RECONSTITUTION OF PHOSPHOLIPID SCRAMBLASE ACTIVITY FROM HUMAN BLOOD-PLATELETS

Cellular activation, accompanied by elevation of cytoplasmic Ca2+ leve ls, can induce a progressive loss of plasma membrane phospholipid asym metry, resulting from increased transbilayer movement (flip-flop) of p hospholipids. While this process has been demonstrated in a variety of different cells, it is most active in blood platelets. In order to te st whether this lipid scrambling process is mediated by a membrane pro tein, platelet membranes were solubilized in cholate and fractionated by anion exchange chromatography, and fractions were reconstituted int o phospholipid vesicles by detergent dialysis in the presence of small amounts of fluorescent (NBD) phospholipids. Using dithionite reductio n to monitor the transbilayer location of NBD phospholipids, it was sh own that addition of Ca2+ and ionomycin to vesicles reconstituted with a particular fraction results in transbilayer movement of the fluores cent phospholipid analogs from the vesicle's inner to outer leaflet. L ipid vesicles reconstituted in the absence of membrane protein, or rec onstituted with another platelet membrane protein fraction, were devoi d of this activity. Heating the active fraction or incubating it with pronase or the SH reagent pyridyldithio-ethylamine markedly diminished the ability of the vesicles to translocate fluorescent phospholipid a nalogs across the bilayer in response to Ca2+ and ionophore. These res ults argue that a membrane protein (or proteins) from blood platelets is required to catalyze Ca2+-induced transbilayer movement of phosphol ipids, suggesting its (or their) involvement in the loss of lipid asym metry that can occur during cellular activation.