{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T05:05:50Z","timestamp":1780376750817,"version":"3.54.1"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.15602.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2020,7,7]],"date-time":"2020-07-07T00:00:00Z","timestamp":1594080000000}},{"DOI":"10.12688\/wellcomeopenres.15602.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2020,7,7]],"date-time":"2020-07-07T00:00:00Z","timestamp":1594080000000}}],"reference-count":29,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2020,7,7]],"date-time":"2020-07-07T00:00:00Z","timestamp":1594080000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MCPC16052"],"award-info":[{"award-number":["MCPC16052"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000691","name":"Academy of Medical Sciences","doi-asserted-by":"publisher","award":["SGL014\\1012"],"award-info":[{"award-number":["SGL014\\1012"]}],"id":[{"id":"10.13039\/501100000691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["095507"],"award-info":[{"award-number":["095507"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["109698"],"award-info":[{"award-number":["109698"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns4:p>\n                    <ns4:bold>Background:<\/ns4:bold>\n                    Sequestration and cytoadherence of\n                    <ns4:italic>Plasmodium falciparum<\/ns4:italic>\n                    -infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by\n                    <ns4:italic>P. falciparum<\/ns4:italic>\n                    erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation\/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Methods:<\/ns4:bold>\n                    We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with\n                    <ns4:italic>P. falciparum<\/ns4:italic>\n                    material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER). \u00a0A selection of PAR1 inhibitors was tested for their ability to reverse the\n                    <ns4:italic>P. falciparum<\/ns4:italic>\n                    and thrombin induced decrease in barrier function.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Results:<\/ns4:bold>\n                    An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, PAR1 inhibitors did not rescue the barrier dysfunction after co-culture with IE lysate.\n                  <\/ns4:p>\n                  <ns4:p>\n                    <ns4:bold>Conclusions:<\/ns4:bold>\n                    The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism.\u00a0 These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.\n                  <\/ns4:p>","DOI":"10.12688\/wellcomeopenres.15602.3","type":"journal-article","created":{"date-parts":[[2020,7,7]],"date-time":"2020-07-07T11:58:37Z","timestamp":1594123117000},"page":"34","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function"],"prefix":"10.12688","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7812-4220","authenticated-orcid":false,"given":"Janet","family":"Storm","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jill","family":"Davies","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6087-9393","authenticated-orcid":false,"given":"Christopher A.","family":"Moxon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0914-6164","authenticated-orcid":false,"given":"Alister G.","family":"Craig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2020,7,7]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"13142","DOI":"10.1038\/s41598-019-49530-1","article-title":"Interplay of Plasmodium falciparum and thrombin in brain endothelial barrier disruption.","volume":"9","author":"M Avril","year":"2019","journal-title":"Sci Rep."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"4016-4029","DOI":"10.1172\/JCI87306","article-title":"Angiotensin receptors and \u03b2-catenin regulate brain endothelial integrity in malaria.","volume":"126","author":"J Gallego-Delgado","year":"2016","journal-title":"J Clin Invest."},{"key":"ref-3","doi-asserted-by":"publisher","first-page":"1883-1899","DOI":"10.1111\/cmi.12479","article-title":"Diverse functional outcomes of Plasmodium falciparum ligation of EPCR: potential implications for malarial 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Wildhagen","year":"2014","journal-title":"Blood."},{"key":"ref-28","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1186\/s12936-018-2445-8","article-title":"Comparative 1D Blue-Native electrophoresis analysis of Plasmodium falciparum and human proteins associated with cytoadherence.","volume":"17","author":"Y Wu","year":"2018","journal-title":"Malar J."},{"key":"ref-29","doi-asserted-by":"publisher","first-page":"1318-1321","DOI":"10.1038\/nm.2053","article-title":"Extracellular histones are major mediators of death in sepsis.","volume":"15","author":"J Xu","year":"2009","journal-title":"Nat Med."}],"container-title":["Wellcome Open Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,7,15]],"date-time":"2020-07-15T07:00:32Z","timestamp":1594796432000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,7]]},"references-count":29,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.15602.3","relation":{"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.17712.r39456","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.17712.r39456","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,7]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.17089.r37975, Maria Bernabeu, European Molecular Biology Laboratory, Barcelona, Spain, 06 Mar 2020, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v1#referee-response-37975","order":0,"name":"referee-response-37975","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Janet Storm<\/b>; \n<i>Posted: 19 May 2020<\/i>; We thank the reviewer for her constructive comments and have addressed these in our reply and in the manuscript.