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



Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors have satisfactorily addressed the concerns. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The authors have been very responsive and have completely addressed my previous concerns 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Re-Review on Bennett et al. 
The authors addressed some of the reviewer’s questions and added new data to the manuscript. 
The revised version now comprises a deeper analysis on Scl44a2-mediated mitochondrial 
metabolism, however, several questions still remain and a few more came up regarding the new 
experiments, thus making the manuscript still improper for publication. 
 
Line 69: How were the mice generated? Are they conditional KOs only lacking Slc44a2 in BM cells? 
Fig 1 c: GAPDH line looks fully oversaturated and artificial. 
Fig 1 f: Representative images showing in vivo thrombus formation on FeCl3-injured mesenteric 
arterioles do not fit the quantification. While there is almost no thrombus formation in the KO 
mouse detectable after 20 min in the representative image, the bar graph shows a mean occlusion 
time of 1100 s. How many mice were not able to form an occlusive thrombus (as the images would 
suggest) within the observation period of 30 min? The data may be displayed as dots with one dot 
representing one arteriole/animal. Did the WT mice really occlude after 200 s? If so, why is this 
not evident in the representative images? 
Fig 1 g/h: Similar comment as for Fig 1f: the blood flow chart (esp. for Ko -> Ko and Ko -> WT) 
does not fit to the quantification in h. The standard deviation is very high in h. How many vessels 
did not occlude within the observation period? You can’t display your results in a bar graph if 
several mice did not occlude. 
Fig 1 e-h: Authors did not include platelets transfusion experiments, which would specify whether 
the observed hemostasis and thrombosis defect is due to altered platelet activation or reduced 
WBC counts (almost 50% reduction in KO mice!). 
Fig 1 i: Thank you for including results on DVT in Slc44a2-deficient mice, which are in line with the 
proposed GWAS studies. A discussion of the contribution of immune cells, most importantly 
neutrophils, to DVT is missing, especially with regard to the altered general blood parameters in 
Slc44a2-deficient mice. How is the function of other immune cells altered influencing thrombosis 
and hemostasis? 
The differences of the used venous/arterial thrombosis models is not taken into consideration in 
the discussion of the data: 
- DVT model: venous vascular bed, involvement of lymphocytes (reduces amount in KO mice), 
stenosis model over longer time period (here 6 h, usually 24-48 h), “red thrombus” 
- Arterial thrombosis model: no involvement of neutrophils, no NET formation, primarily composed 
of platelets -> thrombus composition “white thrombus” 
o Mechanical injury not comparable to stenosis, mechanical model missing 
Line 121: Please specifiy in the text: Slc44a2(KO) platelets also have less activation of GPIIbIIIa 
than Slc44a2(WT)122 platelets (Fig. 2g). KO platelets show diminished integrin αIIbβ3 activation 
upon stimulation with which agonists? 
 
Fig 2a-d: Here, platelet activation is measured and not aggregation! A flow cytometric analysis not 

gated on platelet activation markers does not allow any conclusion on platelet aggregation, which 
is typically measured using light transmission or impedance. Besides this, the gating strategy 
remains unclear due to missing labeling of the y-axis in Fig. 2a. Strong platelet activation as 
achieved with the used thrombin concentration in Fig 2b normally results in over 90% double 
positive (Fibrinogen/CD62/CD63) WT-platelets. However, ADP as weak platelet agonist does not 
result in comparable platelet activation as thrombin. 
Fig 2d: Why is the aggregation response towards ADP not altered, ROS production on the other 
hand though seems to be impaired (Fig 4i). Can you then really conclude that reduced ROS 
generation results in impaired activation of platelets? 
Fig 2 e -f: How do you explain the variable resting levels for untreated WT controls? How do you 
explain the different P-selectin exposure upon stimulation with 0.5 u/ml thrombin in WT platelets 
in 2e and 2f – this is a 2x fold difference! 
Fig 2k: It still remains unclear whether reduced ROS production is cause or consequence of altered 
platelet activation. Therefore, also the conclusion in line 138 f. needs to be attenuated. 
Fig 3b: Were mitochondria per platelet counted? It is not possible to display mitochondria per EM 
field since the number of platelets might differ as in Fig 3b. 
Fig 3d: What is exactly displayed on the left blot? Mitochondria? Fractioned mitochondria? 
Platelets? Please specify. 
Fig 3i: difference in OCR at 0 min -> how does this account for differences after stimulation? 
Fig 4 c/d: Taken into account that the overall ADP content is reduced in KO platelets, is the 
normalized release still reduced or is it simply due to reduced amounts of ADP in dense granules? 
Fig 4g: Resting levels of P-selectin exposure are again not comparable at all to the ones shown in 
Fig 2 e-f? How can the results differ that much? 
Fig 4h-i: Similarly to P-selectin exposure, why do the oxidation values in vehicle-treated 
mitochondria differ that much from the ones shown in Fig. 2i? 
Line 276: Conclusion not addressed by experiments. 
 
 
Minor: 
Data presentation overall is still very poor. Differing fonts and font sized, bad labelling, difficult to 
understand legends. 
 
Fig 1e: label is missing 
Fig 2f: y-axes missing from 700 onwards. 
Fig 3i: x-axes labeling: 0 is somewhere, but not at the axis. 
Fig 5: The model is too basic to be considered useful. The effect of ROS is not mentioned at all, 
which would make sense since the authors propose decreased ROS production to account for the 
observed phenotype.