Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBD-labeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED50 of nearly 100 μm, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance. Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBD-labeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED50 of nearly 100 μm, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance. The appearance of phosphatidylserine in the outer leaflet of the plasma membrane appears to be a universal phenomenon in cells undergoing apoptosis, or programmed cell death (1Martin S.J. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Schie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2562) Google Scholar). Importantly, outer leaflet PS likely serves as a signal in tissues for the noninflammatory engulfment of apoptotic cells (Ref. 2Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar and see “Discussion”), a distinctly different response than the pro-inflammatory events following tissue necrosis. While phosphatidylserine (PS) 1The abbreviations used are: PS, phosphatidylserine; PC, phosphatidylcholine; NBD, [6[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl]; HBS, Hepes-buffered saline; FITC, fluorescein isothiocyanate; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. is actively transported from the outer to the inner leaflet by the aminophospholipid translocase (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar, 4Zachowski A. Favre E. Cribier S. Hervé P. Devaux P.F. Biochemistry. 1986; 25: 2585-2590Crossref PubMed Scopus (186) Google Scholar, 5Sune A. Bette-Bobillo P. Bienvenüe A. Fellmann P. Devaux P.F. Biochemistry. 1987; 26: 2972-2978Crossref PubMed Scopus (97) Google Scholar), the mechanism(s) by which PS appears in apoptosis is not well understood. Verhoven et al. (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar) reported that PS appearance was accompanied by both loss of aminophospholipid translocase activity and enhanced nonspecific transbilayer movement of phospholipids, perhaps due to activation of a “scramblase.” Hence, they proposed that PS may become detectable in the outer leaflet due to the combination of these two events. However, to date, the relative contribution of these two events has not been clarified. Additionally, it is not known what role, if any, other changes occurring during apoptosis (e.g. membrane blebbing and vesiculation) may play in the appearance of PS in the outer membrane leaflet. We hypothesized that the appearance of PS on the surface of the cell undergoing apoptosis would result primarily from enhanced calcium-dependent transbilayer movement of phospholipids (flip-flop) across the plasma membrane. Here we present data that demonstrates that (i) the loss of aminophospholipid translocase activity alone does not result in PS appearance, (ii) the appearance of PS likely results from calcium-dependent phospholipid flip-flop that is nonspecific for head group, (iii) while loss of aminophospholipid translocase activity is insufficient to result in PS appearance, loss of its function is probably a necessary event, and (iv) the appearance of PS at the cell surface can be dissociated from nuclear changes (DNA fragmentation) during apoptosis. The human leukemia cell line HL-60 and the human T cell line Jurkat were obtained from ATCC (Rockville, MD). Both HL-60s and Jurkats were cultured in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 20% fetal bovine serum and maintained at 37 °C in a 5% CO2, humidified atmosphere and harvested while in log phase of growth at approximately 1.5 × 106 cells/ml for HL-60s, and 1 × 106 cells/ml for Jurkat. The cells were washed once in phosphate-buffered saline (pH 7.4) and resuspended as described below. Human peripheral blood neutrophils were isolated by the plasma Percoll density gradient method from healthy donors as described previously (6Haslett C. Guthrie L.A. Kopaniak M.M. Johnston Jr., R.B. Henson P.M. Am. J. Pathol. 