Inhibition of MP-mediated GPIbα and GPV shedding after refrigeration does not improve the survival of transfused mouse platelets. (A) GPIbα and (B) GPV surface expression were assessed by flow cytometry. WT mouse PRP was stored for 0, 24, and 48 hours at 4°C in the presence of DMSO (Control) or 100μM of the MP inhibitor GM6001 (n = 6). Surface expression of GPIbα (C) and GPV (D) was determined by flow cytometry on freshly isolated or 24 and 48 hours refrigerated PRP from Adam17^+/+ and Adam17^ΔZn/ΔZn mice. Results are the mean ± SEM (n = 5). (C, inset) Immunoblot for GPIbα in lysates from Adam17^+/+ and Adam17^ΔZn/ΔZn platelets stored for 3, 24, and 48 hours in the cold. (E) Fluorescently labeled (CMFDA) fresh PRP (room temperature) or platelets from stored platelet rich plasma in the absence (48 hours) or presence of 100μM GM6001 (48 hours + GM6001) were infused into WT mice (10⁸ platelets/10 g of body weight). Blood was drawn at the indicated time points, and platelets were immediately analyzed by flow cytometry. Results are mean percentage of CMFDA-labeled platelets ± SEM. The percentage of CMFDA-positive fresh platelets at 5 minutes after transfusion was set as 100% (n = 5); *P < .05. Cold-stored platelets are compared. (F) Fluorescently labeled (CMFDA) fresh platelets (Adam17^+/+ RT and Adam17^ΔZn/ΔZn RT) or platelets from cold stored PRP (Adam17^+/+ 48 hours and Adam17^ΔZn/ΔZn 48 hours) were infused intravenously into WT mice (10⁸ platelets/10 g of body weight). Blood was drawn at the indicated time points, and platelets were immediately analyzed by flow cytometry. Results are mean percentage of CMFDA-labeled platelets ± SEM. The percentage of CMFDA-positive fresh Adam17^+/+ platelets at 5 minutes after transfusion was set as 100% (n = 5).