Review Reports
by
- Mahtab Khatibi 1,
- José R. Almeida 1,2 and
- Sakthivel Vaiyapuri 1,*
- et al.
Reviewer 1: Anonymous Reviewer 2: Etsuko Oyama Reviewer 3: Anonymous Reviewer 4: Anonymous
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIntroduction: Note that isolated PLA2 from N. naja venom disaggregated platelets & inhibited collagen & thrombin-induced aggregation of platelet-rich plasma (Dutta et al, Biochimie 2015;110:93-106).
Materials & Methods: was the N nivea venom used in these studies from one snake or pooled, how was it stored, and how was proteolysis of venom proteins prevented?
Results: There is an error in line 97: figure 1C is being described rather than figure 1B (serine protease activity), and line 99 should reference figures 1B & 1D). Line 135: there might be subtle effects on the intrinsic & extrinsic systems not detected by the PT & PTT. Better to note: “does not affect these assays.” Figure 6, parts D,E,F are not mentioned in the legend. If you are trying to illustrate differences in morphologic effects on myoblasts vs platelets, higher magnification might be necessary.
Discussion: The first citation of reference 21 is wrong-N. nigricollis was studied (the second time 21 is cited). The discussion overemphasizes the potential for new therapies and might be more cautious; for example, might 3FTX’s be neurotoxic as well as having effects on platelets?
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis thesis is a standard content. However, the health hazard due to this snake venom snakebite is extensive for the resident in this snake kind habitat region.
Therefore, I think that this thesis is useful very much for the establishment of the treatment method for the patient of this snake venom snakebite.
A little restoration is necessary, and after it restores it, you may accept this thesis.
minor comment
page 8 Figure 4A-B
The amount of the protein added because the amount of fibrinogen is too large, and the image of SDS-PAGE is indistinct is good by about 1/2.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsReference: “Effects of Cape Cobra (Naja nivea) venom and its isolated protein on the modulation of platelet activation” Submittted to TOXINS, March, 2026.
General comments: In the manuscript submitted for publication, the authors are studying the biological activities of Naja nivea snake venom, a major snake involved on accidents throughout the African continent and focusing on coagulation function with an emphasis on platelet function, which, according to the authors, is poorly described in the scientific literature. Throughout the manuscript, the authors describe their experimental analyses of the enzymatic profiles and the effects of the venom on hemostasis using human blood as a model. The results show that the venom increases clotting time in rotational thromboelastometry without affecting other coagulation parameters. The venom also inhibits platelet aggregation and activation, but does not exert cytotoxic effects on platelets. Fractions obtained from the venom by reversed-phase high-performance liquid chromatography identified a toxin (three-finger toxin) described by mass spectrometry. This purified fraction showed an inhibitory effect on platelet activity. According to the authors, these findings show that N. nivea venom can induce hemotoxicity, in addition to neurotoxicity. Furthermore, according to the authors the toxin three-fingered may be promising candidates as biotools for bioprospecting and the development of new antithrombotic agents. After reading the text, it is my opinion that the manuscript is written in a clear and understandable manner, discusses a subject that falls within the scope of TOXINS. However, throughout my comments, I indicate pertinent questions that I would like the authors to answer, in addition to suggesting changes for the revised manuscritp throughout the text that will make it more attractive and complete from a scientific point of view.
Specific Comments:
Introduction
1- Between lines 32 to 34, authors wrote ….Blood clot formation is a vital physiological process where platelets and plasma proteins, specifically fibrinogen, form a fibrin mesh to stop bleeding and maintain the integrity of the haemostatic system during injury. Please include one or more references to this sentence!
2- In the line 55 and throughout of the text. Please change the word molecular weight by molecular mass, as molecules have mass instead weight. Although accepted this term is wrong. The author used during their experiments mass spectrometry, but not weight spectrometry, which does not exist!
3- In the line 73 to 75, … In the Western Cape, clinicians have observed that Cape cobra bites, while less common than other snakebites, result in disproportionately high morbidity and mortality. If possible include references about this phrase!
4- In the line 75 …. The rationale of this study is to…. Please open a new paragraph!
About Material and Methods
5- I felt the authors lacked detail regarding how the venom from the Naja nivea snake used in the experiments was obtained. As it seems that the venom was obtained by purchasing from a reliable supplier; data such as the number of animals used in the collection, the sex of the animals, the date of venom collection, and the processing of the venom to obtain the final product should have been included.
