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

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
General comments: 
This is a generally well-written manuscript in which the authors, through a set of cleverly designed 
experiments, demonstrate that the choline transporter Slc44a2 controls platelet activation through 
a mechanism involving mitochondrial production of ATP. This study unravels a new regulator of 
platelet activation that might be eventually used for the modulation of diseases involving an 
abnormal platelet aggregation. To demonstrate their hypothesis, Bennet et al. take advantage of 
the use of a Slc44a2 knockout mice, that has been demonstrated to have impaired platelet 
activation. We highlight the number of different techniques used in the study, from molecular 
biology, to sub-cellular fractioning or mass spectrometry. The order of experiments flows 
reasonably well, allowing the reader to follow the paper easily. Therefore, many of the possible 
questions that arise from a set of results are readily solved in the next figure. However, the 
presentation of the data is poor and somehow confusing, not matching its quality. This is an issue 
that should be improved in the next versions of the manuscript. 
This work provides a well-designed approach for the characterization of a molecular target of 
platelet function beyond the thrombotic pathway, as well as, an endogenous metabolite (ADP) that 
can modulate platelet activation when it is added exogenously. All in all, we do not think that there 
is any major concern that precludes the publication of this article. However, several minor 
concerns should be adequately addressed before its publication. 
 
Minor concerns: 
1. Presentation of data should be improved in a global way. Resolution of figures is low, colors are 
sometimes difficult to distinguish, and doses of some molecules are missed. We encourage the 
authors to improve the quality of figures, facilitating readers to follow them more easily. Also, 
captions should contain more information. In particular, the next points should be addressed: 
- For all the drugs/inhibitors added, the dose should be detailed either in the graph or in the 
caption. This applies at least for figures 2a, 2c, 2f-h, 3-h-j, 4d, 4f-g. 
- Captions information should be enough to minimally understand the experiment. Please, avoid 
descriptions like “Platelet GPIIbIIIa activation” (2d) and similar. 
- Figure 1c→ The WB quality is very low. It seems that you have the spots for the first and third 
condition and a blank hole in the middle. Please, repeat the WB or show a better image. Also, 
explain why HEK293 are used as control in the text or caption. 
- Figure 1d→ There is redundant information (legend and X-axis showing the same information). 
- Figure 1e→ Explain in the text (last paragraph of page2) that you are transplanting bone marrow 
cells between mice. 
- Figure 1f→ Upper panel is impossible to read. Make it bigger. 
- Figure 1g→ Time unit is shown as “sec” when in panel d is shown as seconds. Please unify, or 
even better, show international system notation (s). 
- Figure 1f→ X-axis labeling is not aligned with the bars. Rotate it vertically. 
- Figure 1a→ Impossible to see anything. 
- Figure 2g-h→ Some bars are thicker than others. Very confusing to follow in general. Please, use 
colors to help with the identification of which bar corresponds to which treatment. 
- Figure 3e-f→ If the magnitude measured is supposed to be the same (choline transport), why the 
Y-axis units are different? Please, clarify this. In order to distinguish between the 2 panels, 
indicate that the second one is isolated mitochondria (even when it is expressed in the caption). 
- Figure 3g→ Use different colors for dots that you consider of interest for the project instead of 
using arrows. Show the legend with the color of every metabolite. Make it bigger. 
- Figure 4g→ Use colors for the bars. The second and third one can be barely distinguished. I do 
not know if that grouping is the best way to make your data more impactful. I encourage you to 
figure out another way to make that graph with 3 variables (WT/KO, ADP and Thrombin) easier to 
follow. 
2. A scheme with the proposed working model should be included as new figure 5. Since the data 

