Page 198 - 2019_09-HaematologicaMondo-web

O.V. Kim et al.
stage alterations can be critical for platelet fate and may be important for remodeling and resolution of clots and thrombi. In this work, we tracked delayed structural, bio- chemical, and biomechanical changes in human platelets exposed to thrombin, a potent platelet stimulant in (pro)thrombotic states.
Based on the literature and our own observations,18,28 we hypothesized that after a period of thrombin-induced aug- mented functionality, platelets would become dysfunc- tional and lose their structural integrity. Our present find- ings support this hypothesis and shed light on the mecha- nisms underlying thrombin-induced platelet death. Structurally, a substantial fraction of thrombin-stimulated platelets undergo disintegration into subcellular organelle- containing fragments (Figures 1-3). This effect is specific for thrombin because no fragmentation was induced by collagen or ADP (Online Supplementary Figures S2). Thrombin-induced platelet fragmentation is associated with changes in intracellular Ca2+ concentration and reor- ganization of the platelet cytoskeleton (Figures 4,5). It is also concurrent with cessation of platelet contractility, metabolic ATP depletion, formation of ROS, and mito- chondrial depolarization (Figure 6). Notably, thrombin does not induce detectable activation of the effector cas- pases 3 and 7 in platelets, suggesting a caspase-indepen- dent platelet death pathway (Figure 7).
Thrombin-treated platelets break up into extracellular particles of various sizes, origin and composition. It is tempting to identify the platelet body fragments as exo- somes, but they are distinct in a number of features. First, particles formed during break up of thrombin-stimulated platelets are relatively large and heterogeneous (0.1-1 μm) (Figures 1-3), whereas exosomes are smaller and more uni- form (0.03-0.10 μm).29 Second, the larger platelet fragments are produced by dysfunctional and energetically exhaust- ed platelets, whereas exosomes are generated by metabol- ically active and structurally intact platelets from their endosomal multi-vesicular bodies.29 Therefore, the parti- cles formed during thrombin-induced platelet disintegra- tion comprise a special type of platelet-derived particles looking like “apoptotic cellular bodies” rather than mem- brane-derived microvesicles or secreted exosomes and are likely to be cleared by phagocytes from the blood flow.30 Furthermore, structurally, the changes observed in platelets do not resemble those that occur in necrosis since there is no swelling and no rupture of the cell membrane or that of cellular organelles and platelet fragments all retain intact membrane (Figure 3).
Fragmentation of thrombin-treated platelets is accompa- nied by a dramatic increase in F-actin-related fluorescence intensity and redistribution of F-actin towards the center of platelets with formation of highly fluorescent patches (Figure 4), suggesting polymerization and/or clustering of actin. Remarkably, the smallest platelet fragments do not show F-actin staining, suggesting that actin filaments are depolymerized or destroyed at the late stages of platelet disintegration. The essential role of F-actin dynamics in thrombin-induced platelet fragmentation is confirmed by the complete prevention of platelet disintegration after blocking actin polymerization with cytochalasin D or latrunculin A (Figure 4G,H and Online Supplementary Figure S4). The membrane cytoskeleton formed of microtubules is also involved in platelet disintegration, because inhibition of tubulin dynamics with paclitaxel prevented the break-up of platelets caused by thrombin (not shown).
Platelet disintegration induced by thrombin correlates strongly with energy exhaustion and dysfunction that includes mitochondrial depolarization, a drop in the ATP content, and the loss of contractility, all seen at about 30 min after addition of thrombin (Figure 6). The observed mitochondrial depolarization may be attributed to an increase in cytosolic and mitochondrial Ca2+ upon throm- bin treatment (Figure 5), which can trigger cyclophilin D- dependent mechanisms of the mitochondrial potential collapse.31 This decrease in DΨm was shown to be associat- ed with generation of ROS (Figure 6), which can damage intracellular structures and could cause platelet death.32,33 Remarkably, some mitochondria in activated platelets localize in filopodia and may be released in the extracellu- lar milieu. Although the mechanism of mitochondria translocation toward the tips of platelet filopodia is not clear, ROS-dependent actin polymerization and mito- chondria-bound myosin were shown to participate in intracellular mitochondria transport.34,35 This mechanism may also be important to translocate mitochondria to sites of high ATP utilization, such as contracting filopodia.
The metabolic ATP depletion in platelets may be due to mitochondrial depolarization as well as to impaired gly- colysis, both important sources of ATP in activated platelets.36–38 The insufficiency of ATP is aggravated by its consumption due to energy-demanding platelet functions, such as contractility. Irrespective of the mechanism of the decrease of ATP content, it is a signature of energy exhaus- tion and impaired platelet functionality. Not surprisingly, the significant (~54%) decrease of ATP content in platelets after 30 min of thrombin treatment coincided with the ter- mination of platelet-driven clot contraction that depends on the activity of non-muscle myosin II, which is a major part of the ATP-dependent-cell contractile machinery.39 The inhibitory effect of blebbistatin on platelet fragmenta- tion (not shown) implies that the contractile myosin IIa- actin complex contributes to platelet disintegration, per- haps facilitating mechanical disconnection or dislodging of fragments. It is not clear whether the revealed biochemical and biomechanical alterations associated with platelet thrombin-induced disintegration provide the conditions for the structural decomposition of platelets or comprise its consequences. However, it is evident that the magni- tude and kinetics of the observed late-stage functional and metabolic alterations following thrombin treatment of platelets are distinct from the non-specific changes related to platelet aging or “storage lesions” (see the Online Supplementary Results section).
Neither time-lapse confocal microscopy, nor flow cytometry nor western blotting detected procaspase 3/7 activation in platelets in response to thrombin stimulation (Figure 7), indicating that the thrombin-induced platelet death pathway is caspase-independent and is likely non- apoptotic, which is in agreement with previous results40 that demonstrated no caspase-3 activity in platelets treat- ed with thrombin. Alternatively, our data suggest the involvement of Ca2+-dependent protease calpain in platelet disintegration, perhaps due to calpain-catalyzed cleavage of cytoskeletal proteins.41 Calpain may compensate for the lack of active caspases by cleaving caspase substrates such as gelsolin, protein kinase C-δ and fodrin. It can also cleave other cytosolic proteins and many important regu- lators of apoptosis, including the anti-apoptotic XIAP and Bcl-xL, thereby recapitulating apoptotic events in activat- ed platelets.26,42–44 For most cells, changes in the nucleus are
1876
haematologica | 2019; 104(9)