Abstract
Platelet lifespan is limited by activation of intrinsic apoptosis. Apoptotic platelets are rapidly cleared from the circulation in vivo. ABT-737 triggers platelet apoptosis and is a useful tool for studying this process. However, in vitro experiments lack clearance mechanisms for apoptotic platelets. To determine whether apoptotic platelets progress to secondary necrosis, apoptosis was triggered in human platelets with ABT-737, a BH3 mimetic. Platelet annexin V (AnV) binding, release of AnV+ extracellular vesicles (EVs), and loss of plasma membrane integrity were monitored by flow cytometry. ABT-737 triggered AnV binding, indicating phosphatidylserine exposure, release of AnV+ EVs, and a slow loss of plasma membrane integrity. The latter suggests that apoptotic platelets progress to secondary necrosis in vitro. These responses were dependent on caspase activation and Ca2+ entry. Surprisingly, although intracellular Ca2+ concentration increased, AnV+ EV release was not dependent on the Ca2+-dependent protease, calpain. On the contrary, ABT-737 downregulated the ability of the Ca2+ ionophore, A23187, to trigger calpain-dependent release of AnV+ EVs. This was dependent on caspase activity as, when caspases were inhibited, ABT-737 increased the ability of A23187 to trigger AnV+ EV release. These data suggest that apoptotic platelets progress to secondary necrosis unless they are cleared. This may affect the interpretation of ABT-737-triggered signaling in platelets in vitro. Ca2+-dependent AnV+ EV release is downregulated during apoptosis in a caspase-dependent manner, which may limit the potential consequences of secondary necrotic platelets.
Highlights
Human platelets circulate for around 10 days before they are cleared.[1]
ABT-737induced platelet death was blocked by caspase inhibitors[6,7] and absent in BakÀ/ÀBaxÀ/À mouse platelets,[3] further indicating that it is dependent on the intrinsic apoptosis pathway
Our data indicate that the responses to ABT-737 in vitro are not a single event, but a temporal sequence of stages leading through to secondary necrosis, each regulated differently by intracellular signaling
Summary
Human platelets circulate for around 10 days before they are cleared.[1] the trigger for platelet clearance has not been fully resolved, intrinsic apoptosis delimits platelet lifespan.[2] Platelets lacking proapoptotic Bak/Bax have a prolonged circulating half-life in vivo, whereas platelets lacking prosurvival Bcl-xL have a very short circulating half-life.[3] Understanding platelet apoptosis is an important step in understanding disorders of platelet number and function, for understanding the consequences of drugs that trigger platelet apoptosis. Prosurvival Bcl-2 family proteins, including Bcl-xL, are upregulated in many cancers and have become attractive targets for anticancer therapy.[4,5] Prosurvival Bcl proteins can be inhibited by BH3 mimetics. One BH3 mimetic, ABT-263 (navitoclax), which inhibits Bcl-2 and Bcl-xL, is associated with thrombocytopenia through activation of intrinsic apoptosis in platelets.[6,7] This has been avoided through the development of a more selective Bcl[2] inhibitor, ABT-199 (venetoclax), which received February 16, 2019 accepted after revision June 12, 2019
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