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O.V. Kim et al.
of 3.5 μm2 (Figure 4F). Remarkably, the intensity of F-actin staining decreased in fragments (Figure 4I), so that in many particles that had a cross-sectional area <4 μm2 F- actin staining was not detected at all, suggesting a break- down of the actin network during platelet fragmentation. The high intensity of F-actin staining in thrombin-activat- ed platelets and its extinction in fragments is clearly seen in three-dimensional reconstructions (Figure 4D,E).
Thrombin-induced platelet fragmentation is preceded by Ca2+ influx and is Ca2+-dependent
Because platelet functionality and structural rearrange- ments depend strongly on Ca2+, we correlated thrombin- induced platelet fragmentation with intracellular Ca2+ con- centration using confocal microscopy with a Ca2+-depen- dent fluorophore (Figure 5A,B). The intracellular Ca2+ spiked during the first minutes of thrombin treatment in the presence of extracellular calcium. After spiking, the average platelet calcium content remained constant up to about 30 min, and then decreased greatly, with a strong, inverse correlation with the time course of platelet frag- mentation (r=-0.93, P<0.01). In contrast, in the absence of extracellular Ca2+, the intracellular calcium (released from intracellular depots) increased monotonically 4-fold after 120 min of incubation with thrombin, with no evidence of platelet fragmentation within 2 h (Figure 5C).
Mitochondrial depolarization, ATP depletion, and generation of reactive oxygen species in platelets undergoing thrombin-induced fragmentation
Thrombin induced a time-dependent reduction of the mitochondrial transmembrane potential (DΨm) in platelets. As revealed by time-lapse confocal microscopy, the overall fluorescence intensity of the DΨm-sensitive MitoTracker dye in freshly formed thrombin-initiated PRP-clots dropped 2- and 4-fold after 60 min and 90 min, respective- ly (Figure 6A-D). A similar gradual decrease was observed with another DΨm-sensitive dye, tetramethylrhodamine (data not shown). Remarkably, in activated platelets some mitochondria were translocated towards the platelet periphery, localized within filopodia and either remained inside filopodia-derived vesicles or got released as free mitochondria into the extracellular space (Figure 6E,F). The reducing mitochondrial membrane potential strongly and inversely (r=-0.93, P<0.01) correlated with an increase of the fraction of disintegrated platelets, which reached 55% of the total number of visualized platelets by 90 min (Figure 6G). Remarkably, the initial drop of DΨm was almost concurrent with the beginning of platelet fragmen- tation at about 30 min after thrombin-induced clot forma- tion and platelet activation. This time point also corre- sponded to the termination of contraction of a PRP clot, measured as a decrease by 90% of platelet-generated con- tractile stress (Figure 6H), suggesting that platelet disinte- gration is responsible for the cessation of clot contraction.
To establish whether thrombin treatment disturbs ener- gy metabolism, we performed time-lapse quantification of the ATP content in thrombin-treated platelets using confo- cal microscopy in the presence of an ATP-sensitive fluo- rophore. The intracellular ATP decreased progressively during the 2 h following platelet activation (Figure 6I). Importantly, such steady kinetics is characteristic of the metabolic depletion of ATP rather than ATP secretion, which occurs as a burst within the first seconds or min- utes following platelet activation with thrombin.22
Accordingly, when ATP was measured both in platelet lysates and in activated platelet supernatants during a 3 h incubation of PRP with thrombin, the ATP in the super- natant first increased (over 15 min), reflecting the fast ATP secretion from activated platelets, and then remained con- stant, while the intracellular ATP decreased progressively over time (Figure 6J). These results confirm that the con- tinuing decrease in thrombin-treated platelet ATP is not due to secretion, but results from gradual metabolic exhaustion. The decline of the intracellular ATP level and reduction of DΨm in response to thrombin were concomi- tant with reactive oxygen species (ROS) formation, which occurred at about 30 min after activation (Figure 6K-M). Importantly, the increasing mitochondrial ROS produc- tion showed a strong temporal correlation (r=-0.98, P<0.01) with the platelet disintegration dynamics (Figure 6M).
Lack of caspase activation in platelets undergoing thrombin-induced fragmentation
Previous studies suggested involvement of executor cas- pases in thrombin-induced platelet activation and further dysfunction.23–25 To test whether thrombin-treated platelets underwent a caspase-dependent death pathway, we used time-lapse confocal microscopy of PRP clots and flow cytometry of thrombin-activated isolated platelets pre-incubated with a fluorogenic substrate of caspases 3 and 7. In both experimental settings, there were no signs of caspase activation in response to thrombin stimulation, at least within 1.5 h (Figure 7A-F). Cleavage of the fluoro- genic caspase substrate, as revealed by confocal microscopy, was detected in less than 3% of platelets after 90 min of treatment with thrombin (Figure 7A,C), while treatment for 90 min with calcium ionophore A23187, used as a positive control, led to the activation of caspases 3 and 7 in about one-third of platelets (Figure 7B,C). Flow cytometry also did not reveal any increase of caspase activity in response to thrombin treatment up to 3 h, with less than 0.5% of platelets exhibiting substrate-related flu- orescence, while stimulation of platelets with A23187 led to the activation of caspases in 32% of platelets (Figure 7D,E). It is worth noting that an increase of thrombin con- centration from 1 U/mL to 5 U/mL did not cause caspase activation (data not shown). In addition, no procaspase 3 cleavage was revealed in western blots of platelet lysates obtained from thrombin-treated platelets (Figure 7G). The lack of caspase activation suggests that thrombin-induced platelet dysfunction and fragmentation represent a cas- pase-independent pathway of platelet death.
Calpain activity increases in platelets undergoing thrombin-induced fragmentation
As an alternative to caspases, calpains may also con- tribute to platelet fragmentation by cleaving cytoskeletal proteins such as actin and fodrin.26,27 We evaluated activity of calpains in thrombin-treated platelets incubated with a fluorogenic calpain substrate using both flow cytometry of isolated cells and time-lapse confocal microscopy of platelets in PRP clots. As revealed by flow cytometry, treatment of platelets with thrombin resulted in 1.5- and 2-fold increases of a fluorescent signal of the calpain cleav- age product after 60 min and 180 min incubation, respec- tively, as compared to that in untreated cells (Figure 8A). At the same time, the signal from the calpain cleavage product was 7-fold weaker than that of the strongly
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haematologica | 2019; 104(9)