The discussion above highlights an important conceptual idea that different PS scramblases react to distinct upstream signals to externalize PS. Adding complexity to PS biology, it is now apparent that not all externalized PS is functionally equivalent. In the above-mentioned scenario for Xkr8 and TMEM16 that are activated by caspase 3 and Ca2+, respectively, only the former serves as an eat-me signal for PS receptors and efferocytosis. Indeed, when a mutant TMEM16F was introduced into a mouse lymphoma cell (W3-Ildm) to achieve constitutive PS exposure, PS-positive tumor cells (assessed as annexin V positive) were not engulfed by professional DCs, and only became phagocytosed after activation of caspase 3 and Xkr8 with Fas antibody.25 Thus, the PS externalized by TMEM16 does not provide an eat-me signal, but is sufficient to provide an electrostatic charge to recruit clotting factors via the interactions of their Ca2+-dependent Gla domains. Moreover, the Ca2+-stimulated PS externalization induced by TMEM16F is rapid (within minutes) and reversible upon restoration of Ca2+ homeostasis,22 while Xkr8-mediated PS exposure is slow (within hours) and irreversible (Figure 2).

Figure 2 Models for the different forms of PS externalization: As noted in the text, PS can be externalized under a variety of physiological and patho-physiological conditions that include platelet activation (a) and caspase-dependent apoptosis (b). (a) Activated platelets promote a Ca2+-TMEM16-mediated externalization of PS that serves as a nucleation scaffold for the recruitment of hemostasis factors that initiate blood clotting (indicated by the solid black line in a). (b) Apoptotic cells externalize PS via the caspase 3/7-mediated cleavage of Xkr8 that serves as an eat-me signal for various PS receptors (TAMs, TIMs, and αvβ5 and αvβ3 integrins). Recent studies suggest that during apoptosis, the surface density of the PS may reach a critical threshold that clusters and activates PS receptors. Why PS externalized on apoptotic cells (Xkr8-dependent) serves as a signal for efferocytosis, while PS expressed on stressed and activated cells (TMEM16-dependent) has not been completely elucidated Full size image

With respect to the externalization of PS by Xkr8 during apoptosis, recent evidence suggests that stable and irreversible PS externalization is achieved by a dynamic interplay between Xkr8 and ATPase, class VI, type 11C (ATP11C), a member of the P4-type ATPase family of flipases that redirects PS from the outer membrane leaflet back to the inner leaflet.26 Similar to Xkr8, ATP11C contains a caspase cleavage site, but unlike Xkr8 that is activated by caspase cleavage, ATP11C is inactivated by the same process and prevents return of PS to the inner leaflet. Conversely, when cells express ATP11C with a mutated caspase recognition site, cellular flipase activity remains high, and cells expressing mutant ATP11C do not sustain PS externalization or retain their ability to be engulfed. In the non-apoptotic context, a high Ca2+ concentration activates TMEM16, but does not inactivate ATP11C, possibly explaining the reversibility of TMEM16-mediated PS externalization.

The preceding reasoning suggests that a critical concentration or topology of PS may need to be acquired for recognition as an eat-me signal. A possible explanation as to how PS topology or local density might be recognized differently by PS receptors might also be related to the PS clustering activity exerted by Annexin.27 The combination of low membrane fluidity and consequent low clustering of PS receptors on the phagocytes’ surface due to reduced lateral mobility of the PS molecule may help to distinguish dead/dying from viable PS-exposing cells.11 Receptor clustering is often sufficient to activate intracellular signaling cascades. In apoptotic cells the cytoskeleton and the focal adhesion molecules are early targets of caspases. After death receptor stimulation, active caspase 8 immediately translocates to plectin, a major cytoskeletal cross-linking protein and quantitatively cleaves it at Asp 2395.28 The resulting weakening of the cytoskeleton increases the lateral mobility of PS and might consequently enable cooperative binding of PS ligands or receptors. Furthermore, there is evidence that lipid rafts and PS are mutually exclusive on the membranes of apoptotic cells in contrast to viable and activated cells.29 This suggests that there may be different topologies of PS arranged on the surfaces of apoptotic versus viable cells that engage receptors in distinct ways. Indeed, recent studies examining the effects of ligand-density on the activation of AXL receptor tyrosine kinase (Axl; a PS receptor) support this idea, in which it was concluded that the specific sensing of ligand spatial distribution is a critical feature for PS-dependent (Axl) receptor activation.30

Although the preceding sections have focused on the interplay between scramblases, flipases, and PS externalization, other enzymes and pathways have been implicated in PS externalization including the ATP-binding cassette (ABC) transporter ABC131 and Tat1.32 Moreover, studies by Lee et al.32 suggest that increases in bidirectional membrane trafficking results in PS externalization. In their model, PS is externalized by a two-step process whereby internalization of plasma membrane into cytoplasmic vesicles occurs as cells shrink during apoptosis. This is followed by Ca2+-dependent trafficking of PS-positive vesicles back to the cell surface.32 Whether these specialized forms of PS externalization lead to diverse depots of PS on the membrane is not clear, although the recent development of high-resolution fluorescent probes, such as Disciodin-C2, and GFP-LactadherinC2, should make it more feasible to visualize PS in discrete subcellular membrane domains and topologies.33

The realization that not all externalized PS has the same biological function also highlights the need to better characterize the nature of the molecular species of PS on the cell surface. Identification of the PS fatty acyl composition, its saturation, length, and oxidative status by mass spectrometry might be instructive in determining whether different externalization itineraries lead to discrete species of PS. With respect to the idea of PS oxidation, in which one or more of the acyl chains has unsaturated and oxidized substitutions, there is some evidence that oxidized PS (oxPS) is a more efficient eat-me signal than the non-oxidized molecule.34 Also some PS-binding proteins involved in efferocytosis (i.e., Gas6, milk fat globule-EGF factor 8 protein (MFG-E8), and T-cell immunoglobulin and mucin domain receptor-1 (TIM-1)) appear to bind with higher affinity to oxPS,34 which tends to protrude from the planar layers of cell membranes. This is interesting from a mechanistic view, as one of the proposed pathways for PS oxidation involves cytochrome c-dependent PS oxidation, with cytochrome c acquiring a gain-of-function peroxidase activity once released from mitochondria.35 In this model, cytochrome c released during mitochondrial outer membrane permeabilization would serve two interrelated functions. First, as a central component of the apoptosome, and second, to concomitantly catalyze the oxidation of PS to provide an eat-me assurance signal for efferocytosis.36 As discussed below, one of the most important future goals will be to assess whether all forms of externalized PS are immunosuppressive.