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Role of mitochondrial membrane permeabilization and depolarization in platelet apoptosis

2017; Wiley; Volume: 181; Issue: 2 Linguagem: Inglês

10.1111/bjh.14903

1365-2141

Valery Leytin, Armen V. Gyulkhandanyan, John Freedman,

Cardiac Ischemia and Reperfusion

The intrinsic mitochondria-dependent pathway of platelet apoptosis is well documented (Leytin, 2012; Appendix S1, reference 1). There is, however, no direct evidence that, in contrast to nucleated cells, apoptosis in anucleate platelets can be triggered via the extrinsic apoptosis pathway executed by interaction of cell-surface death receptors with the cognate death ligands, such as Fas-receptor and Fas-ligand (Leytin, 2012). Mitochondrial membrane permeabilization and dissipation of mitochondrial transmembrane potential (ΔΨm depolarization) are frequently used as markers of apoptosis in nucleated cells (Appendix S1, references 2–5) and in anucleate platelets (Leytin, 2012; Gyulkhandanyan et al, 2013, 2014, 2015). Two pathways of mitochondrial permeabilization have been reported in nucleated cells. The first permeabilization pathway (PERM-1) is triggered by the opening of the mitochondrial permeability transition pore in the mitochondrial inner membrane (Appendix S1, references 2–7). The second pathway (PERM-2) is triggered by overexpression of pro-apoptotic Bax (also termed BAX) and Bak (BAK1) proteins versus anti-apoptotic Bcl-xL (BCL2L1) and Bcl-2 (BCL2) proteins followed by permeabilization of the mitochondrial outer membrane (Appendix S1, references 8, 9). In platelets, overexpression of pro-apoptotic Bax protein in comparison with anti-apoptotic Bcl-2 protein was first reported 20 years ago (Vanags et al, 1997) and confirmed by other studies (reviewed by Leytin, 2012). This correspondence aims to elucidate the roles of the PERM-1 and PERM-2 pathways of permeabilization and ΔΨm depolarization in platelet apoptosis, which remain largely unknown at present. Table 1 summarizes the results of eight reports, in which platelet apoptosis has been investigated in three experimental models – in platelets exposed to (i) calcium ionophore A23187, (ii) pro-apoptotic BH3 mimetic ABT-737 and (iii) potassium ionophore valinomycin, together with appropriate control diluent buffers. As shown previously (Gyulkhandanyan et al, 2014, 2015, 2017), ΔΨm depolarization is strongly induced in platelets treated with any of these three agents, when up to 90–95% cells of the total platelet population undergo ΔΨm depolarization (Table 1, platelet response 1). Notably, this strong depolarization may be stimulated with or without participation of mitochondrial permeability transition pore (Table 1, platelet response 2). Furthermore, the effects of A23187, ABT-737 and valinomycin on expression of pro-apoptotic Bax and Bak proteins in platelets and their translocation to mitochondria and oligomerization in the outer membrane have been demonstrated to be different. A23187 and ABT-737 induce these mitochondria-associated apoptotic events in platelets, whereas, in contrast, valinomycin does not trigger Bax and Bak expression; Bcl-xL and Bcl-2 anti-apoptotic proteins are not induced by any of these treatments (Table 1, platelet response 3). Fundamental differences were also observed when the effects of A23187, ABT-737 and valinomycin on molecular and cellular extra-mitochondrial apoptotic responses were investigated. A23187 induces both molecular and cellular extra-mitochondrial events, including caspases-3, -9 and -8 activation, phosphatidylserine exposure (Table 1, platelet responses 4–7), platelet shrinkage and microparticle formation (Table 1, platelet responses 8, 9). On the contrary, ABT-737 induces only molecular (Table 1, platelet responses 4–7) but not cellular extra-mitochondrial events (Table 1, platelet responses 8, 9). Valinomycin is able to trigger only ΔΨm depolarization (Table 1, platelet response 1) rather than other mitochondria-associated (Table 1, platelet responses 2, 3) and extra-mitochondrial platelet responses (Table 1, platelet responses 4, 7–9). Figure 1A–D present the effects of A23187, ABT-737 and valinomycin on platelet apoptosis, as well as results reported previously for nucleated cells. The data indicate that several scenarios are implemented in platelets depending on the nature of triggering stimulus. In the first scenario, in platelets treated with calcium ionophore A23187, mitochondrial membrane permeabilization is executed via two permeabilization pathways, PERM-1 and PERM-2, accomplished by induction of both molecular and cellular manifestations of platelet apoptosis (Fig 1A,D). The PERM-1 pathway includes permeability transition pore opening in the inner membrane, mitochondrial matrix swelling, rupture of inner and outer membranes and release of pro-apoptotic proteins from mitochondrial matrix and intermembrane space between mitochondrial membranes. The PERM-2 pathway is achieved through Bax and Bak overexpression, and their translocation to mitochondria and oligomerization in the outer membrane, followed by opening of mitochondrial apoptosis-induced channel in outer membrane, permeabilization of outer membrane and release of pro-apoptotic proteins from intermembrane space but not from mitochondrial matrix (Fig 1A). The second scenario occurs in platelets treated with pro-apoptotic BH3 mimetic ABT-737. In this case, platelet apoptosis is executed only via the PERM-2 pathway; PERM-1 pathway is not induced. This less-damaging PERM-2 mechanism does not involve permeability transition pore opening in the inner membrane, rupture of mitochondrial membranes and release of pro-apoptotic proteins from mitochondrial matrix to the cytosol and is accomplished by execution of only molecular but not cellular manifestations of platelet apoptosis (Fig 1B,D). Experiments with ABT-737-treated platelets have shown strong ΔΨm depolarization, with 92·4 ± 4·1% cells having dissipated ΔΨm (Gyulkhandanyan et al, 2015) in the absence of permeability transition pore opening in the inner membrane (Table 1) and PERM-1 mechanism of permeabilization (Fig 1D). The third scenario is executed in platelets treated with potassium ionophore valinomycin. In contrast to platelets exposed to A23187 and ABT-737, only strong ΔΨm depolarization is induced by valinomycin (Table 1). However, this strong depolarization was not associated with PERM-1 and PERM-2 pathways of permeabilization and does not promote platelet apoptosis (Fig 1C,D). Experiments with valinomycin-treated platelets (Gyulkhandanyan et al, 2017) indicate that even strong (94·0 ± 1·1%) ΔΨm depolarization per se is not causal for platelet apoptosis, because it is not able to trigger neither molecular nor cellular manifestations of platelet apoptosis in the absence of PERM-1 and/or PERM-2 permeabilization. Activation of caspases-3, -9 and -8 plays essential roles in platelet apoptosis (Mutlu et al, 2012). The analysis of mitochondrial membrane permeabilization and depolarization elucidates the impact of these mitochondrial events on activation of these caspases, which can be induced by PERM-1 and/or PERM-2 permeabilization in A23187- and ABT-737-treated platelets but not by ΔΨm depolarization in the absence of permeabilization in valinomycin-treated platelets (Table 1, Fig 1). In summary, taken together, these findings suggest that platelet apoptosis is driven by mitochondrial membrane permeabilization rather than by ΔΨm depolarization and that pharmacological manipulations of platelet apoptosis should target PERM-1 and/or PERM-2 pathways of permeabilization. This work was supported by a grant from the Platelet Research Fund of Ronya Beskin, Israel. The authors thank D.J. Allen, S. Mykhaylov, A. Mutlu, E. Lyubimov and H. Ni for research cooperation. VL, AVG and JF wrote the paper. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.