Revised Manuscript Received June 19, 1995s abstract: Dithionite reduction of fluorescent (NBD) phospholipids was used as the basis of a continuous assay of transbilayer lipid movement to the cell surface during platelet activation. This assay revealsthat virtually all previouslyinternalized phosphatidylserine passes through the external leaflet of the membrane within 90 s after activation with Ca2+ and ionophore or with thrombin and thapsigargin. We demonstrate that this lipid scrambling is reversible, bidirectional, and insensitive to the lipid headgroup. Prolonged activation gradually results in inactivation of the scramblase. The assay also reveals that activation of the scrambling activity is sensitive to the sulfhydryl reagent pyridyldithioethylamine, suggesting the involvement of a protein in the process of activated transbilayer lipid scrambling.

In erythrocytes, lymphocytes, and platelets, the two leaflets of the plasma membrane bilayer differ in composition, with the aminophospholipids phosphatidylserine (PS) 1 and phos-phatidylethanolamine (PE) concentrated in the inner leaflet and the neutralphospholipids sphingomyelin (Sph) and phosphatidylcholine (PC) concentrated in the outer leaflet [for reviews, see Schroit and Zwaal (1991), Williamson and Schlegel (1994), Devaux and Zachowski (1994)]. This asymmetric transbilayer lipid distribution is regulated by two opposing processes. Under normal conditions an ATP-dependent aminophospholipid transporter (Seigneuret & Devaux, 1984) transports PS andPE to the innerleaflet, maintaining asymmetry. This asymmetry can be abolished by a second mechanism that increases the rate of both inward and outward transbilayer movement of all of the preceding phospholipids. This scrambling is pronounced during platelet activation, where it was first detected (Bevers et al., 1982, 1983), but it also occurs in erythrocytes (Williamson et al., 1985, 1992; Schwartz et al., 1985; Chandra et al., 1987) and lymphocytes (Fadok et al., 1992; Schlegel et al., 1993). The process is induced by increased cytosolic Ca2+(Williamson et al., 1985; Verhoven et al., 1991; Verhallen et al., 1988); although the aminophospholipid translocase is inhibited under these conditions (Bitbol et al., 1987), inhibition of this transporter by itself does not result in rapid lipid rearrange-ment (Haest, 1982; Comfurius et al., 1990; Connor & Schroit,