 47) or control matched healthy volunteers (n = 22) by fluorescence-activated cell sorter (FACS) after annexin-a5 labeling, as well as plasma Hb by spectrophotometry (Abs₅₇₅-Abs₆₀₀), before and after MP depletion by ultracentrifugation (U/C). (B) Whole spectrum analysis of light absorbance in SCD (full lines) and control PFP (dotted lines). Black arrow marks peak of Hb absorbance at 575 nm; gray arrow points at 600 nm. (C) Absorbance (Abs₅₇₅-Abs₆₀₀) in PFP from control volunteers and steady-state SCD patients. *P < .01 vs total in controls; ^$P < .05 vs total in SCD. (D) Circulating erythrocyte MPs were quantified in plasma from control and SCD patients (n = 12) by FACS after phycoerythrin-conjugated anti-CD235a⁺ immunoglobulin G (IgG) labeling; *P = .01. We generated stocks of MPs shed by purified erythrocytes in vitro. SCD and control erythrocytes were incubated in vitro with CD47 agonist peptide 4N1-1, truncated 4N1-2, or mutated 4NGG peptide (25 μM, 30 minutes). (E) Quantification of MPs released by control erythrocytes, by FACS after annexin-a5 labeling. Supernatants were ultracentrifuged to pellet MPs, which were resuspended at similar concentrations in PBS (500 MPs/μ). Spectrophotometric measurements relative to Hb (Abs₅₇₅) (F) and heme (Abs₃₉₈) (G) were gathered. *P < .05 vs control MPs.