1985; 119: 101-110PubMed Google Scholar). HL-60s were plated at 2 × 106 cells/ml in either 35-mm dishes or 12-well plates in RPMI with calcium (550 μm) or without calcium (nominally present at 620 nm) from Life Technologies, Inc. (Gaithersburg, MD) supplemented with 0.25% bovine serum albumin, with or without 5 μm cytochalasin D. Neutrophils were plated at 5 × 106 cells/ml in 12-well plates in RPMI with or without calcium with 0.25% human serum albumin. Apoptosis was induced by UV irradiation at 254 nm for 10 min (neutrophils) or 5 min (HL-60s). Both cell types were then incubated at 37 °C in a 5% CO2humidified atmosphere for 2 h (HL-60) or 4 h (neutrophils) or as indicated to allow the apoptotic phenotypes to develop. Apoptosis was also induced in neutrophils by the addition of mouse anti-human Fas IgM (Upstate Biotechnology, Lake Placid, NY) (400 ng/ml) with continued exposure for 4 h. Jurkat cells were incubated at 2 × 106 cells/ml in Hepes-buffered saline (see below) for 3 h with mouse anti-human Fas IgM (100 ng/ml), or apoptosis was induced by UV irradiation at 254 nm for 10 min. In experiments in which calcium was added back, the cells were initially incubated in RPMI without calcium, and then Ca(NO3)2 added to the concentrations and at the times indicated, and the cells replaced in culture until the end of the incubation period. As stated in the text, in certain experiments, cells were incubated with the following agents: BAPTA/AM (10 μm) was purchased from Calbiochem (San Diego, CA); dl-α-tocopherol (0.06–2 mm) from Roche Vitamins and Fine Chemicals (Nutley, NJ);N-acetylcysteine (5 or 10 mm), dithiothreitol (1–10 mm), ascorbic acid (0.06–2 mm), and EGTA (2 mm) were purchased from Sigma. At the end of the incubation period, cell cultures were subdivided and samples simultaneously stained for surface PS, phospholipid uptake, and DNA degradation as described below. Several experiments were conducted to determine if the calcium flux alone was sufficient to induce annexin binding and a loss of aminophospholipid translocase activity. In these experiments, HL-60 cells (without apoptosis induction) were harvested, washed as above, and then treated with the indicated concentrations of ionomycin (Calbiochem, San Diego, CA) and incubated at 37 °C in a 5% CO2, humidified atmosphere 30 min and stained as described below. Cells bearing PS in the plasma membrane outer leaflet were identified as those binding FITC-labeled annexin V using an Apoptosis Detection Kit (R&D Systems, Minneapolis, MN). The binding of FITC-labeled annexin V to phosphatidylserine on the surface of apoptotic cells closely correlates with the appearance of nuclear and cytoplasmic condensation by light microscopy (7Bratton D.L. Hamid Q. Boguniewicz M. Doherty D.E. Kailey J.M. Leung D.Y.M. J. Clin. Invest. 1995; 95: 211-218Crossref PubMed Google Scholar) and the appearance of hypodiploid DNA (see below). Briefly, 105 cells were pelleted, resuspended in 100 μl of Hepes-buffered saline, and 100 ng of FITC-labeled annexin V and 500 ng of propidium iodide were added. The cells were incubated 15 min at room temperature, then the samples were transferred to ice and the sample volume brought to 0.5 ml. Analysis was done on a (San CA) or and the results were with cells were as described in the Apoptosis Kit by to cells from and cells from those for the FITC-labeled annexin V apoptosis was from the cells than the of the cells was simultaneously uptake was in Hepes-buffered saline 2 5 10 (pH with or without as concentrations of were by the of the calcium and by the method M. J. 1985; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) and was 1 NBD-labeled phospholipids were by 1 of or 1 of in a The were then resuspended in μl of μm with 0.25% bovine serum human serum used in with neutrophils or that these NBD-labeled in and the plasma membrane outer leaflet (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar, D.L. J. PubMed Google Scholar, D.L. J. PubMed Google Scholar). the incubation period, cells were harvested, washed and then resuspended in × The cells (5 × 105 in were incubated with 1 μl of the and 5 μl of propidium iodide for 10 min at room of the plasma membrane outer leaflet to that not the cell was (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar, D.L. J. PubMed Google Scholar, D.L. J. PubMed Google Scholar) with μl of bovine serum or human serum in for an 5 min and then samples were transferred to ice and with μl of and in the cytofluorograph 2 h. experiments demonstrated that the signal from the was during The of the phospholipid were by in a as to cells for propidium iodide or from cell In experiments, uptake of NBD-labeled was used as a measure of D. J. 1995; PubMed Scopus Google Scholar). all three cell types the of we for of the that the was not for of the other phospholipids and we used NBD-labeled phospholipid uptake as a measure of transbilayer phospholipid flip-flop (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar, D.L. J. PubMed Google Scholar, D.L. J. PubMed Google Scholar). As in and was and was for in as is of cells demonstrating aminophospholipid translocase activity A. Favre E. Cribier S. Hervé P. Devaux P.F. Biochemistry. 