6- About 4.1 enzymatic assays, between lines 474 to 502… The authors use a large number of abbreviations that need to be defined in the first citation in the text. What may be trivial to some one, is not necessarily trivial to others interested in the text. This observation is valid for all the text. MP would be Metalloproteases? SP would be Serine proteases? Please define this and other abbreviations throughout of the text!
7- If the authors are working with samples containing proteins, one would expect that the first methodology defined would be the quantification of proteins in the venoms and other samples. Therefore, I suggest that the authors include the methodology used to quantify proteins!
8- In the lines 476 authors wrote … Several concentrations of venom, also in the line 483 and 484 … with different venom concentrations. This generic writing does not follow scientific rigor, and the authors should specify the concentrations evaluated in the different experiments, so that other interested researchers can reproduce these results!
9- In the lines 484 and 485… Fluorescence was measured at specific time points… The same rule of scientific rigor must be followed. The authors need to indicate the experimentation times!
10- 493 to 494 … The caseinolytic activity was assessed using azocasein as the substrate. Please include details of manufacture of azocasein?
11- Line 502… Absorbance of each well was recorded at 440 nm using a plate reader. Include details of plate reader.
12- Regarding item 4.1, where the authors describe the experiments in which enzymatic activities are evaluated in the tested batches of venom, I suggest that more methodological details be included and, if possible, that the authors separate the different activities tested, describing each evaluation separately. Items 4.1, 4.2, 4.3, ....
13- Lines 503 to 512… 4.2. Human blood collection and preparation of plasma, PRP and washed platelets. Lines 509 and 510 … and then incubated at 30 °C before use. It would be better to replace the word "incubated," which gives the idea of being mixed with something, with "maintained," which gives the idea of being left to rest until it is used!
14- Lines 513 and 514 … To isolate platelets, PRP was combined with 3 mL of acid citrate dextrose (ACD) and 10 μL of prostaglandin I2 (PGI2) (125 μg/mL) dissolved in Ethanol. What is the concentration of ACD? Where is PGI2 from?
15- Line 516… was resuspended in Tyrode’s-HEPES buffer. Please define composition and details of Tyrode’s-HEPES buffer?
16- Line 524 … in accordance with standard methods. Authors could include some reference of standard methods!
17- Lines 532 and 533… to measure the effect of N. nivea venom (50 μg/mL) on various aspects of the whole blood clotting process. It would be interesting if the authors explained why they used only this concentration of venom and not others, and whether it is sufficient to obtain reliable data.
18- Line 544… U46619. What is this? An agonist of platelet agregation? Some definition could be included in the revised text.
19- Lines 556 and 557… samples were fixed with 0.2% (v/v) formyl saline. What is formyl saline? Formaldehyde dissolved in PBS please define!
20- Lines 561 to 571… 4.6. Haemolytic assay. Autros wrote … erythrocytes were resuspended in PBS and treated with various concentrations of N. nivea venom or inhibitors. Please Following the principles of scientific rigor, the authors need to indicate details of the concentrations used.
21- Lines 572 and 573, 4.7. Fibrinogenolytic assay N. nivea venom (100 μg/mL) was added to human fibrinogen at 10 mg/mL. The authors need to indicate the reason for the concentrations used. Sometimes they use 50 μg/mL, other times various concentrations, here 100 μg/mL. An explanation of the choice of concentrations used for several experiments needs to be included in the revised version of the text!
22- Lines 598 to 600 …(RP-HPLC; Spectra-Physics P200) was employed to separate N. nivea venom using a BDS Hypersil C18 column (250 × 599 4.6 mm). Include details of the suppliers and countries of origin.
23- Lines 608 to 610 …4.10. Mass spectrometry analysis a small piece of SDS-PAGE gel with the purified and selected protein fraction was sent to Alta Bioscience (Birmingham, UK) for mass spectrometry analysis . In this part of the text, the authors could explain the objectives of the experiment and the regions of the SDS-PAGE chosen.
24- Since the authors used many reagents throughout their experiments, it would be interesting for them to provide a list of the sources of these reagents, such as trifluoroacetic acid, acetonitrile, Coomassie blue, methanol, and several others.
25- Finally, I noticed the authors lacked a description of the methods used to calculate the statistics of the generated data. That's fundamental!
26- The impression given by the text presented in M/M is that the authors prepared a text in haste, without presenting details that need to be included so that other researchers can reproduce these findings.
