Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase

Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase Yingting Huang, Liqin Ling, Shanshan He, Chaonan Liu, Xunbei Huang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6568753/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Inflammation → Version 1 posted 7 You are reading this latest preprint version Abstract Background : P-selectin (P-sel) on activated platelets plays a vital role in inflammation by mediating platelet-neutrophil interactions. However, P-sel surface expression on activated platelets is temporary, with rapid shedding into circulation as soluble P-sel (sP-sel). It is suggested that neutrophils adhesion to P-sel on activated platelets can induce P-sel shedding, however, the exact mechanism remains unclear. This study is to investigate the role of neutrophils in platelet P-sel shedding. Methods : Platelets were activated by thrombin and then incubated with whole blood, peripheral blood mononuclear cells (PBMCs), or neutrophils, with or without neutrophil elastase (NE) inhibitors, myeloperoxidase (MPO) inhibitors, or latrunculin B(LatB). Samples were collected at 0, 1, 2, and 4 hours. P-sel expression on platelets was assessed by flow cytometry and chemiluminescence immunoassay, while sP-sel was quantified by ELISA. Results : When incubated with neutrophils, P-sel expression on activated platelets was reduced (sP-sel was increased) in a time-dependent manner (P-sel-positive platelets, 70.0%→11.1%; fold decrease in CD62P/CD61, 0→0.61; fold increase in sP-sel, 0→6.94), unlike PBMCs. NE inhibitors preincubation with neutrophils could partly reverse the neutrophil-induced P-sel shedding (P-sel-positive platelets, 70.2%→43.4%; fold decrease in CD62P/CD61, 0→0.16; fold increase in sP-sel, 0→4.38), while MPO inhibitors couldn’t. The addition of LatB could also partly reversed the neutrophil-induced P-sel shedding (fold decrease in CD62P/CD61, 0→0.36; fold increase in sP-sel, 0→5.34). Conclusions : The interaction between platelets and neutrophils is mutual. Platelets promote the activation of neutrophils; and in turn, neutrophils induce P-sel shedding via neutrophil elastase, resulting in the irreversible functional downregulation of platelet P-sel-mediated interactions. Platelets neutrophil P-selectin neutrophil elastase Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction P-selectin, a member of calcium‐dependent lectins (glycoproteins) family, is produced in megakaryocytes and endothelial cells[ 1 ].It is stored in the α-granules of platelets and the Weibel‐Palade bodies of endothelial cells when the cells are at resting state. When the cells are activated, P-sel rapidly translocates to the cell surface, forming membrane P-sel[ 2 ]. In addition to serving as a marker of platelet activation, platelet P-sel plays a critical role in leukocyte recruitment and function by mediating their adherence and transmigration to sites of inflammation[ 3 ]. Dysregulated P-sel expression is associated with pathological inflammation and thrombosis in conditions like atherosclerosis, stroke, myocardial infarction, and deep vein thrombosis[ 4 ]. Similar to other platelet receptors (such as GPVI, GPIbα, and GPV), P-sel is rapidly shed from the platelet surface into the plasma shortly after activation, becoming soluble P-sel (sP-sel)[ 5 – 7 ].Unlike P-sel on the membrane surface, which forms dimers or oligomers to enhance leukocyte binding, sP-sel exists as a monomer. sP-sel is unable to effectively interact with its ligands on leukocytes[ 4 ], thus, the overall role of sP-sel in immune responses and hemostasis is not prominent[ 1 ]. From an evolutionary perspective, the shedding of P-sel from activated platelets represents a negative feedback mechanism that regulates platelet P-sel-mediated inflammation. The precise mechanisms underlying P-sel shedding remain poorly understood. Our study aims to explore the mechanisms responsible for P-sel shedding and its role in modulating platelet-mediated inflammatory responses. Materials and methods Blood collection The platelets, leukocytes and red blood cells (RBCs) used in this study were obtained from healthy adult volunteers. Blood was collected into polypropylene tubes containing 0.109 M sodium citrate as an anticoagulant. The samples were processed and isolated in the department of laboratory medicine at West China Hospital, Sichuan University. All procedures were approved by the Ethics Committee of West China Hospital, Sichuan University, and informed consent was obtained from all volunteers. Preparation of platelets, leukocytes, and whole blood Platelet-rich plasma (PRP) was prepared by centrifugation at 200g for 5 minutes. The resulting pellet was washed and resuspended in phosphate buffer saline (PBS). For activation, platelets were treated with thrombin (10602400001, Roche, 1U/mL) for 15 minutes at 37°C (P-sel positive platelet was around 70-80% as evidenced by flow cytometry). Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from whole blood using Polymorphprep™ (Serumwerk, Germany) through density gradient centrifugation at 500 × g for 30 minutes at room temperature with slow deceleration. After centrifugation, the layer above Polymorphprep™ contained only peripheral blood mononuclear cells (PBMCs), while the lower layer contained polymorphonuclear leukocytes (mainly neutrophils). Both PBMCs and neutrophils had a purity greater than 90%. Platelets were removed by centrifugation at 200g for 5 minutes for whole blood preparation. All procedures were at room temperature. Activated platelet co-incubation experiments Platelets isolated from healthy human peripheral blood were activated and incubated with whole blood, PBMCs, or neutrophils under conditions with or without neutrophil elastase (NE) inhibitors, (HY-17443, Sivelastat, 1 mM; Med Chem Express, USA), myeloperoxidase (MPO) inhibitors (475944-1GM, ABAH, 2 mM; EMD Millipore, USA), and Latrunculin B (LatB) (L5288, 5 μM; Calbiochem, Merck Millipore, Germany) at 37°C for 4 hours. Before co-incubating activated platelets with neutrophils, the experimental group received 1 hour of NE inhibitors, MPO inhibitors or LatB pre-treatment, while the control group was not. Samples were taken at four time points: 0, 1, 2, and 4 hours of incubation. After centrifugation, the supernatant was collected, and the cell pellets were fixed with 4% paraformaldehyde. Detection of P-sel on the platelet surface Flow cytometry detection of P-sel on platelets The cell pellets were blocked with 2% Bovine Serum Albumin (BSA) and