Dear Dr. ****:

Your submitted manuscript entitled "Title" has been reviewed by the editors and two experts in the field. While all readers felt that the manuscript approached an interesting area of investigation, both of the expert Reviewers were strongly of the opinion that the studies could have been and should have been done on purified populations of megakaryocytes. Reviewer A believes that potential platelet contamination could have accounted for some of the observations. Reviewer B had two major concerns, including the fact that there is no clear delineation between what your group has previously published and the new data concerning potassium channel current contained in this manuscript. Furthermore, Reviewer B felt that there was little obvious connection between 5-HT production and platelet production.

Based on the recommendations of the Reviewers, I regret to inform you that your manuscript has been judged unacceptable for publication in the Journal. Based on this decision, we are returning all copies of your manuscript to you along with the detailed comments of each of the two Reviewers.

While I very much regret the adverse editorial decision, I would like to thank you and your colleagues for sending your work to the Journal for our consideration.

Sincerely Yours,

A

Comments:

Introduction: What is meant by 'periodic activation of Ca2+- dependent K+ current'. Explain or define.

Methods: How were megakaryocytes identified? What criteria were used for their selection? There is no description of efforts to remove platelets from the bone marrow suspensions, for the studies of 5-HT release. Was potential platelet contamination of the bone marrow suspensions quantified? The calculation of potential platelet contribution to 5-HT release in the bone marrow suspensions(bottom of page 8) does not rule out significant platelet contribution to 5-HT levels. Why do the authors apparently believe it does?

Results: Some introductory explanation of the relationship and significance of the measurements must be provided either at the beginning of Results or in Methods.

5-HT release: Why do the authors state that thrombin causes megakaryocytes to produce platelets? Provide evidence or cite literature that documents this statement. Indicate how 100% release of 5-HT in bone marrow cell suspensions was determined(page 9).

Table 1: Define Imax and frequency. Specify their units.

Page 10, Morphological changes: How were the megakaryocytes isolated? Page 8 stated that these cells were not isolated from the bone marrow. Specify conditions under which the experiments described in Fig. 4 were performed, either in Methods or as legend to Fig. 4.

Fig. 4: Identify the apparent cells that are adherent to the megakaryocytes In 4C and 4D. Use arrows to illustrate the villi-like surface structures and filopodium described in the text. 4C is referred to twice in the text, but for apparently different conditions. Please correct.

Page 12, Iast para.: What is evidence for statement that 'megakaryocytes underwent self-degeneration which was partially inhibited by activating A-kinase'? Provide data or remove this conclusion.

Fig 1, Iegend: The correct word is 'row'.

Fig 2, Iegend: Since the authors have emphasized the fragility of megakaryocytes, why was a 60 min incubation time used(Panel B) rather than the 5 min used for platelets? Provide exact technique for preparation of washed platelets in Methods section.

References: Topp is mispelled. Remove the word 'The' from J Biol Chem citation.

Major Comments

1. Much methodological detail is missing and required.

2. The Introduction or beginning of Results should briefly review for the reader the physiological relationships between adenylate cyclase, protein kinase C, cAMP, Ca2+ flux, A-kinase, etc.

2. The experimental conditions under which the experiments shown in Fig. 4 were performed are critical and must be described in detail.

3. The authors must more convincingly establish that the platelet contamination of the bone marrow preparations did not account for their observations, or repeat the experiments with populations of enriched megakaryocytes, from which platelets have been removed. This latter experiment would be much superior and is feasible since these cells will not be used for the electrical measurements.

4. The authors must have a colleague who is fluent in English assist them in preparing this paper for an English language journal. The grammatical usage in the current manuscript is not satisfactory.

B

Comments

This manuscript, Title , by Author et al describes experiments investigating calcium currents and serotonin release in ATP, ADP and thrombin treated megakaryocytes. In addition the authors also made observations on the morphology of megakaryocytes treated with these platelet agonists as well as with H8, a protein kinase inhibitor.

This is an interesting manuscript, but there are several problems that need to be addressed by the authors.

General Considerations

l. It appears that the data on the effect of ATP and ADP on potassium channgel currents and the effect of IBMX and forskolin on these currents may have already been adequately discussed in previous publications by the same authors (*** et al, J. Biol. Chem. 268,168-174 and ** et al, J. Physiol. 470,731-749) , yet this is a major part of this current manuscript. The authors should indicate clearly what potassium channel current data in this manuscript provides information that is something not dealt with in earlier publications.

2 . In the 5-HT section of the results (page 8) the authors state that ,,Our aim is to quantitate platelet production by megkaryocyte, but at present, we have no appropriate technique to count produced platelet under thrombin stimulation. Thus we determined released 5-HT instead of platelet. ,, This does not make any sense. There is no reason to believe that there is any connection between 5-HT release from megakaryocytes and platelet formation. Although it does not change the data, it makes one wonder if the experiments that were performed were done so simply because they were technically possible, but not because they were meant to answer any particular question.

3 . Although I am sympathetic to the difficulties of writing in a foreign language this manuscript has an unusually large number of errors in spellinq and grammar. At times it is almost impossible to understand what the authors are trying to say.

SPECIFIC CONSIDERATIONS

l. The authors say that pure megakaryocytes could not be used because they are unresponsive to activating agents after isolation. Previous studies cited by the authors show clearly that isolated megakaryocytes do respond to platelet activators. It is known that the cells sirnply need to be incubated overnight to recover their reponse to stimulators. Therefore these studies could no doubt be done with purified cells.

2 . I do not understand the reason for the very long time periods required to detect 5-HT release. This is particularly a concern for the experiment shown in Figure 3B in which 5-HT release is measured after one hour. I do not know how one can distinguish the affect of the drugs on release and on uptake of 5-HT over such a length of time. It would be necessary to do look at 5-HT uptake under the same conditions.

3 . I would agree that the change in morphology observed with H8 is suggestive of a role of cAMP in the control of morphology. I believe that the result is somewhat speculative in the absence of confirming data, such as measurement of cAMP in the cells.