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Dysfunction and disintegration of platelets
tional consequences of platelet activation and the survival of these cells are related to fundamental aspects of cell biol- ogy, including death pathways of anucleate cells.
One of the main platelet activators is thrombin, which causes exposure of activated adhesive proteins (integrins aIIbβ3, aνβ3, a2β1, P-selectin, ephrins, etc.), secretion, contrac- tion, and changes in energy metabolism.7–11 Stimulation leads to the platelets changing from a discoid shape to a star-like cell with multiple protrusions, with this morpho- logical change being driven by reorganization of actin, tubulin, spectrin, and filamin.12–16 Another major conse- quence of platelet activation is the release of microvesicles segregated into plasma membrane-derived ectosomes and exosomes, originating from intracellular structures, both of which have important biological functions.17,18
Apoptosis plays an essential role in the survival of platelets in vivo.19 The role of apoptosis in platelet lifespan was discovered as a result of the use of BH3-mimetic anti- cancer drugs, which induce apoptosis in cancer cells, but also cause thrombocytopenia.20 The role of apoptosis in platelet lifespan has been delineated mainly through vari- ous mouse models.19 In contrast, the fate of platelets after activation is still mired in controversy21 and, therefore, deserves further study.
In this study we tested the hypothesis that thrombin- induced platelet activation later results in metabolic exhaus- tion, dysfunction, and breakup of platelets. Using light and electron microscopy combined with flow cytometry and rheometry, we revealed dynamic alterations of platelet morphology and cytoskeletal rearrangements that accom- pany biochemical and biomechanical changes in platelets treated with thrombin. Thrombin causes delayed agonist- specific dose-dependent platelet dysfunction and fragmen- tation associated with reorganization of actin. Thrombin- induced exposure of phosphatidylserine and active integrin aIIbβ3, as well as Ca2+ influx characteristic of initial platelet activation, are followed by mitochondrial depolarization, formation of reactive oxygen species and metabolic ATP depletion concomitant with platelet disintegration and acti- vation of calpain, but not effector caspases, suggesting a cal- pain-dependent pathway of platelet death.
Methods
Blood was collected and processed in accordance with a proto- col approved by the University of Pennsylvania Institutional Review Board and in compliance with the Helsinki Declaration of ethical principles for medical research involving human subjects.
Formation of platelet-rich plasma (PRP) clots, distinct fluores- cent labeling of components of the PRP clots for confocal microscopy, dynamic rheometry of contracting clots, platelet iso- lation, staining of actin and other intracellular structures, scanning and transmission electron microscopy, flow cytometry, measure- ment of ATP and mitochondrial transmembrane potential, caspase and calpain activity, western blot, image analysis and statistical analyses are described in the Online Supplementary Methods.
Results
Thrombin-induced fragmentation of platelets revealed with confocal microscopy and flow cytometry
Confocal microscopy of platelets in fresh, hydrated plas- ma clots formed with thrombin and Ca2+ revealed that, fol-
lowing shape changes characteristic of platelet activation, many platelets and small platelet aggregates fell apart into subcellular particles in a time-dependent manner. One hour after addition of thrombin, a substantial fraction (~25%) of platelets underwent disintegration, revealed as various stages of fragmentation (Figure 1A,B). After 3 h of throm- bin-induced activation, multiple platelet fragments (>90% of platelets) were dispersed within the fibrin network (Figure 1C). Notably, the platelet fragments displayed a bimodal size distribution with two distinct peaks at about 200 nm and 900 nm (Figure 1D), which are much smaller than intact platelets (2-4 μm). The platelet fragmentation observed by microscopy started ~30 min following throm- bin treatment and progressed linearly (Figure 1E). The degree of platelet fragmentation was dose-dependent in the range of 0.1-10 U/mL thrombin (Online Supplementary Figure S1A) and was partially inhibited by rivaroxaban, an inhibitor of factor Xa (Online Supplementary Figure S1B), sug- gesting the combined destructive effect on platelets of added and endogenously generated thrombin. Remarkably, the onset of platelet fragmentation was the same at 1 U/mL and 10 U/mL concentrations of thrombin, suggesting that platelet disintegration is a delayed process that occurs only after activation is completed and platelets enter a new (dys)functional stage. Flow cytometry of thrombin-treated platelets revealed that the number of CD41-positive frag- ments smaller than 1 μm increased 10- to 20-fold after 60 min (Figure 1F). Concomitantly, the number of platelets (gated as CD41-positive particles >1 μm) decreased 2.5- to 3-fold compared to the number of untreated platelets (Figure 1G).
Time-lapse confocal microscopy of platelets in PRP clots revealed that after initial activation, individual platelets and their aggregates attached to fibrin and extended filopodia to pull on fibrin fibers, causing clot contraction that lasted about 30 min. Subsequently, platelets began decomposing into subcellular fragments (Figure 2). There were two types of platelet-derived particles, which differed in size and cel- lular origin. One type separated from the ends of the filopo- dia, while the other type arose from the cell bodies under- going fragmentation. The tips of filopodia formed smaller platelet fragments (Figure 2A), while much larger subcellu- lar fragments parted from the platelet bodies (Figure 2A,B).
Ultrastructural visualization of thrombin-induced platelet fragmentation
To see structural details of thrombin-induced platelet fragmentation, isolated platelets treated with thrombin for 15 and 60 min and control untreated platelets were imaged using transmission and scanning electron microcopy (Figure 3). Resting platelets displayed a typical discoid shape with a smooth plasma membrane and cytoplasm containing mitochondria, glycogen granules, lysosomes, an open canalicular system, and dense and α-granules (Figure 3A,C). In contrast, many platelets treated with thrombin for 15 min broke apart into more or less separated membranous particles, some of which contained inclusions, such as mito- chondria, glycogen granules and vacuoles (Figure 3B, see fragments 1-4). Scanning electron microscopy of thrombin- treated platelets revealed multiple blebs on the surface, like- ly reflecting fragmentation of the cell body (Figure 3E), quite differently from the untreated control (Figure 3G). At the longer incubation time with thrombin (60 min), platelets underwent further disintegration, producing small frag- ments (Figure 3D,F) with size distributions peaking at 430
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