then stained with CD42b (303934, Brilliant Violet 510 TM anti-human CD42b, Biolegend, USA) and CD62P antibodies (304905, PE-anti-human CD62P, Biolegend, USA) for 1 hour at room temperature, protected from light. After washing with PBS buffer, the cells were analyzed using a flow cytometer (BD FACSCanto II, BD Biosciences). At least 50,000 events were analyzed for each sample. Data were analyzed using FlowJo software (FlowJo, LLC) to determine the percentage of CD62P positive platelet (%). Chemiluminescence immunoassay detection of P-sel on platelets The expression of platelet P-sel was detected in real-time using a homogeneous chemiluminescence immunoassay instrument (HSCL-5000, Poclight Biotech, China). After sampling at the corresponding time points, the samples were centrifuged and washed with PBS to obtain the cell pellets. Homogeneous chemiluminescence immunoassay is a two-site (“sandwich”) chemiluminescence immunoassay based on chemiluminescence (CL) resonance energy transfer (CRET) technology. The acridinium ester (AE) emits a strong CL signal in the presence of H₂O₂. Graphene oxide (GO) serves as an efficient quencher, creating a “signal off” state via CRET between GO and AE-labelled DNA3. Upon sample application, DNA1-coated CD61 antibody, DNA2-coated CD61 antibody, and AE-labelled DNA3 form a sandwich complex, detaching DNA3 from GO and inhibiting CRET. This produces a CD61-CL signal, mimicking quantitative platelet count detection. For P-sel expression on platelets, the same principle is applied using DNA1-coated CD62P antibody and DNA2-coated CD41 antibody. The sandwich complex forms with CD62P-positive platelets, detaching AE-labelled DNA3 from GO and generating a CD62P-CL signal, mimicking quantitative P-sel positive platelet count detection. The CL intensity ratio of CD62P to CD61 is used to represent the P-sel-positive intensity. Due to methodological limitations, chemiluminescence immunoassay could not be used to analyze whole blood samples. Detection of sP-sel sP-sel in the supernatants was determined using an ELISA kit (Boster Bio, China) according to the manufacturer’s instructions. The absorbance was measured at a wavelength of 450 nm using a microplate reader. Statistics Data were analyzed using GraphPad Prism 9.0. The normality of the data was assessed using the Shapiro-Wilk test. For normally distributed data, results are presented as mean ± SEM. A two-way ANOVA was conducted to evaluate differences among groups across various time points (0, 1, 2, and 4 hours). Tukey’s multiple comparisons test was used to analyze differences between groups at each time point, as well as changes from 0 hour to 4 hours measurements within each group. For the groups involving co-incubation of activated platelets with neutrophils and LatB, where other variables were controlled in the control group, one-way ANOVA and paired t-tests were performed to analyze P-sel expression and sP-sel concentration. Statistical significance was defined as P < 0.05. Results P-sel shedding from activated platelets occurred in whole blood but not in buffer After 1h incubation in whole blood, the percentage of P-sel positive platelets already decreased to a very low level (0h→1h, 79.5%→9.7%), while the percentage of P-sel positive platelets incubated in buffer did not change significantly over a period of 4 hours (0h→4h, 74.7%→68.9%) (Fig.1a). Correspondingly, an increase in sP-sel fold change was already evident at 1h incubation in whole blood (6.77 fold increase), while there was only a slight but nonsignificant increase in sP-sel over a period of 4 hours in buffer (1.40 fold increase) (Fig.1b). At 4h, compared to the group where platelets incubated in buffer, the two parameters were statistically different in the group where platelets incubated in whole blood (Fig.1c-d). Neutrophils were involved in the shedding of platelet P-sel After 1h incubation with neutrophils, the percentage of P-sel positive platelets already decreased to a very low level (0h→1h, 70.0%→24.3%), while the percentage of P-sel positive platelets incubated with PBMCs did not significantly change until 4h incubation (0h→4h, 69.4%→56.4%) (Fig.2a). Similarly, the fold decrease in CD62P /CD61 ratio was already evident at 1h when incubated with neutrophils (0.28 fold decrease), but not evident until 2h when incubated with PBMC (0.28 fold decrease) (Fig.2b). Correspondingly, the fold increase in sP-sel was already evident at 1h when incubated with neutrophils (2.89 fold increase), but not evident until 2h when incubated with PBMCs (2.51 fold increase) (Fig.2c). At 4h, compared to the group where platelets incubated with buffer or PBMCs, all the above-mentioned parameters were statistically different in the group where platelets incubated with neutrophils (Fig.2d-f). Role of neutrophil elastase in platelets P-sel shedding The addition of NE inhibitors partly reversed neutrophils-induce P-sel shedding, the percentage of P-sel positive platelets did not significantly change until 2h incubation (0h→2h, 70.2%→46.4%); while it was already evident at 1h when incubated with neutrophils alone or in the presence of MPO inhibitors (0h→1h, 70.0%→24.3% or 73.0%→11.2%) (Fig.3a). Similarly, the fold decrease in CD62P /CD61 ratio was not evident until 4h when incubated with neutrophils in the presence of NE inhibitors (0.16 fold decrease), while it was already evident at 1h when incubated with neutrophils alone or in the presence of MPO inhibitors (0.28 fold decrease or 0.74 fold decrease) (Fig.3b). Correspondingly, the fold increase in sP-sel was not evident until 4h when incubated with neutrophils in the presence of NE inhibitors (3.62 fold increase), while it was already evident at 2h when incubated with neutrophils alone (4.18 fold increase) (Fig.3c). At 4h, compared to the group where platelets incubated with neutrophils alone or in the presence of MPO inhibitors, all the above-mentioned parameters were statistically different in the group where platelets incubated with neutrophils in the presence of NE inhibitors (Fig.3d-f); compared to the group where platelets incubated with neutrophils alone, fold decrease in CD62P /CD61 ratio and fold increase in sP-sel were different in the group where platelets incubated with neutrophils in the presence of MPO inhibitors (Fig.3d-f). LatB treatment partly reversed neutrophil-induced platelets P-sel shedding LatB treatment did not affect NE release from neutrophils (Fig.4a), but compared to the group where platelets incubated with neutrophils alone, fold decrease in CD62P /CD61 ratio (0.36 