Dear Dr ***

MS: ***

I write on behalf of the Chairman to nform you that your manuscript on "Title" has been carefully considered for publication in the Joumal. Your study is clearly of interest but for various reasons acceptance of your manuscript has been precluded.

You will see from the enclosed criticisms that a number of important problems have been identified. Havimg taken into account the weight of the objections raised and the amount of additional work that would be required to justify giving further editorial consideration to your manuscript, I do not believe that the difficulties can be satisfactorily resolved by simple revision. I regret to have to inform you that your manuscript has, therefore, not been accepted for publication.

Thank you, nevertheless, for having let us see your work.

Yours sincerely,

Editor's Report

In their manuscript Author et al have studied the effects of thrombin-induced Ca2+-dependent currents in rat megakaryocytes.

Major concerns

1 . It is not unlikely that, the measured current is indeed a calcium-activated K+-current. However, the evidence as stated in the frrst paragraph of the results section is rather weak (BAPTA diminished the current. . .n=2). Unless this has been demonstrated in detail in previous papers, a record of a current voltage relation to the thrombin-induced current would be required.

2. Methods: How was the frequency of the response to thrombin analysed when the intervals between different current transients were not equal? This point must be clarified.

3 . The quality of the English language was unacceptably poor and the manuscript should be corrected by a native speaker.

Referee's Report

ms ***

Author et al

"Title . . . "

The authors study thrombin-induced Ca2+_dependent K+ currents in rat megakaryocytes with the perforated-patch clamp technique. These currents probably represent the intracellular free calcium concentration, although this has not been rigorously tested in these cells. As previously reported, thrombin-induced currents exhibited oscillations. The manuscript deals with the frequency of these oscillations as well as the latency (time from beginning of stimulation to appearance of first current development) and wash-out time ofthe currents. It is disappointing that the study does not go beyond a mere description of the phenomenology. No conclusions are mentioned in the summary; the concluding remark in the discussion is extremely vague. The mechanisms underlying some of the observed phenomena should have been experimentally addressed rather than only discussed.

General:

I do not think that the experimental model is well suited to draw conclusions about the thrombin signalling in platelets. After all, the required concentrations of thrombin are two orders of maginitude larger than in platelets. I doubt that the authors of previous studies on platelets would agree that "the kinetic studies ofthe [thrombin] response were not well performed" (p. 3). The fact that the response to thrombin is delayed in comparison to that to ATP and TRAP is very well established and has frequently been attributed to the time required for cleavage of the thrombin receptor. The authors' explanation for the delayed decline of the response leaves me unconvinced (see below).

Specific:

l . The authors report that there was no difference in washout time between thrombin and TRAP and they mention that washout of thrombin but not of TRAP was modffied by SBTI.

a) Why should it be unexpected that the apparent washout time of TRAP is not modified by SB TI?

b) The data do not support the authors' assertion that the currents after washout of thrombin are caused by thrombin left bound to its receptor. They rather suggest that the washout is simply uncomplete. Some thrombin may also be unspecifically bound to the cell membrane and released later on. Note that a comparison ofthrombin with ATP is not conclusive because ATP may be rapidly degraded such that an incomplete exchange of the bath would not matter. What happens if a stable ATP analog is used? What If the cells are continously superfused?

2. I found it remarkable that the frequency of oscillations induced by InsP3 is independent of extracellular Ca2+, whereas those induced by thrombin are. The authors should carefully study other work on the effect of Ca2+ influx on oscillations of [Ca2+]i, e.g. Girard et al. Science l 993 .

a) What is the Ca2+ concentration ofthe pipette solution that contains only ATP as Ca2+ chelator? What is the range of the actual Ca2+ concentration in the cell during the perfusion experiment?

b) Can we assume that the cytosolic InsP3 concentration is high and constant over the time of the experiment? I assume that Fig. 7b represents experiments in conventional whole-cell mode although the graph on the top left side of the figure would indicate cell-attached experiments. What is the size of the cell and the aceess resistance?

c) If the currents reflect changes in [Ca2+]i and the InsP3 concentration is constant, then the cells seem to regulate [Ca2+]i Without requiring Ca2+ influx. This would be a quite unique finding, rather unexpected in view of the situation in p]atelets. Thus, these experiments deserve more consideration than glven in this manuscript.

d) If the frequency of current oscillations depends on "Ca2+ mobilizing potency" ofthe agonists, why should the potency of thrombin (in the presence of extemal Ca2+) be stronger than that of 30 ｵM InsP3?

e) It should certainly be explicitly tested whether the effect of extracellular Ca2+ is due to altered binding of thrombin to its receptor, altered receptor cleavage, or Ca2+ influx. Note that Ca2+ influx can be inhibited by other means than removal of Ca2+, e.g. changing the membrane potential or anorganic Ca2+ entry blockers such as Gd3+

As it stands, the manuscript only describes phenomeua without an attempt to explain them.

Minor points

l . Title: I do not think that the manuscript provides a pharmacological characterization of the thrombin receptor. It deals with some kinetic parameters of thrombin signalling.

2. The term "fine time resolution" is inadequate.

3. p.5 Iine 12 f. Meaning unclear.

4. p, 6. It is not helpful to mention 2 experiments with BAPTA/AM as proof that the authors are dealing with Ca2+-dependent currents. A reference to a previous study would be better.

5. p. 11 Iine 2 ff. As it stands, the meaning of this sentence is the opposite of what the authors probably want to say.

6. p. 12. Why is the washout time of TRAP discussed under the subheading "Latency"?