Page 263 - Haematologica April 2020

Programmed necrosis of platelets in WAS
tors and PS exposure by WAS platelets induced by fibrino- gen attachment) goes so far that the phenomenon of pre- dominant necrosis by platelets with fewer mitochondria was observed here for TRAP-6- or thrombin-stimulated healthy platelet activation as well. This is interesting in itself and might have implications beyond the scope of the present study: although several previous studies attempt- ed to identify properties of platelets that predispose them to procoagulant formation such as age or resting calcium concentration,18,33,39 the effects were much less than those of the number of mitochondria, and it has been generally assumed in the field that it is unclear which platelets become necrotic and which do not.
Although the data of the present study clearly character- ize the immediate molecular cause and sequence of events leading to spontaneous PS exposure in WAS platelets, they are more limited with regard to linking this phenomenon to the genetic cause of the disease or clinical consequences. While a decreased number of mitochondria in the WAS platelets (natural because of their decreased size) is likely to contribute to their tendency to undergo necrosis, this difference by itself is not great enough to have such drastic consequences. Non-stimulated WAS platelets undergo necrosis more efficiently than non-stimulated healthy platelets having the same number of mitochondria (Figure 5), so there should be additional mechanisms. The most promising one is disruption of cytosolic calcium balance (even without regard to mitochondria) simply because of the greater surface-to-volume ratio in WAS platelets: com- puter systems biology simulations indicated that this mechanism alone would be sufficient to explain the
platelets’ necrosis. This hypothesis is supported by the sen- sitivity of the PS+ fraction to xestospongin C and a decrease in extracellular calcium, as well as by the ability of the cal- cium pump inhibitor thapsigargin to promote WAS platelet necrosis much more rapidly than that of healthy donor platelets. The inability of mitochondrial function antago- nists to cause necrosis by itself is also in line with the pro- posed picture of events. However, we did not show a causal relationship between size and calcium in direct experiments, and cannot exclude participation of other contributing factors. Furthermore, although it is tempting to speculate that increased PS exposure may promote platelet clearance by macrophages,10,11 the statistics of the present study are not sufficient to confirm or disprove a relationship between clinical severity and the tendency of platelets to expose PS, despite showing a relationship between platelet count and platelet size. Involvement of impaired actin cytoskeleton dynamics due to WAS protein mutations in the programmed cell death of WAS platelets cannot be excluded either, and requires additional research.
Acknowledgments
We thank Prof. A.V. Mazurov and Prof. R.W. Farndale for their kind gifts of monafram and CRP. The project was supported by a grant from the endowment foundation "Doctors, Innovations, Science for Children", and by the Russian Foundation for Basic Research grants 17-00-00141 (17-00-00138/17-00-00139/17- 00-00140), 17-04-01309 and 18-34-20026. Electron microscopy was supported by the Russian Science Foundation grant 17-15-01290. Computer modeling was supported by Russian Science Foundation grant 17-74-20045.
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