v.s. 0.59) and fold increase in sP-sel level (2.96 v.s. 5.34) were different in the group where platelets incubated with neutrophils in the presence of LatB (Fig.4b-c). Discussion Due to the role of P-sel in mediating platelet-neutrophil interaction, understanding the mechanism of platelets P-sel shedding is essential for studying platelet-mediated inflammatory responses and identifying new biomarkers for thromboinflammation. In this study, we demonstrated that neutrophils promote P-sel shedding from activated platelets, evidenced by decreased P-sel positive platelet rate, decreased CD62P /CD61 ratio and increased sP-sel concentration. This finding is supported by another notable study which highlights that the presence of P-sel glycoprotein ligand-1 (PSGL-1), primarily expressed on neutrophils, is essential for P-sel shedding [ 7 ]. Specifically, P-sel was retained longer on activated platelets in PSGL-1 −/− mice compared to wild-type mice[ 7 ]. Considering the important role of neutrophils in platelet P-sel shedding, we further investigated the specific mechanisms underlying this process. We found that NE is one of the key molecules driving this process, as NE inhibitors could partly reverse the neutrophil-induced platelet P-sel shedding. It has been suggested that purified P-sel serves as a substrate for neutrophil elastase[ 8 ]. Moreover, we found that inhibiting the formation of neutrophil extracellular traps (NETs) by LatB (inhibiting actin polymerization) without reducing protease release[ 9 ], could also partly reverse the neutrophil-induced platelet P-sel shedding, suggesting that localized high concentrations of NE in NETs might be important for NE to induce platelet P-sel shedding. Additionally, we found that MPO inhibition can reinforce neutrophil-induced platelet P-sel shedding. This effect can be explained by established studies showing that the MPO-H₂O₂-Cl⁻ system potently inhibits NE activity through HOCl-mediated oxidative modification, as evidenced by both in vivo animal experiments and in vitro cellular studies[ 10 – 11 ]. Together, these data establish MPO as a critical regulator of platelet P-sel shedding through its modulation of NE activity. Interestingly, platelet P-sel plays a vital role in NETs formation. Activated platelets stimulate neutrophil activation through P-sel-PSGL-1 interaction, therefore inducing NET formation, leading to the release of functional molecules[ 12 ]. Combining with our finding that concentrated NE in NETs might play a role in platelet P-sel shedding, we can conclude that platelet P-sel shedding is related to a negative feedback mechanism balancing pro-thromboinflammatory and anti-thromboinflammatory effects within the body. Specifically, the interaction between platelets and neutrophils is mutual. Platelets promote the activation of neutrophils; and in turn, neutrophils induce P-sel shedding from activated platelets via neutrophil elastase, resulting in the irreversible functional downregulation of platelet P-sel-mediated interactions, including platelet-leukocyte interaction, platelet-endothelium interaction, therefore limiting excessive thromboinflammatory response. So far, most existing studies focus on how platelets affect neutrophils, whereas our study is the first to propose a reverse effect of neutrophils on platelets. This novel finding offers a new insight into the complicated interplay between platelets and neutrophils, and provides a fresh perspective on the mechanisms underlying platelet P-sel shedding. Currently, this study examines the mechanism under normal conditions only, but the process of platelet P-sel shedding may differ in various disease states, such as sepsis or cardiovascular diseases. Future research could use animal models to investigate how P-sel shedding changes in different disease contexts and its prognostic implications. Additionally, further exploration is needed to understand how locally high concentrations of NE mediate P-sel shedding through specific signaling pathways and whether other cytokines or receptors also play a role in this process. Furthermore, once P-sel sheds from activated platelets, it enters the bloodstream as soluble sP-sel. In recent years, the study of sP-sel has become a focus in areas like inflammation, thrombosis, and cardiovascular diseases, such as coronary artery disease, hypertension, and atrial fibrillation [ 13 ]. Some of them suggest that elevated sP-sel is linked to the activity and/or severity of these diseases[ 13 ]; some of them suggest not[ 4 ]. Our data provide a unifying explanation for the above-mentioned paradoxical clinical observations regarding sP-sel. Since sP-sel is a result of platelet activation (pro-thromboinflammation) and neutrophil-mediated platelet P-sel shedding (anti-thromboinflammation), it is inappropriate to relate sP-sel alone to the activity and/or severity of the diseases, besides, endothelial-derived sP-sel can further complicate the interpretation. Considering the interaction of platelets and neutrophils is mutual, it might be more appropriate to interpret sP-sel with other molecules related to platelet-neutrophil interactions, nevertheless, further studied are still needed. Conclusions Here we demonstrate a mutual interaction between platelets and neutrophils: while platelets potently activate neutrophils, activated neutrophils in turn shed platelet surface P-sel through NE. This novel finding offers a new insight into the complicated interplay between platelets and neutrophils, and provides a fresh perspective on the mechanisms underlying platelet P-sel shedding. Declarations Funding This work was supported by grants from National Natural Science Foundation of China (82302601 to LL). Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Authors Contributions All authors contributed to the study conception and design. Conceptualization: YH, LL. Data curation: LL, YH. Formal analysis: SH, CL. Investigation: LL, SH, YH. Methodology: YH, JZ. Project administration: ZY, SW. Resources: LL, CL. Supervision: YH, LL, SH. Validation: YH, LL, JZ. Visualization: YH, SW, XH. Writing–original draft: YH, JZ. Writing–review & editing: YH, LL. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ethics Approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of West China Hospital, Sichuan University (ID：2024-215). Consent to Participate Informed consent was obtained from all individual participants included in the study. Consent to publish Not applicable. Availability of data and materials All data are included in this article. 