TSP1 triggers MP shedding via CD47. (A) We injected recombinant TSP1 intravenously to wild-type (WT) and SAD mice (1 mg/kg). Within 10 minutes, we collected platelet-free plasma to quantify circulating PS⁺ MPs by FACS after annexin-V labeling. (*P < .05 vs controls, #P < .05 vs wild-type +TSP1. (B) We determined the surface expression of CD47 on WT and SAD RBC by FACS, expressed in percent (left). Plot of CD47-labeling in Fl-1 (right). (C-D) SAD and WT RBCs were placed in polyvinylpyrrolidone. Shear rate was applied with an ektacytometer (1500 seconds⁻¹; 2.5 minutes) with or without addition of TSP1 (20 μg/mL). RBCs were prepared for scanning electron microscopy. (C) Biconcave discocytes, spicule-covered echinocytes, and elongated sickle cells were quantified by phase-contrast microscopy (SAD in solid bars; *P < .05 vs WT controls, none; #P < .05 vs SAD control, none). (D) Supernatant PS⁺ MPs were quantified by FACS after annexin-V labeling (*P < .05 vs SAD controls; #P < .05 vs wt+TSP1). In other experiments, WT and SAD RBCs were treated with 4N1-1, or control peptides 4N1-2 and 4NGG (10μM; 10 minutes) in RPMI-1640. (E) SAD and WT erythrocytes treated with 4N1-1. (F) Biconcave discocytes, spicule-covered echinocytes, and elongated sickle cells were quantified (SAD in solid bars; *P < .05 vs WT controls, none; #P < .05 vs WT+TSP1 control, none). (G) Spicules on WT and SAD echinocytes were counted (*P < .05 vs control wild-type RBC, none; #P < .05 vs SAD controls). (H) Supernatant PS⁺ MPs were quantified by FACS after annexin-V labeling (*P < .05 vs controls, none; #P < .05 vs WT+TSP1).