1986; 25: 2585-2590Crossref PubMed Scopus (186) Google Scholar, 5Sune A. Bette-Bobillo P. Bienvenüe A. Fellmann P. Devaux P.F. Biochemistry. 1987; 26: 2972-2978Crossref PubMed Scopus (97) Google Scholar). In a not transported by the aminophospholipid and used as a for transbilayer movement of phospholipids, was by cells and for (3Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (608) Google Scholar, 4Zachowski A. Favre E. Cribier S. Hervé P. Devaux P.F. Biochemistry. 1986; 25: 2585-2590Crossref PubMed Scopus (186) Google Scholar, 5Sune A. Bette-Bobillo P. Bienvenüe A. Fellmann P. Devaux P.F. Biochemistry. 1987; 26: 2972-2978Crossref PubMed Scopus (97) Google Scholar, D.L. J. PubMed Google Scholar, D.L. J. PubMed Google Scholar, D.L. Kailey J.M. E. V.A. Henson P.M. J. Scholar, J. J. PubMed Google Scholar, P. PubMed Scopus Google Scholar). Additionally, HL-60s, neutrophils and as well as the blebbing by all three cell the of blebbing and in the appearance of PS and of phospholipid flip-flop cytochalasin 5 was added to J.M. Green D.R. 1992; Google Scholar) and the of cytochalasin on blebbing and or without cytochalasin the results of uptake were blebbing or as an event for either enhanced uptake or the appearance of of the NBD-labeled not during the incubation and was demonstrated by of by the and method used previously D.L. E. Kailey J.M. V.A. Henson P.M. J. Immunol. 1992; 148: Google Scholar). DNA degradation was by the appearance of a hypodiploid in propidium Neutrophils and HL-60 cells (5 × were pelleted, washed then resuspended in phosphate-buffered saline (pH which was then brought to The cells were then at °C Jurkat cells were washed once in phosphate-buffered saline, then in 15 min on The cells were then washed once in phosphate-buffered saline, and resuspended in and at 4 cells were pelleted, and treated with for 30 min at 37 °C and stained with 500 propidium The cells were then incubated at 4 °C for at an in the changes were and DNA fragmentation identified by in (7Bratton D.L. Hamid Q. Boguniewicz M. Doherty D.E. Kailey J.M. Leung D.Y.M. J. Clin. Invest. 1995; 95: 211-218Crossref PubMed Google Scholar). calcium in HL-60s was by the method of et al. Campbell Clin. Exp. Immunol. 1992; PubMed Scopus Google Scholar). Briefly, the cells were incubated with 1.5 μm for the 30 min of the incubation in a 37 °C with to the cells in The cells were then washed in or RPMI with or without calcium, and resuspended at 2 × 106 cells/ml in 3 at 37 °C in a in a and were and of the to calcium, or for 15 min following calcium addition in the experiments, was added to the cells and determine the on of the was by addition of EGTA 10 mm) in 4 The of outer leaflet PS was associated with both loss of aminophospholipid translocase activity and of nonspecific phospholipid flip-flop in all three cells types and with both apoptotic As for HL-60s and neutrophils, cells undergoing apoptosis were to be for PS in the outer leaflet a of approximately 1 h 1 in these cultures of HL-60s and of neutrophils stained for FITC-labeled annexin V by 2 and 3 h of PS uptake was for cultures of HL-60s followed in all by The appearance of PS followed loss of aminophospholipid translocase activity as by uptake of the of extracellular calcium in the of transbilayer movement of plasma membrane phospholipids D.L. J. PubMed Google Scholar, J. J. PubMed Google Scholar, D.L. Kailey J.M. Henson P.M. PubMed Scopus Google Scholar, P. A. A. Schlegel R.A. Devaux P.F. Biochemistry. 1992; PubMed Scopus Google Scholar) and a in the of the aminophospholipid translocase A. Favre E. Cribier S. Hervé P. Devaux P.F. Biochemistry. 1986; 25: 2585-2590Crossref PubMed Scopus (186) Google Scholar, M. Devaux P.F. S. A. PubMed Scopus Google Scholar), we extracellular calcium is necessary for the appearance of As in of calcium (nominally present at 620 nm) from the incubation in of outer leaflet PS in both HL-60s and Jurkat cells neutrophils, not which was to was in to cells undergoing apoptosis in the of Importantly, of PS appearance in the outer leaflet from loss of aminophospholipid translocase activity of PS was in that PS uptake was completely calcium was added (550 μm) or present nm) The data also dissociated the appearance of PS from the nuclear changes of apoptosis calcium not the of hypodiploid DNA of membrane changes during apoptosis of HL-60s and The appearance of PS FITC-labeled annexin V, is as of of FITC-labeled annexin was and of cells for HL-60s and neutrophils, NBD-labeled PS and uptake at the various is as of as appearance of PS of cells binding FITC-labeled