CONTINUOUS ANALYSIS OF THE MECHANISM OF ACTIVATED TRANSBILAYER LIPID MOVEMENT IN PLATELETS

Dithionite reduction of fluorescent (NBD) phospholipids was used as th e basis of a continuous assay of transbilayer lipid movement to the ce ll surface during platelet activation. This assay reveals that virtual ly all previously internalized phosphatidylserine passes through the e xternal leaflet of the membrane within 90s after activation with Ca2and ionophore or with thrombin and thapsigargin. We demonstrate that t his lipid scrambling is reversible, bidirectional, and insensitive to the lipid headgroup. Prolonged activation gradually results in inactiv ation of the scramblase. The assay also reveals that activation of the scrambling activity is sensitive to the sulfhydryl reagent pyridyldit hioethylamine, suggesting the involvement of a protein in the process of activated transbilayer lipid scrambling.