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Dysfunction and disintegration of platelets
fold decrease in the rate of accumulation of the calpain cleavage product (Figure 8B). The residual calpain activity in thrombin-treated platelets exposed to ALLN was still higher than that in untreated platelets because ALLN did not suppress calpain completely, while in the resting platelets calpain was not activated at all. Importantly, pre- treatment of platelets with ALLN prior to platelet activa- tion caused a significant initial increase in fluorescence of the DΨm-sensitive MitoTracker by about 35% in freshly formed PRP clots (Figure 8C). Furthermore, the time- dependent drop of DΨm was attenuated by ALLN; after 90 min of thrombin-induced platelet activation in the pres- ence of ALLN, DΨm was reduced by only 20%, while without the calpain inhibitor the reduction of DΨm was 65% (P<0.05).
To establish whether active calpain is involved in platelet fragmentation, platelets were observed over time in PRP clots in the absence or presence of ALLN (Figure 8D). Calpain inhibition caused an ~30 min delay in the commencement of thrombin-induced platelet fragmenta- tion, which was followed by an increase in the number of
AB
fragmented cells at about the same rate as in the absence of ALLN (27%/min vs. 28%/min with and without ALLN, respectively), suggesting that calpain is involved in platelet fragmentation during the first 30 min, followed by seem- ingly calpain-independent platelet disintegration. After 90 min of incubation, the fraction of thrombin-activated frag- mented platelets pre-treated with ALLN was 55% smaller than the fraction of disintegrated platelets in the absence of ALLN. Thus, inhibition of calpain partially protects thrombin-treated platelets from a progressive decrease of DΨm and delays fragmentation, suggesting involvement of calpain in thrombin-induced platelet dysfunction and dis- integration.
Discussion
While the early stages of platelet activation have been studied extensively,2,7,10 alterations of platelets occurring at the later stages, after their functions have been fulfilled, are much less well understood. Meanwhile, such late-
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Figure 8. Calpain activation in thrombin-treated platelets and the effects of calpain inhibition on the mitochondrial membrane potential and platelet fragmenta- tion. (A) Flow cytometry-based measurement of calpain activity assessed by the fluorogenic substrate. Isolated platelets were analyzed after 15, 60, and 180 min incubation with thrombin and the calcium ionophore A23187 (positive control) in the presence of 2 mM Ca2+ (mean ± standard deviation, n=6). The data were nor- malized by the values for untreated platelets (negative control) at the corresponding time points (mean ± standard error of mean, n=4). *P<0.05, **P<0.01, ****P<0.0001 (two-tailed Mann-Whitney U test). (B) Confocal microscopy-based time-lapse fluorescence intensity of the calpain cleavage product, reflecting calpain activity or the rate of enzymatic reaction (dashed curves, comprising the first derivative of the experimental plots). Platelets in re-calcified platelet-rich plasma (PRP) were activated with thrombin in the presence or absence of ALLN, a calpain inhibitor, and compared with untreated platelets in PRP (mean ± standard error of mean, n=4). (C) Time-lapse confocal microscopy of the MitoTracker fluorescence intensity, reflecting the platelet mitochondrial transmembrane potential in the presence or absence of ALLN during 90 min incubation with thrombin and Ca2+ at 37°C (mean ± standard error of mean, n=4). (D) Confocal microscopy-based platelet fragmen- tation dynamics in the PRP clots tracked over time in the absence or presence of ALLN (mean ± standard error of mean, n=4). P<0.05 (B-D) for the difference between thrombin-treated platelets and thrombin-treated platelets in the presence of ALLN (two-tailed Mann-Whitney U test).
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