re: Principle of detecting apoptosis with Annexin V

Tue, 22 Oct 1996 15:26:43 +0000 (EZT)

Phosphatidylserine (PS) is a normal constituent of the plasma membrane (PM). The viable cell treats this
phospholipid species in a specific manner by localizing PS predominantly in the membrane leaflet facing
the cytosol. The outer leaflet of the PM facing the environment is almost devoid of PS. Aminophospholipid
translocases are thought to be responsible for this asymmetric distribution of PS. The molecular
identitie(s) of the translocases have not been resolved sofar (for a recent review see Diaz, C. and
Schroit, A.J. J.Membrane Biol. 1996,151, 1-9). Translocase activity has been measured in all viable
cell types tested sofar. During apoptosis the equilibrium distribution of PS changes by an increased
appearance in the outer leaflet. Also in this case the machineries responsible for the transbilayer
movement have not been identified. It is accepted though that specific intrinsic membrane proteins
facilitate this movement. As was firstly shown by Valerie Fadok (Fadok, V. et al. J.Immunol 1992, 148,
2207-2216, J.Immunol. 1992, 149, 4029-4035) cell surface exposure of PS occurs during apoptosis of
leukocytes. This exposed PS serves the function of recognition and phagocytosis by phagocytes. Hence,
cell surface exposure of PS likely forms a functional and, consequently, integral part of the apoptotic
program. From this perspective it is an attractive target for measuring apoptosis.
The apoptosis associated loss of PM phospholipid asymmetry was firstly probed flow cytometrically
using Merocyanine 540 (Fadok, V. et al. J.Immunol. 1992, 148.2207-2216). Valerie's papers triggered
us to investigate Annexin V as a probe for cell surface exposed PS during apoptosis. Annexin V was
subject of investigation in our laboratory for over a decade and was appreciated for its ability to bind to
PS. Contrasting MC540, which intercalates in the bilayer, Annexin V binds to the PS exposing surface
extrinsically in a calcium dependent manner. The first papers showed that Annexin V is powerful tool to
measure apoptosis flow cytometrically (Koopman et al. Blood, 1994, 84, 1415-1420, Homburg et al.
Blood, 1995, 85, 532-540, Vermes, et al. J.Immunol.Methods 1995, 184, 39-51). These papers also
showed that an additional probe, like propidium iodide, should be included in order to discriminate
between cells with intact and compromised PM integrity. These papers learned that PS exposure occurs
during a phase of apoptosis where the PM integrity is still intact.
The cell surface exposed PS likely originates from the existing pool of the cell. Whether PS overall
content increases during apoptosis by synthesis is not known and has, to my knowledge, not been
addressed sofar. It is a very interesting question though.
Comprehensive studies of Seamus Martin learned that PS exposure precedes nuclear changes, occurs
downstream of the Bcl-2 and Abl checkpoint and is a ubiquitous process of suspended cell types
irrespective of the apoptosis initiating trigger (J.Exp.Med. 1995, 182, 1545-1557). Using Annexin V
Van Engeland et al. (Cytometry, 1996, 24,131-139) showed that PS exposure also occurs during
apoptosis of adherent cell types in culture. The state of the art is that cell surface exposure of PS is a
ubiquitous and conserved part of apoptosis. This exposure can be simply assayed using Annexin V. It
binds rapidly and with high affinity to the PS exposing cell.
We have reached the point to consider seriously cell surface exposure of PS as the general Hallmark of
apoptosis. I reckon that interesting and stimulating discussions on this subject are about to take place.

Chris Reutelingsperger
Department Biochemistry
University Maastricht
the Netherlands

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