Peer review of: The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating (2020)


Reviewers' Comments: 
 
Reviewer #3: 
Remarks to the Author: 
The authors addressed most of the reviewer’s questions and added new data to the manuscript 
strengthening the overall quality. Other than the reviewer`s comments on mitochondrial 
metabolism part, the platelet part of the paper could still be improved – data are displayed 
incorrectly, new data is poorly discussed. There are a lot of careless and small errors. 
 
Comments: 
 
• The authors conclude that KO platelets have a defective ADP/ATP secretion, while on the other 
hand showing that only alpha-granule release is impaired upon stimulation, whereas dense granule 
release is unaltered. ADP/ATP are stored in dense granules. How do the authors explain these 
contradicting findings? 
Figure 1: 
• 1b: Which proteins are shown? Please label the lanes. 
• 1d: y-axis labeling cut of the numbers (1 of 1000s is missing). labeling (WT and KO) is doubled. 
• 1d, e,f: Please include the whole measuring time in your axis: 20 min for tail bleeding time, 30 
min for thrombus formation and indicate non-occluded vessels as dots above the “measuring-
stopped” line. 
• 1f: Thank you for providing dots to display the results with each dot representing one animal. 
Unfortunately, the methods description and the answers to the reviewer comment do not fit with 
the graph. In the methods, an observation period of 30 min is indicated for in vivo thrombus 
formation. Here, only 20 min are depicted (1200 s). Besides this, in the response to the reviewers 
the authors also use the term bleeding time for Fig. 1f. Fig 1f, however, displays in vivo thrombus 
formation in mesenteric arterioles. 
• 1h: Same as for Fig 1f: Please adjust your methods part to 20 min observation period – and not 
30 min (if this is, how it was done). 
• 1f, h: data is still displayed incorrectly: the authors need to distinguish between occluding and 
non-occluding vessels. Accordingly, they also need to analyze significances using either two-tailed 
Student’s t-test (for occluding vessels) or Fisher’s exact test (for non-occluding vessels compared 
to occluding vessels) and shouldn’t display a mean for all data points (it is statistically not correct 
to calculate mean values and SD of non-occluding vessels). 
• Platelet transfusion experiments are new Fig 1l and 1m (not 2l and 2 m, as stated in the rebuttal 
letter) 
• 1d, l: What is the difference between these two panels? It looks the same for me - bleeding time 
of WT and Slc44a2 Ko mice. In the text, the authors conclude that transfusion of WT platelets into 
KO mice partially restores hemostasis, nonetheless they do not show a corresponding statistical 
comparison between Fig. 1l + m. It would therefore be advisable to merge l + m and compare 
mean bleeding times between WT and WT to KO as well as KO and KO to KO. 
• I would like to encourage the authors to change the order of the figures and display all tail 
bleeding graphs after one another. Please also change the order in the text correspondingly. 
Fig. 3: 
• b and c: thank you for including a second measurement for mitochondrial content. It still needs 
to be highlighted that counting mitochondria per EM field is incorrect. In addition, the y axis 
labeling in c is wrong.