Restored response to ADP downstream of purinergic P2Y12 receptor in apheresis platelets after pathogen-reducing xenon flash treatment.

Abstract

Our previous study revealed that pathogen-reducing filtered xenon flash-treated platelets (fXe-PLTs) showed sustained aggregation in response to adenosine diphosphate (ADP), but apheresis-collected PLTs (Aph-PLTs) showed reversible aggregation. Aph-PLTs, fXe-PLTs, and freshly prepared PLTs (PRP-PLTs) from whole blood were used to investigate the following responses to ADP: concentration response and effects of ADP receptor antagonists on aggregation, the cytosolic calcium (Ca2+ ) flux downstream of P2Y1 receptor signaling, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and signaling intermediate protein Akt downstream of the P2Y12 receptor. The aggregation of Aph-PLTs by ADP (10 µM) changed from reversible to sustained in an fXe flash dose-dependent manner. The concentration-response curve of Aph-PLTs showed a fivefold higher 50% effective concentration compared with PRP-PLTs, and fXe treatment decreased it to threefold. While the basal Ca2+ level was higher both in Aph- and fXe-PLTs than in PRP-PLTs, the increase of cytosolic Ca2+ by ADP remained unchanged in Aph- and PRP-PLTs, but was slightly reduced in fXe-PLTs. Although the forskolin-induced VASP phosphorylation was significantly reduced in Aph-PLTs, and partially restored by the fXe treatment, ADP stimulation attenuated this phosphorylation to an equivalent extent among the three PLT types. The ADP-stimulated time-dependent Akt phosphorylation was weak in Aph-PLTs, whereas fXe-PLTs and PRP-PLTs showed a marked increase. These results indicate that the reversible aggregation of Aph-PLTs is the consequence of insufficient Akt phosphorylation. The fXe treatment restores the increase of phosphorylated Akt, resulting in the sustained aggregation of fXe-PLTs similar to those of PRP-PLTs.