Results 27- To characterise the enzymatic profile of N. nivea venom, SVMP, SVSP, PLA2 and caseinolytic assays were performed using DQ gelatin, BAAMC,… What has already been written above regarding the lack of definitions for abbreviations applies to this text and other parts of the manuscript!
28- About figure 1, page 4. In the line 119 introduce the word ….colorimetry before the word chromogenic substrates
29- About figure 1, … The authors could add subtitles to figures 1A, 1B, 1C, 1D, ... nowadays literature values figures that are self-explanatory, without the need for detailed reading of the legend.
30- About figure 1 …Was there any particular reason for the authors to test for cazeinolytic activity in the venom? Especially since they had already tested for metalloproteinase and serine protease activities, and casein doesn't seem to be a natural substrate for the venom.
31- They could have tested more natural substrates present in tissue structures involved in hemorrhagic activity, such as laminin, fibronectin, collagen, entactin, thrombospondin, and other molecules.
32- From what this reviewer can see, the data presented indicate low metalloproteinase activity, absence of serine protease activity, and absence of caseinolytic activity in the venom. Phospholipase activity predominates.
33- Finally, I noticed the lack of a zymogram using gelatin as a substrate in the proteolytic activity assessments of the venom, which would give an idea of the molecular masses of the enzymes present in the venom with proteolytic activity. This experiment could be done in the presence or absence of specific protease inhibitors. Zymography is a gold standard and easy experiment for detecting proteolytic activity in different secretions or extracts.
34- Finally, I would like the authors to indicate the novelties detected in the experiments shown in Figure 1. Since these proteolytic and phospholipase activity data are not already described in the literature for Naja spp. snake venoms?
35- About figure 2, page 6…In Figure 2, the authors present results regarding the influence of the venom on blood coagulation tests. The data are contradictory, as they indicate an absence of alterations in several coagulation parameters, but the presence of alterations in the rotational thromboelastometry experiment. Given the discrepancies, wouldn't it have been better for the authors to test the venom in vivo using animal models, seeking to verify if the alterations seen in vitro are actually sufficient to alter coagulation in vivo? and do they have any significance in the context of envenoming?
36- About figure 3, page 7… If the venom causes inhibition of platelet aggregation, shouldn't in vivo experiments using animal models like mouse show increased bleeding times after treatment with the venom?
37- About figure 3 …If venom components truly have a direct effect on platelets, then rather than using platelet-enriched plasma, the authors should purify platelets and, using washed platelets free of plasma components, test the venom or purified components to verify direct action.
38- About figure 3 … In addition to ADP as a platelet aggregation agonist, have other agonists such as collagen and epinephrine been tested? Where might the mechanism of action be different?
39- About figure 3…Finally, I felt the need for a fluorescence reaction using platelets exposed to the venom and incubated with antivenom antibodies to verify a direct binding of venom components to the platelets (confocal microscopy and cell cytometry for example).
40- Regarding Figure 3, the experiments shown in graphs A and B are not the same thing, only shown in two different ways. Therefore, could one of them be removed?
41- Figure 3… Once again, the suggestion made for the previous figures, to add subtitles to the images so that they are self-explanatory without reading the legends could be followed.
42- The data shown in figures 3 E, F, G, H, I and J are very interesting, as they indicate that higher concentrations of the venom inhibit fibrinogen binding on the platelet surface and selectin exposure, stimulated by 3 different agonists. However, to support the authors' hypothesis that there is altered expression/activation of the αIIbβ3 integrin, RT-qPCR or immunofluorescence reactions with antibodies to activated integrin could be performed.
43- About figure 4, page 8. Please change MW on the left of figures to MMM, as molecules have mass instead weight, as previously discussed. While some less discerning publications accept this terminology, this cannot be the case for TOXINS, which adheres to the strict principles of science.
44- About figure 4, page 8. This is not an unscientific situation, showing the controls in the absence of fibrinogen treatment with venom in a separate figure, but following the classic rule, the controls should be placed side-by-side with the treated samples. Thus, figures A and B should incorporate figure C, with the respective controls at times 0, 1, 3, 6, 9, and 24 hours.
45- Initially, it would be interesting for the authors to repeat the experiments using an SDS-PAGE less concentrated in Acrylamide, such as 10% or even 7.5%, as this could generate gels where the FG chains could be more separated.
46- Also, less substrate (fibrinogen) could be used, since there is an overlap of the bands due to excess protein.
