Cold storage primes murine platelets to shed GPIbα and GPV on rewarming. (A) Expression of murine VWFR components (GPIbα, GPIbβ, GPIX, GPV), GPVI, and αIIbβ3 was measured by flow cytometry before and after platelet storage in the cold. Results are expressed as means ± SDs; n = 5. Glycoprotein expression on freshly isolated platelets was set as 100%. (B) Fresh mouse platelets (Fresh RT) were surface-biotinylated, refrigerated in plasma for 48 hours, and rewarmed (48 hours cold). The integrity and/or release of the GPIbα and GPV ectodomains were assessed by immunoprecipitation with the use of specific anti-GPV and anti-GPIbα Abs. Input, indicates platelets and corresponding plasma; SHED, material released into the supernatant fraction after removal of the platelets by centrifugation. Shedding of GPV induced by treating fresh platelets with thrombin (Thr) is also shown. Blots are representative of 3 independent experiments. (C) Immunoblot for GPIbα supernatant lysates from fresh room temperature (RT) or refrigerated mouse platelets (2, 24, and 48 hours cold). The immunoblot is representative of 3 independent experiments.
