Mitochondria and Platelet Cell Death

We read with great interest an article by Alberio et al[1] recently published in Thrombosis and Haemostasis. In the article, the authors have successfully demonstrated that COAT (also known as procoagulant) platelets, now well established as a subpopulation of activated platelets, are formed in a stepwise manner going through a transitional state of a proaggregatory phenotype which binds PAC1, before proceeding to a procoagulant state. This exploration advances our understanding of thrombus formation by developing a new element of spatio-temporal regulation to thrombogenesis.

Procoagulant platelets are a subpopulation of activated platelets that are maximally generated upon costimulation of protease-activated receptor (PAR) and glycoprotein VI (GPVI), possess high externalized phosphatidylserine (PSer) and retain granule proteins on their surface. In the past few years, evidence has continued to accumulate regarding the importance of procoagulant platelets in thrombogenesis.[2] [3] [4] [5] [6] Undoubtedly, understanding the molecular pathways of procoagulant platelet formation would unveil novel potential pharmacologic targets for both haemostatic and antithrombotic therapies. We would like to add few points of clarification for some of the concepts proposed in this article regarding platelet cell death.

Two mitochondrial mechanisms can lead to cell death in platelets. First, mitochondrial permeability transition (mPT) and associated mPT pore formation can lead to either apoptosis or necrosis. In an ATP-depleted cell, mPT-mediated death proceeds in a necrotic pattern, whereas in a cell with preserved ATP production, mPT-mediated death proceeds at least in part through an apoptotic mechanism mediated by mitochondrial osmotic swelling, outer mitochondrial membrane (OMM) rupture and subsequent cytochrome c release–induced activation of caspase-dependent apoptosis. Second, an apoptotic pathway can be initiated by antagonism of Bcl-XL, oligomerization of Bax and Bak pro-apoptotic proteins and permeabilization of the OMM. In the setting of platelet costimulation with a PAR and GPVI agonists,[7] cyclophilin D (CypD)-regulated mPT is crucial in procoagulant platelet formation, whereas an apoptotic mechanism mediated by Bax and Bak plays no essential role ([Fig. 1A]). The finding presented in the article by Dr. Alberio's group of no change in levels of the pro-apoptotic Bcl-2 proteins Bax and Bak provides further evidence of the importance of mPT-mediated processes in this setting. The authors' thorough investigation of activated cell-death mechanisms, both apoptotic and mPT mediated, in the procoagulant platelet revealed an elevation of caspase-3 activity. While the authors provided no comment on the mechanistic import of this observation, we offer the hypothesis that the increase in caspase-3 activity observed by Alberio et al is perhaps best explained by the fact that some mitochondria did undergo osmotic swelling followed by OMM rupture and cytochrome c release. However, mPT formation in the vast majority of mitochondria within the costimulated platelet limits production of ATP and therefore those platelets undergo necrotic cell death even in the presence of active caspase-3.

Fig. 1 Platelet phosphatidyl serine (PSer) exposure. Isolated murine (A) and human (B) platelets were undergone stimulation with thrombin and convulxin for 7 minutes at 37°C or with the indicated reagent for 30 minutes. Platelets were then stained with annexin V and fixed. Annexin V–positive cells were measured by flow cytometry and expressed as percent of total. 2,4-DNP, 2,4-dinitrophenol; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; CVX, convulxin; Thr, thrombin. *p < 0.05 vs. wild type (panel A) and vs. Unstim (panel B), n ≥ 3.

Finally, numerous publications including the publication by Alberio et al have suggested loss of mitochondrial transmembrane potential (ΔΨ_m) as a mechanism mediating platelet cell death and procoagulant platelet formation. Generation and maintenance of ΔΨ_m is dependent upon a proton-impermeable inner mitochondrial membrane, the integrity of which can be violated either through the use of a mitochondrial uncoupler or by physical disruption of the inner mitochondrial membrane as occurs upon formation of the mitochondrial transition pore. When induced using either a mitochondrial uncoupler such as carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and 2,4-dinitrophenol (2,4-DNP) or the potassium ionophore valinomycin, loss of ΔΨ_m does not lead to PSer exposure ([Fig. 1B]). In contrast, mPT-mediated loss of ΔΨ_m, which occurs as a result of physical disruption, closely coincides with PSer exposure.[7] [8] Such distinction of the precise mechanism by which the process of platelet cell death occurs will be crucial in the evaluation and development of therapies targeting this essential and regulated physiologic and pathophysiologic event.

• References

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• 2 Daskalakis M, Colucci G, Keller P. , et al. Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis. Cytometry B Clin Cytom 2014; 86 (06) 397-409
• 3 Hua VM, Abeynaike L, Glaros E. , et al. Necrotic platelets provide a procoagulant surface during thrombosis. Blood 2015; 126 (26) 2852-2862
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