47- The degradation of FG seems clear, but the conclusion that only the Aα chain is degraded does not seem obvious to me, since the degradation product of the Aα chain can colocalize in the location of the Bβ or gamma chains, and the Bβ or gamma chains can also be degraded, originating smaller products not shown in the gels.
48- In this case, a Western blotting reaction using antibodies that specifically recognize the Aα , Bβ or γ chains could clarify the facts.
49- It would be helpful if the authors included indications of the Aα , Bβ or γ chains locations on the negative control gel, so that readers less familiar with FG chains could follow the results more easily.
50- The results of FG degradation would be more compelling if the authors performed a zymogram co-polymerized with fibrinogen and applied the crude venom to identify the protease(s) that degrade FG. With the aid of inhibitors, they could finally confirm the proteolytic nature of these molecules.
51- In the line 245…. 2.6. Purified protein from N. nivea venom was identified as a three-finger toxin. … It seems to me that the identification and purification of the toxin characterized as a three-finger toxin is not new (UniProt accession no. P01456), so the authors could change the subtitle to... The three-finger toxin described in the N. nivea venom is involved in the binding of fibrinogen and the externalization of P-selectin on platelets.
52- About figure 5, page 10 and 11. Regarding the SDS-PAGE shown in Figure 5B, as the authors demonstrate the presence of a protein band in the 10 kDa region, which is at the lower limit of the gel, it is impossible to know if molecules with smaller masses are present in the purified fractions. They should create a gel with a higher concentration of acrylamide, or a linear gradient of 3-20, which is the most suitable in this case!
53- Please as suggested for figure 4, change MW by MMM on the left of gels!
54- In the lines 250 to 252… Subsequent SDS-PAGE analysis of the collected fractions revealed concentrated protein bands around 10 kDa, particularly in fractions 12-21 (Figure 5B). But what about the fractions with higher molecular masses seen in fractions 22 to 25, which have molecular masses more compatible with metalloproteinases? Why the authors not comment on this?
55- In the lines 266 and 267 … Overall, these findings suggest that fraction 12 is a pure cytotoxin or three-finger toxin with a significant inhibitory effect on platelet reactivity. It would be elegant and authors finally would demonstrate this claim, by performing an immunofluorescence reaction using antibodies that recognize this toxin and verify if it binds to the surface of the platelets.
56- In the lines 289 and 290 ….2.7. N. nivea venom and its purified protein are cytotoxic towards AB1190 cells, but not for platelets. I would name the toxin... and its purified three-finger toxin are cytotoxic towards ….
57- About figure 6, page 11 and 12… Regarding Figure 6A, where the authors evaluate the cytotoxicity of crude venom and purified fraction on washed platelets, it would also be beneficial to perform a metabolic assessment assay on mitochondria in platelets, which contain these organelles. This would complement the plasma membrane viability assay performed by LDH. The toxin activities could act on organelles instead of the plasma membrane, making the data more robust.
58- Is there any particular reason why the authors didn't perform the MTS assay to evaluate the cytotoxicity of the venom and purified fraction on platelets? Platelets contain mitochondria, and the assay could be very conclusive!
59- In the legend for Figure 6, the authors do not explain what the experiments described in letters D, E, and F are; this is only explained in the text. In my opinion, the images described in figures 6D, 6E, and 6F could be improved by refining the contrast and brightness, as well as by using larger zoom levels, since it is difficult to visualize the changes indicated. The authors could also add arrows and other symbols pointing out the changes seen!
60- As pointed for other figures, add subtitles to the images so that they are self-explanatory without reading the legends!
61- Throughout some parts of the results, the authors show the presence of proteolytic activity in the venom; in others, they show the presence of the three-finger toxin with activity in platelets and myoblasts. What was missing was the relationship between the two, if such a relationship exists?
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe article raises an interesting topic concerning the effect of cobra venom on platelet function and blood clotting. Considering that the venom of these snakes is mainly studied for its neurotoxic and cytotoxic properties, the results presented in the article offer a fresh perspective. I have no serious comments on the content or concept, but the article contains many shortcomings and ambiguities that, in my opinion, need to be improved. Below is a list of comments:
- I suggest using generally accepted abbreviations for enzymes found in venoms, e.g., SVMP or SVSP. Introducing new abbreviations, especially without explaining them, as was done in section 2.1, makes it very difficult to understand the content.
- Nowhere in the article did I find information on how the positive control from Crotalus atrox venom was prepared. There is no information on the origin of the venom and, above all, the concentration used.