presented in this manuscript supports a complex model of platelet activation through a tightly 
regulated mitochondrial function, the inclusion of a figure with the model proposed for Slc44a2 will 
clearly help readers to have a global idea without paying much attention to the results. 
3. In figure 1a, we missed the expression of Slc44a2 in bone marrow cells. Especially, because 
bone marrow cell transplants are carried out in other experiments. Please include it. We would also 
like to have an explanation of why Slc44a2 expression is that high in heart. Could it be because β-
actin is lower in heart? Do you think that using β-actin as housekeeping was the best choice for 
this experiment? 
4. In figure 2c, different agonists are used to stimulate platelets, observing that ADP has an 
impact in KO mice. Have you included any reference that explains why ADP is pivotal for platelet 
activation? Otherwise, it seems that the fact of including ADP was a random choice. 
In the line 10 of page 8, it is stated that “adding exogenous ADP should rescue the platelet 
activation decrease”. In that particular form it seems that the idea of adding ADP arises as a 
consequence of the previous results (which is true in part). But also, it is perfectly known that ADP 
is a key player in platelet activation. Please include this fact at any point of the text. We also 
request the authors to show the effect of an analogous non-hydrolysable molecule of ATP in 
platelet activation, since the rationale to add ADP is the same to add ATP. 
5. In the second line of page 6, please replace “spectrometric” by “spectrometry”. 
6. In the first paragraph of page 6, it is stated that “Mice lacking Slc44a2 have decreased choline 
and its metabolites such as betaine and dimethylglycine”. Observing the figure 3g, it seems that 
betaine change is not statistically significant. With our interpretation of the Y-axis unit, when -
logP<1, the pvalue is greater than 0.1, so betaine is not (by far) statistically significant. Is this 
correct? If so, please modify the text. Actually, I would say that betaine is not changed at all (fold 
change of 0.95 cannot be considered relevant). 
7. In the second paragraph of page 7, FCCP is not introduced. It should be specified why you are 
using this compound for your assay. 
8. In general, the results are powerful, but their discussion is vague and for some points is even 
incomplete. For instance, is it possible to find a connection between choline (and 
phosphatidylcholine phospholipids) metabolism and cardiolipins? Cardiolipins are very important 
lipids in the inner mitochondrial membrane and they have been demonstrated as key molecules for 
the oxidative phosphorylation. Also, patients with anti-cardiolipin antibodies can present 
thrombotic events. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This is an interesting and in general well-preformed study. Overall, the data is of high quality and 
supportive of the overall conclusions. I have a few suggestions the authors might wish to consider. 
The authors state that ‘Taken together, our data suggest that Slc44a2 control mitochondrial 
production of ROS during platelet activation’. This conclusion is based I believe on the data Figure 
2g where the KO platelets do not produce an increase in ROS levels following thrombin 
stimulation. However, I would caution the authors from this strong of a conclusion since the KO 
cells do not respond at all to thrombin (i.e. no P-selectin, fibrinogen, etc.). As such, it is unclear 
whether there is a direct effect of Slc44a2 on ROS levels or a more general effect on thrombin-
mediated activation. Looking at mitochondrial ROS levels in response to ADP might resolve this 
issue since Slc44a2 KO platelets respond to this stimulus. Secondly, while the overall data quality 
is high, I have two issues the authors can hopefully resolve. First, the change in ATP and ADP in 
Fig 3g is not significant while in Figure 4 it is. Is there a reasonable explanation for this? Secondly, 
the response to choline in Fig 3j looks quite modest. Is this significant/reproducible? Finally, the 
data in Figure 3g is of interest and if possible should be shown in a more usable form as a 
Supplemental Table as well as in its current volcano plot. 
 
 
 

Reviewer #3: 
Remarks to the Author: 
In this study entitled, “The choline transporter Slc44a2 controls platelet activation and thrombosis 
by regulating mitochondrial function”, Bennett and colleagues propose the choline transporter 
Slc44a2 as a novel therapeutic target based on the observation that Slc44a2-deficient mice display 
a prolonged time to in vivo thrombus formation. Furthermore, the authors link mitochondrial 
metabolism to platelet activation. 
In general, the data shown in this manuscript is interesting but at the present stage considered 
preliminary. Further, the conclusions are not always fully supported by the presented data. I have 
the follwing major concerns: 
1. An Abstract summarizing the main findings of the manuscript is missing. 
2. A detailed discussion of the results in the context of the current literature is missing. Further, 
the discussion of the clinical relevance of the findings is too short and incomplete. In addition, 
there is no correlation to the mentioned patients’ data. Since the authors refer to patients carrying 
mutations in SLC44A2, a comparison of the human disease manifestations to the described knock-
out mouse would be mandatory. 
3. Since the authors mention a correlation between deep vein thrombosis and variants in 
SLC44A2, exploring the phenotype of Slc44a2-/- mice, especially in VTE experiments is essential. 
4. As shown in supplemental table 1, the full knock-out of Slc44a2 dramatically alters WBC and 
lymphocyte counts, which is not addressed in this manuscript. How do the reduced counts affect 
DVT or in vivo thrombosis? 
5. The performed BMC transfer experiments indicate a role of Slc44a2-deficient hematopoietic cells 
in the regulation of hemostasis. However, this manuscript does not provide evidence for are role of 
platelet-derived Slc44a2 in hemostasis. Were platelet transfusion experiments performed to test 
whether the observed phenotype is platelet-dependent? 
6. The authors show results from in vivo thrombosis models: FeCl3-induced injury of the A. carotis 
and of mesenteric arterioles. Both models use FeCl3 as a chemical agent to induce endothelial 
injury and therefore rely on exposed tissue factor to a great extent. Mechanical injuries rather lead 
to the exposure of collagen with a lower overall contribution of tissue factor. Do Slc44a2 mice 
display a comparable prolongation of thrombus formation in such models? 
7. The here displayed platelet activation and aggregation studies were only performed after 
thrombin stimulation. Do Slc44a2-/- platelets present with similar activation defects when 
stimulated with(hem)ITAM-coupled or other agonists? Were different thrombin concentrations 
tested for the different assays? Can the observed defects be overcome by increasing thrombin 
concentrations? 
8. Were alterations in the granule content observed? How are dense granules and mitochondria 
linked? Are granule numbers also altered? 
9. Statistics: Please clarify your significances: Which groups are compared to each other (e.g. 
Figure 2c: Asterisks on ADP-stimulated samples (WT and KO) – compared to which sample?)