In vitro aging of mouse platelets induces profound shedding of GPIbα and platelet clearance. (A) Freshly isolated (0 h) or in vitro-aged (16 h) PRP was washed twice and platelets were labeled with calcein. Platelets (1.5 × 108 per 15 g body weight) were infused intravenously into mice and blood was drawn at the indicated time points. Blood platelets were stained for GPIIbIIIa and analyzed by flow cytometry. Results are shown as percent calcein-labeled platelets ± SEM, n = 5. (B) Surface expression of P-selectin, PS (annexin V), GPIbα, and GPIbβ was determined by flow cytometry on fresh PRP (shaded area) and PRP aged for 16 hours at 37°C (black curve). Results are representative of 5 experiments. (C) Platelets in PRP were aged for 16 hours, washed once, and resuspended in modified Tyrode buffer containing 1 mM CaCl2 (thrombin) or plasma (U46619). Platelet responses were tested in standard aggregometry by adding 0.5 U/mL thrombin (upper panel) or 10 μM thromboxane A2 analog U46619 (lower panel). The gray line represents platelets from fresh PRP, the black line platelets from aged PRP. The bar indicates 5 minutes along the x axis. Results are representative of 3 separate experiments.