- Please mark on the gels in Figure 4 which bands correspond to which fibrinogen chains. Please also add a mass marker lane.
- The results of the MS analysis are unsatisfactory (as is the lack of a description of the procedure in the methodology; it is not even clear what instrument was used to perform the analysis). In terms of results, I would expect at least scores and sequence coverage percentages. Although Figure 5E shows that 100% sequence coverage was achieved, it is difficult to believe this, given that lysine and arginine appear side by side in the presented sequence and the 58th amino acid is lysine, which means that trypsin cleavage will result in a dipeptide of cysteine and asparagine, which I cannot imagine seeing in the MS spectrum. And if it were visible in the spectrum, it would be ignored by the algorithms searching the databases that identify sequences. Please also correct the text in line 258 and indicate that cytotoxin 1 was identified, not cytotoxin.
- The caption for image 6 does not provide any information about what is in photos D-F, apart from the fact that nothing can be seen in these photos.
- Please standardize the text to clarify whether the object of the study is CapeCobra (1, 78, 354, 368) or Cape cobra (7, 11, 63, 366, 371).
- Section 4.1 lacks information on: substrate concentration, venom concentration range, incubation time, time points
- In section 4.6, please specify the concentration range of venom and inhibitors.
- In section 4.7, please specify the ratio of venom and fibrinogen solution used in the reaction mixture.
- Section 8 describes the preparation of samples for electrophoresis: 2x RSTB was added to 60 µg of venom. Which photos show the gels from this experiment? What kind of electrophoresis required such a large amount of protein? If this description refers to fraction electrophoresis, how were the protein concentrations in the fractions measured? If it refers to the fibrinogen experiment, in what ratio were the venom and fibrinogen mixed so that there were 60 µg of protein in the 30 µl collected?
- There is no description of the preparation of chromatographic fractions for electrophoresis. Were they standardized in any way in terms of concentration?
- The description of the preparation of samples for MS analysis and the analysis itself needs to be significantly detailed.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsAnswer to Authors.
Dear authors, after reading the revised version of the text and the reply letter sent, addressing the questions and suggestions raised by the reviewer, it is my opinion that you were competent in producing a new text and convincing answers. In the reply letter, you were competent and polite, responding to all the items indicated by the reviewer, resulting in a clear and complete document. In the revised text, you made several modifications, making the revised version more complete, with an emphasis on materials and methods, which became much more detailed and comprehensive, facilitating the readers' understanding. In this regard, it is my opinion that this revised version meets TOXINS' quality standards to be accepted and published by the Editorial Board. Congratulations on your effort and hard work.
Author Response
We thank the reviewer for their positive comments and recommending this manuscript for publication.
Reviewer 4 Report
Comments and Suggestions for AuthorsThank you very much for incorporating my comments into the revised version of the article. Unfortunately, however, the issue of the MS analysis has still not been satisfactorily clarified. I understand that this analysis was performed as a commissioned study, but you must obtain the necessary information from that laboratory. First of all, I still do not know what equipment was used, or what the LC and MS parameters were. Information regarding protein reduction and alkylation should be added to the sample preparation procedure, as I find it hard to imagine that this was not done prior to digestion. Speaking of digestion, the description indicates that trypsin was used—that is, only a single enzyme. Therefore, there is no possibility for sequentially overlapping peptides to form. And this, as I mentioned in the previous review round, raises significant doubts regarding the identified protein. You must obtain data from the company specifying exactly which peptides were identified and prove that these peptides are specific to cytotoxin 1. Given that the amino acid sequences of cytotoxins are very similar to one another, MS analysis identifies many peptides that can be attributed to literally dozens of proteins, simply because they are identical. The specific identity of a protein can be determined based on peptides that are characteristic and unique to each cytotoxin. Prove that it is not cytotoxin 5 or 8, which share 90% of the sequence with cytotoxin 1. This can only be done by showing the sequences that were actually detected.
Author Response
We thank the reviewer for this important comment about our mass spec data and we also would like to get this right in the final version of the manuscript.
We have now received further method details from the company which performed the mass spec analysis, and included detailed information in the methods section.
We have also uploaded the excel spreadsheet with all the mass spec data with coverage levels, sequences identified and score information for the reviewer's attention.
As we are not experts in mass spec analysis, we are happy to alter the results section based on the reviewer's further comments after reviewing this data.
We look forward to hearing from the reviewer.