annexin in cultures of and HL-60s and Jurkats in the absence or of extracellular were incubated with or without calcium for 2 h (HL-60) or 3 h as described and and stained with FITC-labeled annexin as uptake as of and the appearance of hypodiploid DNA as in cells UV and maintained in calcium and HL-60s were incubated in RPMI with and without were incubated with calcium and were different from incubated without as of calcium has been to apoptosis in HL-60s PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), it has been to apoptosis of neutrophils S.J. C. J. Clin. Invest. PubMed Scopus Google Scholar). it was necessary to determine calcium during apoptosis. the of al. Campbell Clin. Exp. Immunol. 1992; PubMed Scopus Google Scholar), we that the of culture in HL-60s undergoing apoptosis demonstrated of calcium as by the calcium of calcium not the of calcium flux across the plasma we the of extracellular calcium by UV neutrophils and HL-60s in RPMI without calcium, for 1 and then different concentrations of As for HL-60s, incubation calcium in a calcium of which was followed by 2 of calcium extracellular calcium was added as in both the appearance of PS and uptake of phospholipid flip-flop) nearly identical requirements for extracellular The appearance of PS as by of FITC-labeled annexin V binding was to be at 105 μm μm the calcium of uptake was nearly identical and to be at μm μm closely the calcium of both PS and calcium during calcium was in and HL-60s incubated in calcium for the of by the as were and in a of PS appearance in HL-60s and uptake in neutrophils following UV Cells were UV in RPMI without calcium and then calcium added at various concentrations 1 h of binding and uptake were as described and as of response in cells UV and maintained in calcium for an incubation of 2 h for HL-60s and 4 h for UV maintained in calcium as as calcium was in and HL-60s incubated in calcium for the of by the as were and Taken together, these results strongly suggest that flux of extracellular calcium during apoptosis, in the absence of of calcium, is for the appearance of outer leaflet PS during apoptosis and that outer leaflet PS results from nonspecific flip-flop of data however, calcium flux is sufficient for the appearance of HL-60s were treated with ionomycin nm to 2 mm) which in the of calcium to 1 μm, nearly 10 times that during apoptosis. However, the appearance of outer leaflet PS not and PS uptake was 30 min following with ionomycin calcium flux alone is insufficient to the membrane events of apoptosis. Furthermore, these data suggest the that if the aminophospholipid translocase is PS is not in the outer leaflet despite calcium flux and enhanced phospholipid flip-flop D.L. E. Kailey J.M. V.A. Henson P.M. J. Immunol. 1992; 148: Google Scholar). In of it was in the of these experiments that incubation of HL-60s in the of ionomycin in of the apoptotic as has been previously J. PubMed Scopus Google Scholar) with the appearance of PS, loss of uptake, enhanced uptake, and DNA degradation not determine the relative of aminophospholipid translocase activity with nonspecific phospholipid flip-flop in the appearance of PS, were to aminophospholipid translocase activity during apoptosis. the aminophospholipid translocase has been to be to P. Biochemistry. PubMed Scopus Google Scholar) a of were used to activity. ascorbic and (see and and in were used without to aminophospholipid translocase activity. As calcium has also been reported to aminophospholipid translocase activity at concentrations A. Favre E. Cribier S. Hervé P. Devaux P.F. Biochemistry. 1986; 25: 2585-2590Crossref PubMed Scopus (186) Google and extracellular calcium were of PS uptake was in the of demonstrating that loss of aminophospholipid activity is calcium In the absence of or of the aminophospholipid as an we to both the calcium used and the to loss of the aminophospholipid translocase to determine the of aminophospholipid translocase function on the appearance of these experiments, HL-60s were UV and incubated in RPMI without calcium to PS appearance until calcium (550 μm) addition at either 1 or 2 h. was is PS uptake at by a aminophospholipid translocase of while at 2 h is translocase activity (10 of At both times uptake as a measure of nonspecific phospholipid flip-flop is We hypothesized that if aminophospholipid translocase activity is in the appearance of PS by PS to the inner leaflet, then nonspecific phospholipid we would to outer leaflet PS at 1 h than at 2 h following UV In of the appearance of PS as both of FITC-labeled annexin V cells and