\n<b>The authors used the same parasitemia and hematocrit for long-term culture or to prepare the IEs lysate and they obtained significantly differences in barrier dysfunction. Did the authors observe merozoite egress after long term IE incubation? <\/b>In similar co-culture experiments (in EC culture medium) the parasites are still healthy looking after 6-7 hours. However, after overnight co-culture they do not develop properly and we hardly ever saw newly formed rings. In using IE, we are specifically looking at the effect of adhesion rather than the release of parasite material.&nbsp;\n<b>Are the molecules released on a mid-trophozoite lysate a good proxy of schizont molecules released after rupture<\/b>\n<i>? <\/i>To mimic the release of schizont molecules, we wanted to use lysate of schizonts and we spent considerable time optimising culture methods to obtain parasites in schizogony. This proved to be rather difficult and therefore we made lysates of trophozoites in a late as possible stage. The cultures were regularly synchronised with sorbitol to keep them synchronous. In the methods section, preparation of IE and lysates has been altered to mid-late trophozoite stage. We did notice that lysates with a higher proportion of schizonts had a greater effect on the cell index, but for the experiments described in this manuscript it was not investigated further.&nbsp;\n<b>Is the concentration used \u201cphysiologically\u201d present in the cerebral malaria microvasculature. Although I agree that the concentration used in this study might be necessary to observe a major barrier breakdown in in vitro cultures, it might not be accurately mimic the pathogenic mechanisms on patients. A sentence in the discussion reflecting the limitations of the model might improve the interpretation of the paper by readers outside the field.<\/b>The reviewer is correct in pointing out that the \n<i>in vitro<\/i> conditions might not mimic the\n<i> in vivo<\/i> situation accurately, but the release of parasite molecules at sequestration sites could result in high local concentrations of these parasites factors. Measurements from patient plasma may only partially reflect these local concentrations, but hopefully new 3D models of cytoadherence may be able to resolve this issue. A comment has been added to the discussion to reflect this.\n<b>Long term incubation with both IE or IE lysate showed 1) reduced surface expression of EPCR and reduced HBMEC cell index. However, the reduced cell index might not be a direct consequence of loss of EPCR in the surface. As the authors accurately describe in the discussion, loss of barrier in their experiments is more likely a consequence of the presence of parasite histones, HRP2 or other soluble mediators. However, the hypothesis exposed in the introduction might be misleading for the average reader. <i>\u201cOur hypothesis is that, through both of these mechanisms, the Thrombin-PAR1 coagulation\/inflammation axis plays a significant role in barrier loss in CM, and that adjunct treatments targeting this might alleviate mortality or post-CM neurological sequelae in CM. Since the activation of PC may be prevented by either of these mechanisms of EPCR disruption (steric inhibition or receptor cleavage) it is important to identify treatments that could be barrier stabilising to EPCR abrogation by either mechanism.\u201d<\/i> Since we don\u2019t understand quite well the molecular mechanisms that induce loss of endothelial integrity and surface EPCR, the authors should be more careful while establishing associations.<\/b>The reason to test the PAR1 inhibitors was to test our initial hypothesis that the Thrombin-PAR1 axis plays a significant role in barrier loss in CM. If true, the PAR1 inhibitors should affect the parasite-induced disruption of barrier function. &nbsp;Our results show that this is not the case and the implications of these data are discussed. We have changed the wording at the start of this paragraph to indicate the context of the hypothesis as a starting point. We agree with the reviewer that we do not understand all the mechanisms that contribute to endothelial dysfunction in severe malaria.&nbsp;&nbsp;\n<b>I would recommend to include both 1) representative traces of cell index and bar plots in figure 3, 4, 5 and 6 in a similar way than in figure 2. Or include it in supplementary data. <\/b>Cell index traces have been added to figure 5 and 6. In figure 5B, the plot shows the effect of Vorapaxar on thrombin induced disruption of barrier function in the absence and presence of lysate. Figure 5A depicts the same traces but over a longer time frame, also showing the effect of Vorapaxar on IE lysate induced decrease in cell index. Figure 6A then shows the effect of all four inhibitors on the lysate induced decrease in cell index.Note that the calculations are also provided in the raw data files \u201cCombined analysis data for fig5_TEER_includes normalisation_2020-02-04.csv\u201d and \u201cCombined analysis data for fig6_TEER_includes normalisation_2020-02-04.csv\u201d.&nbsp;Figure 4 already depicts TEER traces, but a panel (4B) has been added on request from both reviewers. It shows that HBMEC exposed to IE lysate recovers slowly from thrombin induced barrier disruption.&nbsp;Figure 3 shows the EPCR expression data, derived from flow cytometry.&nbsp;\n<b>Was the experiment shown in figure 4 done on independent days?