LOSS OF PHOSPHOLIPID ASYMMETRY IN HUMAN PLATELET PLASMA-MEMBRANE AFTER 1-12 DAYS OF STORAGE - AN ESR STUDY

We used paramagnetic analogs of endogenous phospholipids to study modi fication of phospholipid distribution in platelet plasma membranes dur ing aging. Asymmetrical distributions and translocation kinetics were very different for spin-labeled phosphatidylserine and spin-labeled ph osphatidylcholine in fresh platelet plasma membranes. In freshly prepa red platelets and up to day 7, spin-labeled phosphatidylserine very ra pidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin-labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin-labeled ph ospholipids at equilibrium. Inhibition of translocase activity and mod ification of membrane stability accounted for these transformations. T he rapid re-exposition of spin-labeled phosphatidylserine after stimul ation by the calcium ionophore A23187, measured in fresh platelet conc entrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppr ession of beta-N-acetyl glucosaminidase secretion and vesicle formatio n after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these resul ts showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet acti vation. In addition, all of the observed events occurred simultaneousl y between day 7 and day 9. These results highlight the importance of m aintaining plasma membrane asymmetry to increase the hemostatic effect iveness of transfused platelet concentrates.