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Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Inflammation → Version 1 posted Editorial decision: Revision requested 19 May, 2025 Reviews received at journal 19 May, 2025 Reviewers agreed at journal 10 May, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 30 Apr, 2025 Submission checks completed at journal 30 Apr, 2025 First submitted to journal 30 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6568753","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453618435,"identity":"01ab8ce4-a8e9-45ac-b0be-9c83e440847a","order_by":0,"name":"Yingting Huang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yingting","middleName":"","lastName":"Huang","suffix":""},{"id":453618437,"identity":"231fd932-47b3-4c0a-b548-0e72c3de32c7","order_by":1,"name":"Liqin Ling","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Liqin","middleName":"","lastName":"Ling","suffix":""},{"id":453618438,"identity":"817b1cb0-8095-49b0-9fac-1725a744ef94","order_by":2,"name":"Shanshan He","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"He","suffix":""},{"id":453618439,"identity":"8a895a50-50d0-46d3-a0ed-169c84112798","order_by":3,"name":"Chaonan Liu","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Chaonan","middleName":"","lastName":"Liu","suffix":""},{"id":453618440,"identity":"04339f9e-0a91-423f-94ee-9974249f4313","order_by":4,"name":"Xunbei Huang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xunbei","middleName":"","lastName":"Huang","suffix":""},{"id":453618441,"identity":"5e8b4594-9699-4c3c-9bf8-560d1bbde7f9","order_by":5,"name":"Shuang Wang","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Wang","suffix":""},{"id":453618442,"identity":"c628131a-6824-4f04-9ae1-763320b48d4b","order_by":6,"name":"Zhiyu Yu","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyu","middleName":"","lastName":"Yu","suffix":""},{"id":453618443,"identity":"5d95cbfd-0cc4-487a-81fb-ae983f752bab","order_by":7,"name":"Jing Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCRA2YGAwAHESKiTk5EnUcsbC2LCBGC0gANbC2FaRyHCAgA752c3HJCwK7thtl0h+JvFwnkQCYwPzw0c38GhhnHMsTULC4FnyzhlpxgaJ2yTy2BnYjI1z8GhhlsgxA2o5nGxwI8HwAVBLMWMDD5s0Pi1sCC3pHw4kzpFIbDhAQAsPVIudwY0coC0NRGiRkEhLtgBqSTA486bYIOGYhLFhMwG/yM9IPnhb4s9he4Pj6dskf9TUycmzNz98jE8LCDAD4yaxAcEloBwEGD8wMNgToW4UjIJRMApGKgAAsYJIaS+mRCAAAAAASUVORK5CYII=","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-05-01 03:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6568753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6568753/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10753-025-02350-0","type":"published","date":"2025-07-28T16:39:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82360796,"identity":"94c83c62-bb3c-47ea-adad-61d4e49509a6","added_by":"auto","created_at":"2025-05-09 11:39:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":664249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated platelets incubated with whole blood or buffer\u003c/strong\u003e When activated platelets were incubated with buffer or whole blood, (a, c) percentage of P-sel-positive platelets (n=5); (b, d) fold increase in sP-sel levels (n=5). Data represent mean ± SEM. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6568753/v1/406637a9772335cc8adc1d85.png"},{"id":82361203,"identity":"0a76a501-8f48-46fe-ac78-79c30fb6c4f5","added_by":"auto","created_at":"2025-05-09 11:47:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":888373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated platelets incubated with neutrophils or PBMCs \u003c/strong\u003eWhen activated platelets were incubated with buffer, PBMCs or neutrophils, (a, d) percentage of P-sel-positive platelets (n=5); (b, e) fold decrease in CD62P/CD61ratio (n=5); (c, f) fold increase in sP-sel level (n=5). Data represent mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6568753/v1/546945dbe14801db5a2385f8.png"},{"id":82360797,"identity":"bd52668e-8d42-4792-a335-f64753782d0e","added_by":"auto","created_at":"2025-05-09 11:39:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":852426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated platelets incubated with neutrophils alone or in the presence of NE or MPO inhibitors\u003c/strong\u003e When activated platelets were incubated with neutrophils alone or in the presence of NE or MPO inhibitors, (a, d) percentage of P-sel-positive platelets (n=5); (b, f) fold decrease in CD62P/CD61ratio (n=5); (c, f) fold increase in sP-sel level (n=5). Data represent mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6568753/v1/ba64127b72ec007aa212a044.png"},{"id":82360799,"identity":"a2662934-f72c-4324-a287-d214310bd0fd","added_by":"auto","created_at":"2025-05-09 11:39:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":439170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of LatB on NE release and neutriphils-induced P-sel shedding \u003c/strong\u003eActivated platelets were incubated with neutrophils with or without LatB under identical conditions (cell source, quantity, and treatment duration), (a) NE concentration in supernatants (n=5); (b) fold decrease in CD62P/CD61ratio (n=6); (c)fold increase in sP-sel level (n=5). Data represent mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6568753/v1/999088f7dd2a1c1044708f71.png"},{"id":88272985,"identity":"ecc6bfc8-c115-4f17-b372-e5e20409548b","added_by":"auto","created_at":"2025-08-04 17:24:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2848802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568753/v1/5232c5f5-601b-4f8f-9e3c-c0703ad0120c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase","fulltext":[{"header":"Introduction","content":"\u003cp\u003eP-selectin, a member of calcium‐dependent lectins (glycoproteins) family, is produced in megakaryocytes and endothelial cells[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].It is stored in the α-granules of platelets and the Weibel‐Palade bodies of endothelial cells when the cells are at resting state. When the cells are activated, P-sel rapidly translocates to the cell surface, forming membrane P-sel[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to serving as a marker of platelet activation, platelet P-sel plays a critical role in leukocyte recruitment and function by mediating their adherence and transmigration to sites of inflammation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Dysregulated P-sel expression is associated with pathological inflammation and thrombosis in conditions like atherosclerosis, stroke, myocardial infarction, and deep vein thrombosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar to other platelet receptors (such as GPVI, GPIbα, and GPV), P-sel is rapidly shed from the platelet surface into the plasma shortly after activation, becoming soluble P-sel (sP-sel)[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].Unlike P-sel on the membrane surface, which forms dimers or oligomers to enhance leukocyte binding, sP-sel exists as a monomer. sP-sel is unable to effectively interact with its ligands on leukocytes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], thus, the overall role of sP-sel in immune responses and hemostasis is not prominent[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. From an evolutionary perspective, the shedding of P-sel from activated platelets represents a negative feedback mechanism that regulates platelet P-sel-mediated inflammation.