&nbsp;<\/b>Yes, the data shown in figure 4 A, B and C (note that there is an additional panel) are from three different experiments.&nbsp;\n<b>Although TEER measurements are highly variable depending on the day of the experiment, box plots and dot plots might better represent data distribution than bar graphs.<\/b>The bar graphs in figure 5 and 6 are showing mean &plusmn; SD, with relatively low SD values. We do not think depicting it as box plots or dot plots will change the interpretation of the data.\n<b>Avril <i>et al.<\/i>, 2019 showed using the same methodology that schizont-stage parasites (EPCR binding strains) prolonged thrombin-induced barrier disruption in resting and activated HBMEC, and HBMEC do not get back to baseline after IE incubation. Thrombin and IE colocalization is frequent in the microvasculature in cerebral malaria autopsies. As the effects seen in Avril <i>et al.<\/i> are likely to occur in patients this experiment is highly relevant. Is the experiment in Figure 5 equivalent to Avril <i>et al.<\/i>, 2019 but with trophozoites lysates expressing CD36-binding PfEMP1?&nbsp;At which time-scale was the CI measured in figure 5? A representative cell index trace in Figure 5 will clarify that.<\/b>We thank the reviewer for their comment and we observed the same delayed recovery from thrombin treatment after co-culture with IE lysate. We have added a representative cell index trace to show this in figure 5 (A and B) and added text to the results and discussion sections to acknowledge this.&nbsp;A similar cell index trace was also added to figure 4 to show all three observations with HBMEC in one figure:A) Barrier function disruption by thrombin with a fast recovery. This can partly be rescued by the addition of aPC.B) Slow recovery from thrombin treatment after co-culture with IE lysate.C) Disruption of barrier function by IE lysate.\n<b>\u201cHowever, in the IE lysate co-culture system we identified a mechanism that slowly reduces barrier function and which is insensitive to PAR1 inhibitors.\u201dPlease, rephrase to \u201cHowever, PAR1 inhibitors did not rescued the barrier dysfunction after long-term co-culture with IE lysate\u201d. The molecular mechanisms has not been described in this manuscript.<\/b>The reviewer is correct and we have changed this in the abstract.\n<b>Which amount of IE is used in the long-term co-culture in Figures 3, 4 and 5? The authors give the parasitemia, the hematocrit but as the volume is missing, the quantity or concentration of IE cannot be calculated. &nbsp;<\/b>This has been added in the methods, it is 300 &micro;l. The number of IE (or equivalent for lysates) has also been added, as requested by the second reviewer.\n<b>Why did the authors test the rescue after short-term EPCR-PfEMP1 steric inhibition on EA.hy.926 cells (HUVEC) (behavior A) and the long-term parasite effects on HBMEC (behavior B)? Is the rescue seen on Figure 2 also reproducible on HBMEC?<\/b>The initial screening experiments were done with a cell line to simplify the approach, and compounds that were identified from this screen were entered into more complex investigations using primary human brain endothelial cells.&nbsp; We recognise that the screen may miss some details but we used this as a cost-effective way to down-select the number of experiments needed.&nbsp; As others have reported on the steric effects of PfEMP1, we did not replicate this work on HBMEC.&nbsp;\n<b>A visual timeline of the experiments in each figure might improve the clarity. The experiments performed are quite complex and difficult to interpret and to compare with the current figure legends. &nbsp;<\/b>A timeline has been added to figure 2, 4, 5 and 6.\n<b>Kessler et al., 2017 is not well cited in the first paragraph as a review of aPC-PAR1 axis. Is this a typo?<\/b>We had included it to give full coverage but the reviewer is correct and we have removed the reference at this point \u2013 it is cited later.\n<b>In the current manuscript Avril et al., 2019 is only being cited in the introduction along with 5 other papers. The paper should be introduced in the result section <i>\u201cBehaviour B has been reported previously by several investigators using a range of co-culture <\/i><\/b> \n<b><i>systems (<u>Tripathi&nbsp;et al., 2007<\/u>) (<u>Gillrie&nbsp;et al., 2012<\/u>), with either IE or parasite material affecting <\/i><\/b> \n<b><i> barrier integrity.\u201d<\/i><\/b>\n<b> Given the similarities and complementarity between Storm et al., 2020 and Avril et al., 2019, it might be beneficial to discuss it more extensively in the discussion.<\/b>The reference to \n<i>Avril et al<\/i> has been added to the results and discussion section, specifically after adding TEER traces to figure 4 and 5 describing a similar observation with delayed thrombin recovery after IE lysate exposure.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3#referee-comment-4176","order":1,"name":"referee-comment-4176","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.17712.r39456, Ana Rodriguez, Department of Microbiology, New York University, New York, NY, USA, 15 Jul 2020, version 3, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/5-34\/v3#referee-response-39456","order":2,"name":"referee-response-39456","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was funded by a Wellcome Trust Senior Investigator Award (ref: 095507) and a Medical Research Council, Confidence in Concept Award (ref: MCPC16052) to AGC and a Wellcome Trust Seed Award (ref: 109698\/Z\/15\/Z) and an award from the Academy of Medical Sciences (SGL014\\12) to CAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":3,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}