ABT-737 Triggers Caspase-Dependent Inhibition of Platelet Procoagulant Extracellular Vesicle Release during Apoptosis and Secondary Necrosis In Vitro

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.

Fig. 1

ABT-737 triggers Ca²⁺-dependent phosphatidylserine (PS) exposure. (A) Washed human platelets were stimulated with ABT-737 (10 μM, 3 hours), after which samples were stained with anti-CD41-PerCP-Cy7, and annexin V (AnV)-fluorescein isothiocyanate (FITC) to detect PS exposure. PerCP-Cy7 fluorescence was used to trigger acquisition of CD41+ events. The panels show density plots of events from low density (blue) to high density (red) of forward scatter (FSC-A) and FITC fluorescence. Unstimulated platelets have high FSC-A and low annexin V-FITC binding (AnV^neg). Stimulation with ABT-737 triggers PS exposure at two levels (AnV^med and AnV^high). The vertical line separating left and right was defined by the FSC-A of 1 μm silica beads. The density plots are representative of data from 5 different donors. The percentage of platelets with indicated AnV binding in the presence or absence of extracellular CaCl₂, or in platelets treated with 20 μM BAPTA-AM (n = 5) is shown in (B) (n = 5; **p < 0.01, ***p < 0.001 for AnV^high compared with platelets with CaCl₂; †††p < 0.001 for AnV^med compared with platelets with CaCl₂). (C) Fluo-4-loaded platelets were stimulated with ABT-737 (or dimethyl sulfoxide [DMSO] as vehicle control) for the indicated times. The Fluo-4 fluorescence of platelets (CD41 + , > 1 μm) is shown normalized to the fluorescence of platelets prior to stimulation (*p < 0.05, **p < 0.01, ***p < 0.001 compared with DMSO-treated; †p < 0.05, ††p < 0.01 for no CaCl₂ compared with + CaCl₂ condition). (D) Number of AnV⁺ extracellular vesicles (EVs) following stimulation with ABT-737 for the indicated times in the presence or absence of extracellular CaCl₂ or in BAPTA-AM treated platelets (n = 5) (**p < 0.01, ***p < 0.001 compared with DMSO-treated; †††p < 0.001 for ABT-737 + BAPTA-AM compared with ABT-737 + CaCl₂; for clarity, comparison of ABT-737 no CaCl₂ with ABT-737 + CaCl₂ or with DMSO-treated is not shown).

Fig. 2

ABT-737 treatment leads to secondary necrosis. (A) Platelets were loaded with calcein, treated as indicated and stained with anti-CD41-PerCP-Cy7, and annexin V-APC. Density plots are gated on platelets (CD41 + , > 1 μm) and are representative of 5 independent experiments. The percentage of calcein negative (-ve) platelets is shown in (B) (n = 5; ***p < 0.01 for CaCl₂ vs. no CaCl₂; †††p < 0.001 for CaCl₂ vs. BAPTA).

Fig. 3

ABT-737-induced platelet apoptosis is caspase-dependent whereas secondary necrosis requires calpain. (A, B) Platelets were treated with the caspase inhibitor, Q-VD-Oph (50 μM), the calpain inhibitor, calpeptin (140 μM), or the vehicle (dimethyl sulfoxide [DMSO]) prior to stimulation with ABT-737 for the indicated times. The percentage of platelets with the indicated level of annexin V (AnV) binding is shown in (A) (n = 5; **p < 0.05, ***p < 0.01 for AnV^high compared with DMSO-treated platelets; ††p < 0.01, †††p < 0.001 for AnV^med compared with DMSO-treated platelets). The number of AnV⁺ extracellular vesicles (EVs) detected is shown in (B) (n = 5; **p < 0.01, ***p < 0.001 compared with ABT-737-treated platelets). (C, D) Calcein-loaded platelets were treated with Q-VD-Oph or calpeptin then with ABT-737. The mean percentage of calcein -ve platelets is shown in (C), and representative density plots are shown in (D) (n = 5; ***p < 0.01 compared with ABT-737-treated platelets).

Fig. 4

Calpain-dependent release of annexin V-positive (AnV⁺) extracellular vesicle (EVs) is downregulated during apoptosis. (A) Scheme of the experiment. Platelets were treated with Q-VD-Oph (or dimethyl sulfoxide [DMSO] as vehicle control; 30 minutes) prior to ABT-737 (10 μM; or DMSO) in the absence of extracellular CaCl₂. After 3 hours of ABT-737, platelets were treated with various concentrations of Ca²⁺ ionophore, A23187, with 2 mM CaCl₂, for 10 minutes. Representative density plots are shown in (B). The number of AnV⁺ EVs detected is shown in (C), and total platelet AnV binding (AnV^med + AnV^high) is shown in (D) (n = 5; **p < 0.01, ***p < 0.001 compared with DMSO-treated platelets). In (E), platelets were treated with ABT-737 for 10 or 180 minutes in the same manner as for (A–D). Subsequent A23187 stimulation (10 μM, 10 minutes) led to cleavage of the calpain substrate, talin, which is unaffected by either ABT-737 or Q-VD-Oph. The blot was stripped and reprobed for CD41, as a loading control, and is representative of 5 independent experiments.

Fig. 5

ABT-737 triggers apoptosis and secondary necrosis. ABT-737 triggers a sequence of responses in platelets that can be distinguished by their sensitivity to different inhibitors. Medium phosphatidylserine (PS) exposure (annexin V [AnV^med] binding) is triggered in a caspase-dependent manner. There is a slow progression to higher PS exposure (AnV^high binding), which is blocked by chelation of intracellular Ca²⁺ by BAPTA. These platelets release AnV-positive (AnV⁺) extracellular vesicles (EVs) in a calpain-independent manner. Slowly, platelets begin to lose plasma membrane integrity, becoming secondary necrotic. This is dependent on calpain. Caspase activation is required for all these responses and downregulates the rapid release of AnV⁺ EVs in response to A23187 and an acute increase in intracellular Ca²⁺. Thus, platelet apoptosis slowly progresses to secondary necrosis if it is not cleared. During the early apoptotic phase, caspases also protect platelets from the effects of prothrombotic stimuli by downregulating the rapid release of prothrombotic EVs.