\u003c/p\u003e \u003cp\u003eThe precise mechanisms underlying P-sel shedding remain poorly understood. Our study aims to explore the mechanisms responsible for P-sel shedding and its role in modulating platelet-mediated inflammatory responses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBlood collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe platelets, leukocytes and red blood cells (RBCs) used in this study were obtained from healthy adult volunteers. Blood was collected into polypropylene tubes containing 0.109 M sodium citrate as an anticoagulant. The samples were processed and isolated in the department of laboratory medicine at West China Hospital, Sichuan University. All procedures were approved by the Ethics Committee of West China Hospital, Sichuan University, and informed consent was obtained from all volunteers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of platelets, leukocytes, and whole blood\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlatelet-rich plasma (PRP) was prepared by centrifugation at 200g for 5 minutes. The resulting pellet was washed and resuspended in phosphate buffer saline (PBS). For activation, platelets were treated with thrombin (10602400001,\u0026nbsp;Roche, 1U/mL) for 15 minutes at 37\u0026deg;C (P-sel positive platelet was around 70-80% as evidenced by\u0026nbsp;flow cytometry). Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from whole blood using Polymorphprep\u0026trade; (Serumwerk, Germany) through density gradient centrifugation at 500 \u0026times; g for 30 minutes at room temperature with slow deceleration.\u0026nbsp;After centrifugation, the layer above Polymorphprep\u0026trade; contained only peripheral blood mononuclear cells (PBMCs), while the lower layer contained polymorphonuclear leukocytes (mainly neutrophils). Both PBMCs and neutrophils had a purity greater than 90%. Platelets were removed by centrifugation at 200g for 5 minutes for whole blood preparation. All procedures were at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivated platelet co-incubation experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlatelets isolated from healthy human peripheral blood were activated and incubated with whole blood, PBMCs, or neutrophils under conditions with or without neutrophil elastase (NE) inhibitors, (HY-17443, Sivelastat, 1 mM; Med Chem Express, USA), myeloperoxidase (MPO) inhibitors (475944-1GM, ABAH, 2 mM; EMD Millipore, USA), and Latrunculin B (LatB) (L5288, 5 \u0026mu;M; Calbiochem, Merck Millipore, Germany)\u0026nbsp;at 37\u0026deg;C for 4 hours. Before co-incubating activated platelets with neutrophils, the experimental group received 1 hour of NE inhibitors, MPO inhibitors or LatB pre-treatment, while the control group was not. Samples were taken at four time points: 0, 1, 2, and 4 hours of incubation. After centrifugation, the supernatant was collected, and the cell pellets were fixed with 4% paraformaldehyde.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of P-sel on the platelet surface\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFlow cytometry detection of P-sel on platelets\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe cell pellets were blocked with 2% Bovine Serum Albumin (BSA) and then stained with CD42b (303934, Brilliant Violet 510\u003csup\u003eTM\u003c/sup\u003e anti-human CD42b, Biolegend, USA) and CD62P antibodies (304905, PE-anti-human CD62P, Biolegend, USA) for 1 hour at room temperature, protected from light. After washing with PBS buffer, the cells were analyzed using a flow cytometer (BD FACSCanto II, BD Biosciences). At least 50,000 events were analyzed for each sample. Data were analyzed using FlowJo software (FlowJo, LLC) to determine the percentage of CD62P positive platelet (%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChemiluminescence immunoassay detection of P-sel on platelets\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe expression of platelet P-sel was detected in real-time using a homogeneous chemiluminescence immunoassay instrument (HSCL-5000, Poclight Biotech, China). After sampling at the corresponding time points, the samples were centrifuged and washed with PBS to obtain the cell pellets. Homogeneous chemiluminescence immunoassay\u0026nbsp;is a two-site (\u0026ldquo;sandwich\u0026rdquo;) chemiluminescence immunoassay based on chemiluminescence (CL) resonance energy transfer (CRET) technology. The acridinium ester (AE) emits a strong CL signal in the presence of H₂O₂. Graphene oxide (GO) serves as an efficient quencher, creating a \u0026ldquo;signal off\u0026rdquo; state via CRET between GO and AE-labelled DNA3. Upon sample application, DNA1-coated CD61 antibody, DNA2-coated CD61 antibody, and AE-labelled DNA3 form a sandwich complex, detaching DNA3 from GO and inhibiting CRET. This produces a CD61-CL signal, mimicking quantitative platelet count detection.\u0026nbsp;For P-sel expression on platelets, the same principle is applied using DNA1-coated CD62P antibody and DNA2-coated CD41 antibody. The sandwich complex forms with CD62P-positive platelets, detaching AE-labelled DNA3 from GO and generating a CD62P-CL signal, mimicking quantitative P-sel positive platelet count detection. The CL intensity ratio of CD62P to CD61 is used to represent the P-sel-positive intensity.