following calcium addition at irradiation was than that at 2 h irradiation The appearance of PS at 2 h was not to of the cells the in calcium was calcium was added at 1 h than at 2 h irradiation not of calcium was added or 2 if cells were with FITC-labeled annexin V at 3 h all demonstrated identical and appearance of outer leaflet PS and DNA degradation not is to play a in of apoptosis S. J. PubMed Scopus Google Scholar, P. E. PubMed Scopus Google Scholar). in an in calcium is to a which to DNA and in of calcium with the of can induce apoptosis PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, S. P. S. Exp. PubMed Scopus Google Scholar). these the with of apoptosis, may in by of calcium from and S. J. PubMed Scopus Google Scholar). to the of apoptosis likely S. J. PubMed Scopus Google Scholar), A. A. M. S. A. PubMed Scopus Google Scholar, V.A. R.A. PubMed Scopus Google Scholar), and the Cohen J.J. J. Immunol. Google Scholar) and the S.J. D.M. Green D.R. J. PubMed Scopus Google Scholar, S. 1995; PubMed Scopus Google Scholar, M. S. PubMed Scopus Google Scholar). a the appearance of outer leaflet PS activation S.J. D.M. Green D.R. J. PubMed Scopus Google Scholar, M. PubMed Google Scholar) to experiments various Several the of apoptosis, but were not in the events to the appearance of the other calcium flux alone is insufficient to the plasma membrane changes of apoptosis and D.M. J. PubMed Scopus Google Scholar). Furthermore, calcium does not to be in cells undergoing apoptosis in a of and see S. P. S. Exp. PubMed Scopus Google Scholar) and may as a of the apoptotic Campbell Clin. Exp. Immunol. 1992; PubMed Scopus Google Scholar). calcium can apoptosis in neutrophils S.J. C. J. Clin. Invest. PubMed Scopus Google Scholar). we that the appearance of PS in the outer leaflet is inhibited in the absence of extracellular calcium and can be dissociated from the other events of apoptosis, DNA degradation and loss of the aminophospholipid translocase activity. data strongly suggest that calcium in the absence of a of in the of calcium is a necessary event in the appearance of The data using the calcium that extracellular calcium of 100 μm in calcium of 4 is for of PS in the plasma membrane outer leaflet of HL-60s, neutrophils, and Jurkats undergoing apoptosis 3 and We previously demonstrated a extracellular calcium for enhanced transbilayer movement of phospholipids in the and 100 μm D.L. J. PubMed Google Scholar, D.L. J. PubMed Google Scholar), and a described isolated from the and in a for calcium activity and μm) J. PubMed Scopus Google Scholar). been from the of van A. A.J. P. van PubMed Scopus Google Scholar) may be the which can be inhibited by the calcium to both uptake and the appearance of PS with were in neutrophils, Jurkat, and in HL-60s in the latter of were used that phospholipid is to be in these cells undergoing apoptosis. that may transbilayer movement of phospholipids D.L. J. PubMed Google Scholar) and membrane as which the of Importantly, we that loss of the aminophospholipid translocase activity, DNA is not on extracellular calcium and that the nuclear events of apoptosis can be completely dissociated from the appearance of While we were to that loss of aminophospholipid translocase activity alone was insufficient to result in the appearance of PS, we not been to the to which loss of activity is necessary for the appearance of to activity with were a aminophospholipid translocase has been Schlegel R.A. Williamson P. PubMed Scopus Google Scholar), to its activity likely be in the data using HL-60s suggest that activity of the aminophospholipid translocase can the appearance of PS in the outer leaflet The PS at the may be to the of the where outer leaflet PS is not in the of aminophospholipid translocase activity despite enhanced nonspecific transbilayer movement D.L. E. Kailey J.M. V.A. Henson P.M. J. Immunol. 1992; 148: Google Scholar). in the outer leaflet PS can be demonstrated despite enhanced aminophospholipid translocase activity, due to PS from flip-flop in of PS by the aminophospholipid translocase P. PubMed Scopus Google Scholar). We also that while the appearance of outer leaflet PS in HL-60s, and neutrophils undergoing apoptosis was accompanied by both loss of aminophospholipid translocase activity and enhanced nonspecific phospholipid uptake, were in the of loss of aminophospholipid translocase activity and the of nonspecific flip-flop. it is that in the relative contribution of these to the appearance of PS and phospholipid flip-flop in different cells undergoing apoptosis or We for of the