Transverse Redistribution of Phospholipids during Human Platelet Activation: Evidence for a Vectorial Outflux Specific to Aminophospholipids

Abstract

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.

Tracking down lipid flippases and their biological functions

“…This has been ascribed to the activation of the lipid scramblase, a putative membrane protein facilitating a rapid bi-directional movement of all major phospolipid classes between the leaflets of the plasma membrane (Smeets et al, 1994;Williamson et al, 1995). However, for PS and PE a vectorial efflux from the inner to the outer leaflet upon activation of a scramblase activity has been reported (Gaffet et al, 1995;Hamon et al, 2000).…”

“…This has been ascribed to the activation of the lipid scramblase, a putative membrane protein facilitating a rapid bi-directional movement of all major phospolipid classes between the leaflets of the plasma membrane (Smeets et al, 1994;Williamson et al, 1995). However, for PS and PE a vectorial efflux from the inner to the outer leaflet upon activation of a scramblase activity has been reported (Gaffet et al, 1995; Hamon et al, 2000).A potential phospholipid scramblase (PLSCR1) has been isolated and cloned from human erythrocytes (Sims and Wiedmer, 2001). However, upon reconstitution in liposomes the protein exhibits a very low calcium-activated phospholipid scrambling activity (Bassé et al, 1996).…”

Sphingolipid transport in eukaryotic cells

“…First of all, an activity has been noted in erythrocytes and blood platelets that scrambles the asymmetric distribution of the phospholipids in the plasma membrane, and an abundantly expressed`scramblase' has been puri¢ed and its cDNA cloned [241,242]. Evidence has been presented that scramblase action £ips SM to the cytosolic surface [243], whereas others have not observed this [244]. As a result of some stimuli SM must reach the cytosolic lea£et of the plasma membrane to serve as a substrate for the neutral sphingomyelinase (see Section 3.1).…”

Involvement of extracellular calcium in phosphatidylserine exposure during apoptosis

“…E-mail: mark.hampton@imm.ki.se 4bbreviations: PS, phosphatidylserine; TNF, tumour necrosis factor; EGTA, ethylene glycol-bis(~-aminoethylether)-N,N,N',N'-tetraacetic ~cid; BAPTA-AM, acetoxymethyl ester of 1,2-bis(2-aminophenoxy)-:~'thane-N,N'-tetraacetic acid; FITC, fluorescein isothiocyanate; ICE, nterleukin-l[3 converting enzyme; PARP, poly(ADP-ribose) polymertse; AMC, amino-4-methylcoumarin; cmk, chloromethylketone; CHX, cycloheximide; FSC, forward scatter; PMSF, phenylmethylsulronyl fluoride vates the aminophospholipid translocase which normally constrains PS to the inner leaflet [13], but this alone is insufficient for exposure. Another protein is proposed to mediate the scrambling of membrane phospholipids [14], although there is debate as to whether there is a general phospholipid scrambling or a specific translocation of PS [15]. Reconstituted membranes have been used to study these mechanisms [16], and recently a 37 kDa protein has been extracted from red cell ghosts that can transport labelled phospholipids upon triggering with Ca…”

“…Ca 2+ inacti- vates the aminophospholipid translocase which normally constrains PS to the inner leaflet [13], but this alone is insufficient for exposure. Another protein is proposed to mediate the scrambling of membrane phospholipids [14], although there is debate as to whether there is a general phospholipid scrambling or a specific translocation of PS [15]. Reconstituted membranes have been used to study these mechanisms [16], and recently a 37 kDa protein has been extracted from red cell ghosts that can transport labelled phospholipids upon triggering with Ca…”