\u003c/p\u003e\n\u003cp\u003eDue to methodological limitations, chemiluminescence immunoassay could not be used to analyze whole blood samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of sP-sel\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esP-sel in the supernatants was determined using an ELISA kit (Boster Bio, China) according to the manufacturer\u0026rsquo;s instructions. The absorbance was measured at a wavelength of 450 nm using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using GraphPad Prism 9.0. The normality of the data was assessed using the Shapiro-Wilk test. For normally distributed data, results are presented as mean \u0026plusmn; SEM. A two-way ANOVA was conducted to evaluate differences among groups across various time points (0, 1, 2, and 4 hours). Tukey\u0026rsquo;s multiple comparisons test was used to analyze differences between groups at each time point, as well as changes from 0 hour to 4 hours measurements within each group. For the groups involving co-incubation of activated platelets with neutrophils and LatB, where other variables were controlled in the control group, one-way ANOVA and paired t-tests were performed to analyze P-sel expression and sP-sel concentration. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eP-sel shedding from activated platelets occurred in whole blood but not in buffer\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 1h incubation in whole blood, the percentage of P-sel positive platelets already decreased to a very low level (0h\u0026rarr;1h, 79.5%\u0026rarr;9.7%), while the percentage of P-sel positive platelets incubated in buffer did not change significantly over a period of 4 hours (0h\u0026rarr;4h, 74.7%\u0026rarr;68.9%) (Fig.1a). Correspondingly, an increase in sP-sel fold change was already evident at 1h incubation in whole blood (6.77 fold increase), while there was only a slight but nonsignificant increase in sP-sel over a period of 4 hours in buffer (1.40 fold increase) (Fig.1b). At 4h, compared to the group where platelets incubated in buffer, the two parameters were statistically different in the group where platelets incubated in whole blood (Fig.1c-d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeutrophils were involved in the shedding of platelet P-sel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 1h incubation with neutrophils, the percentage of P-sel positive platelets already decreased to a very low level (0h\u0026rarr;1h, 70.0%\u0026rarr;24.3%), while the percentage of P-sel positive platelets incubated with PBMCs did not significantly change until 4h incubation (0h\u0026rarr;4h, 69.4%\u0026rarr;56.4%) (Fig.2a). Similarly, the fold decrease in CD62P /CD61 ratio was already evident at 1h when incubated with neutrophils (0.28 fold decrease), but not evident until 2h when incubated with PBMC (0.28 fold decrease) (Fig.2b). Correspondingly, the fold increase in sP-sel was already evident at 1h when incubated with neutrophils (2.89 fold increase), but not evident until 2h when incubated with PBMCs (2.51 fold increase) (Fig.2c). At 4h, compared to the group where platelets incubated with buffer or PBMCs, all the above-mentioned parameters were statistically different in the group where platelets incubated with neutrophils (Fig.2d-f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of neutrophil elastase in platelets P-sel shedding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe addition of NE inhibitors partly reversed neutrophils-induce P-sel shedding, the percentage of P-sel positive platelets did not significantly change until 2h incubation (0h\u0026rarr;2h, 70.2%\u0026rarr;46.4%); while it was already evident at 1h when incubated with neutrophils alone or in the presence of MPO inhibitors (0h\u0026rarr;1h, 70.0%\u0026rarr;24.3% or 73.0%\u0026rarr;11.2%) (Fig.3a). Similarly, the fold decrease in CD62P /CD61 ratio was not evident until 4h when incubated with neutrophils in the presence of NE inhibitors (0.16 fold decrease), while it was already evident at 1h when incubated with neutrophils alone or in the presence of MPO inhibitors (0.28 fold decrease or 0.74 fold decrease) (Fig.3b). Correspondingly, the fold increase in sP-sel was not evident until 4h when incubated with neutrophils in the presence of NE inhibitors (3.62 fold increase), while it was already evident at 2h when incubated with neutrophils alone (4.18 fold increase) (Fig.3c). At 4h, compared to the group where platelets incubated with neutrophils alone or in the presence of MPO inhibitors, all the above-mentioned parameters were statistically different in the group where platelets incubated with neutrophils in the presence of NE inhibitors (Fig.3d-f); compared to the group where platelets incubated with neutrophils alone, fold decrease in CD62P /CD61 ratio and fold increase in sP-sel were different in the group where platelets incubated with neutrophils in the presence of MPO inhibitors (Fig.3d-f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLatB treatment partly reversed neutrophil-induced platelets P-sel shedding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLatB treatment did not affect NE release from neutrophils (Fig.4a), but compared to the group where platelets incubated with neutrophils alone, fold decrease in CD62P /CD61 ratio (0.36 v.s. 0.59) and fold increase in sP-sel level (2.96 v.s. 5.34) were different in the group where platelets incubated with neutrophils in the presence of LatB (Fig.4b-c).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the role of P-sel in mediating platelet-neutrophil interaction, understanding the mechanism of platelets P-sel shedding is essential for studying platelet-mediated inflammatory responses and identifying new biomarkers for thromboinflammation. In this study, we demonstrated that neutrophils promote P-sel shedding from activated platelets, evidenced by decreased P-sel positive platelet rate, decreased CD62P /CD61 ratio and increased sP-sel concentration. This finding is supported by another notable study which highlights that the presence of P-sel glycoprotein ligand-1 (PSGL-1), primarily expressed on neutrophils, is essential for P-sel shedding [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Specifically, P-sel was retained longer on activated platelets in PSGL-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to wild-type mice[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the important role of neutrophils in platelet P-sel shedding, we further investigated the specific mechanisms underlying this process. We found that NE is one of the key molecules driving this process, as NE inhibitors could partly reverse the neutrophil-induced platelet P-sel shedding. It has been suggested that purified P-sel serves as a substrate for neutrophil elastase[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, we found that inhibiting the formation of neutrophil extracellular traps (NETs) by LatB (inhibiting actin polymerization) without reducing protease release[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], could also partly reverse the neutrophil-induced platelet P-sel shedding, suggesting that localized high concentrations of NE in NETs might be important for NE to induce platelet P-sel shedding. Additionally, we found that MPO inhibition can reinforce neutrophil-induced platelet P-sel shedding. This effect can be explained by established studies showing that the MPO-H₂O₂-Cl⁻ system potently inhibits NE activity through HOCl-mediated oxidative modification, as evidenced by both in \u003cem\u003evivo\u003c/em\u003e animal experiments and in \u003cem\u003evitro\u003c/em\u003e cellular studies[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Together, these data establish MPO as a critical regulator of platelet P-sel shedding through its modulation of NE activity.\u003c/p\u003e \u003cp\u003eInterestingly, platelet P-sel plays a vital role in NETs formation. Activated platelets stimulate neutrophil activation through P-sel-PSGL-1 interaction, therefore inducing NET formation, leading to the release of functional molecules[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Combining with our finding that concentrated NE in NETs might play a role in platelet P-sel shedding, we can conclude that platelet P-sel shedding is related to a negative feedback mechanism balancing pro-thromboinflammatory and anti-thromboinflammatory effects within the body. Specifically, the interaction between platelets and neutrophils is mutual. Platelets promote the activation of neutrophils; and in turn, neutrophils induce P-sel shedding from activated platelets via neutrophil elastase, resulting in the irreversible functional downregulation of platelet P-sel-mediated interactions, including platelet-leukocyte interaction, platelet-endothelium interaction, therefore limiting excessive thromboinflammatory response. So far, most existing studies focus on how platelets affect neutrophils, whereas our study is the first to propose a reverse effect of neutrophils on platelets. This novel finding offers a new insight into the complicated interplay between platelets and neutrophils, and provides a fresh perspective on the mechanisms underlying platelet P-sel shedding.\u003c/p\u003e \u003cp\u003eCurrently, this study examines the mechanism under normal conditions only, but the process of platelet P-sel shedding may differ in various disease states, such as sepsis or cardiovascular diseases. Future research could use animal models to investigate how P-sel shedding changes in different disease contexts and its prognostic implications. Additionally, further exploration is needed to understand how locally high concentrations of NE mediate P-sel shedding through specific signaling pathways and whether other cytokines or receptors also play a role in this process.\u003c/p\u003e \u003cp\u003eFurthermore, once P-sel sheds from activated platelets, it enters the bloodstream as soluble sP-sel. In recent years, the study of sP-sel has become a focus in areas like inflammation, thrombosis, and cardiovascular diseases, such as coronary artery disease, hypertension, and atrial fibrillation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Some of them suggest that elevated sP-sel is linked to the activity and/or severity of these diseases[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; some of them suggest not[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Our data provide a unifying explanation for the above-mentioned paradoxical clinical observations regarding sP-sel. Since sP-sel is a result of platelet activation (pro-thromboinflammation) and neutrophil-mediated platelet P-sel shedding (anti-thromboinflammation), it is inappropriate to relate sP-sel alone to the activity and/or severity of the diseases, besides, endothelial-derived sP-sel can further complicate the interpretation. Considering the interaction of platelets and neutrophils is mutual, it might be more appropriate to interpret sP-sel with other molecules related to platelet-neutrophil interactions, nevertheless, further studied are still needed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHere we demonstrate a mutual interaction between platelets and neutrophils: while platelets potently activate neutrophils, activated neutrophils in turn shed platelet surface P-sel through NE. This novel finding offers a new insight into the complicated interplay between platelets and neutrophils, and provides a fresh perspective on the mechanisms underlying platelet P-sel shedding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from National Natural Science Foundation of China (82302601 to LL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Conceptualization: YH, LL. Data curation: LL, YH. Formal analysis: SH, CL. Investigation: LL, SH, YH. Methodology: YH, JZ. Project administration: ZY, SW. Resources: LL, CL. Supervision: YH, LL, SH. Validation: YH, LL, JZ. Visualization: YH, SW, XH. Writing\u0026ndash;original draft: YH, JZ. Writing\u0026ndash;review \u0026amp; editing: YH, LL. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of West China Hospital, Sichuan University (ID：2024-215).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in this article. Further enquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePurdy, Megan, Andrea Obi, Daniel Myers, and Thomas Wakefield. 2022. P‐ and E‐ selectin in venous thrombosis and non‐venous pathologies. \u003cem\u003eJournal of Thrombosis and Haemostasis\u003c/em\u003e 20: 1056\u0026ndash;1066. https://doi.org/10.1111/jth.15689.\u003c/li\u003e\n\u003cli\u003eDole, Vandana S., Wolfgang Bergmeier, Heather A. Mitchell, Sarah C. Eichenberger, and Denisa D. Wagner. 2005. Activated platelets induce Weibel-Palade\u0026ndash;body secretion and leukocyte rolling in vivo: role of P-selectin. \u003cem\u003eBlood\u003c/em\u003e 106: 2334\u0026ndash;2339. https://doi.org/10.1182/blood-2005-04-1530.\u003c/li\u003e\n\u003cli\u003eWang, Dehao, Pei Zhao, Yan Lv, Jing Ming, Ziqing Wang, Erpeng Yang, Yumeng Li, et al. 2023. Proteomic-Based Platelet Activation-Associated Protein SELP May Be a Novel Biomarker for Coagulation and Prognostic in Essential Thrombocythemia. \u003cem\u003eJournal of Clinical Medicine\u003c/em\u003e 12: 1078. https://doi.org/10.3390/jcm12031078.\u003c/li\u003e\n\u003cli\u003ePanicker, Sumith R., Padmaja Mehta-D\u0026rsquo;souza, Nan Zhang, Arkadiusz G. Klopocki, Bojing Shao, and Rodger P. McEver. 2017. Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice. \u003cem\u003eBlood\u003c/em\u003e 130: 181\u0026ndash;191. https://doi.org/10.1182/blood-2017-02-770479.\u003c/li\u003e\n\u003cli\u003eMontague, Samantha J., Robert K. Andrews, and Elizabeth E. Gardiner. 2018. Mechanisms of receptor shedding in platelets. \u003cem\u003eBlood\u003c/em\u003e 132: 2535\u0026ndash;2545. https://doi.org/10.1182/blood-2018-03-742668.\u003c/li\u003e\n\u003cli\u003eHellen, Nicola, Gregory I. Mashanov, Ianina L. Conte, Sophie le Trionnaire, Victor Babich, Laura Knipe, Alamin Mohammed, et al. 2022. P-selectin mobility undergoes a sol-gel transition as it diffuses from exocytosis sites into the cell membrane. \u003cem\u003eNature Communications\u003c/em\u003e 13: 3031. https://doi.org/10.1038/s41467-022-30669-x.\u003c/li\u003e\n\u003cli\u003eDole, Vandana, Wolfgang Bergmeier, Ian Patten, Junichi Hirahashi, Tanya Mayadas, and Denisa Wagner. 2007. PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets. \u003cem\u003eThrombosis and Haemostasis\u003c/em\u003e 98: 806\u0026ndash;812. https://doi.org/10.1160/TH07-03-0207.\u003c/li\u003e\n\u003cli\u003eGardiner, Elizabeth E., Mariagrazia De Luca, Tracy McNally, Alan D. Michelson, Robert K. Andrews, and Michael C. Berndt. 2001. Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G. \u003cem\u003eBlood\u003c/em\u003e 98: 1440\u0026ndash;1447. https://doi.org/10.1182/blood.V98.5.1440.\u003c/li\u003e\n\u003cli\u003eSprenkeler, Evelien G. G., Anton T. J. Tool, Stefanie S. V. Henriet, Robin Van Bruggen, and Taco W. Kuijpers. 2022. Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements. \u003cem\u003eBlood\u003c/em\u003e 139: 3166\u0026ndash;3180. https://doi.org/10.1182/blood.2021013565.\u003c/li\u003e\n\u003cli\u003eZhang, Nan, Xiahenazi Aiyasiding, Wen-Jing Li, Hai-Han Liao, and Qi-Zhu Tang. 2022. Neutrophil degranulation and myocardial infarction. \u003cem\u003eCell communication and signaling: CCS\u003c/em\u003e 20: 50. https://doi.org/10.1186/s12964-022-00824-4.\u003c/li\u003e\n\u003cli\u003eHirche, Tim O., Joseph P. Gaut, Jay W. Heinecke, and Azzaq Belaaouaj. 2005. Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase: effects on host defense. \u003cem\u003eJournal of Immunology (Baltimore, Md.: 1950)\u003c/em\u003e 174: 1557\u0026ndash;1565. https://doi.org/10.4049/jimmunol.174.3.1557.\u003c/li\u003e\n\u003cli\u003eNeri, Tommaso, Dario Nieri, and Alessandro Celi. 2020. P-selectin blockade in COVID-19-related ARDS. \u003cem\u003eAmerican Journal of Physiology - Lung Cellular and Molecular Physiology\u003c/em\u003e 318: L1237\u0026ndash;L1238. https://doi.org/10.1152/ajplung.00202.2020.\u003c/li\u003e\n\u003cli\u003eEtulain, Julia, Kimberly Martinod, Siu Ling Wong, Stephen M. Cifuni, Mirta Schattner, and Denisa D. Wagner. 2015. P-selectin promotes neutrophil extracellular trap formation in mice. \u003cem\u003eBlood\u003c/em\u003e 126: 242\u0026ndash;246. https://doi.org/10.1182/blood-2015-01-624023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Platelets, neutrophil, P-selectin, neutrophil elastase","lastPublishedDoi":"10.21203/rs.3.rs-6568753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6568753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: P-selectin (P-sel) on activated platelets plays a vital role in inflammation by mediating platelet-neutrophil interactions. However, P-sel surface expression on activated platelets is temporary, with rapid shedding into circulation as soluble P-sel (sP-sel). It is suggested that neutrophils adhesion to P-sel on activated platelets can induce P-sel shedding, however, the exact mechanism remains unclear. This study is to investigate the role of neutrophils in platelet P-sel shedding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Platelets were activated by thrombin and then incubated with whole blood, peripheral blood mononuclear cells (PBMCs), or neutrophils, with or without neutrophil elastase (NE) inhibitors, myeloperoxidase (MPO) inhibitors, or latrunculin B(LatB). Samples were collected at 0, 1, 2, and 4 hours. P-sel expression on platelets was assessed by flow cytometry and chemiluminescence immunoassay, while sP-sel was quantified by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eWhen incubated with neutrophils, P-sel expression on activated platelets was reduced (sP-sel was increased) in a time-dependent manner (P-sel-positive platelets, 70.0%→11.1%; fold decrease in CD62P/CD61, 0→0.61; fold increase in sP-sel, 0→6.94), unlike PBMCs. NE inhibitors preincubation with neutrophils could partly reverse the neutrophil-induced P-sel shedding (P-sel-positive platelets, 70.2%→43.4%; fold decrease in CD62P/CD61, 0→0.16; fold increase in sP-sel, 0→4.38), while MPO inhibitors couldn’t. The addition of LatB could also partly reversed the neutrophil-induced P-sel shedding (fold decrease in CD62P/CD61, 0→0.36; fold increase in sP-sel, 0→5.34).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eThe interaction between platelets and neutrophils is mutual. Platelets promote the activation of neutrophils; and in turn, neutrophils induce P-sel shedding via neutrophil elastase, resulting in the irreversible functional downregulation of platelet P-sel-mediated interactions.\u003c/p\u003e","manuscriptTitle":"Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 11:39:08","doi":"10.21203/rs.3.rs-6568753/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-19T16:03:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T15:50:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296641664427733753827787960692390962727","date":"2025-05-10T22:35:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T15:43:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-01T03:50:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-01T03:46:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2025-05-01T02:55:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e30b31ec-4936-498c-b54d-211e65868722","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:48:10+00:00","versionOfRecord":{"articleIdentity":"rs-6568753","link":"https://doi.org/10.1007/s10753-025-02350-0","journal":{"identity":"inflammation","isVorOnly":false,"title":"Inflammation"},"publishedOn":"2025-07-28 16:39:16","publishedOnDateReadable":"July 28th, 2025"},"versionCreatedAt":"2025-05-09 11:39:08","video":"","vorDoi":"10.1007/s10753-025-02350-0","vorDoiUrl":"https://doi.org/10.1007/s10753-025-02350-0","workflowStages":[]},"version":"v1","identity":"rs-6568753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6568753","identity":"rs-6568753","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}