Thrombosis and Platelet Adhesion and Aggregation in the Third Trimester of Pregnancy under Arterial Shear Strip

Thrombosis and Platelet Adhesion and Aggregation in the Third Trimester of Pregnancy under Arterial Shear Strip | 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 Article Thrombosis and Platelet Adhesion and Aggregation in the Third Trimester of Pregnancy under Arterial Shear Strip cui he, haidong ma, xuemei gao, xiaojing huang, surong deng, yu liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4203479/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Platelet adhesion and aggregation effect increases in third-trimester wemen, and the risk of thrombosis increases, so how to achieve early diagnosis is particularly important.In this study, microfluidic chip technology was used to study the adhesion and aggregation behavior of platelets in third-trimester under different arterial shear rates (1000s-1, 1500s-1, 4000s-1). Flow cytometry was used to analyze platelet surface activation markers (PAC-1 and P-selectin CD62P), and to explore the diagnostic value of different platelet function assessment methods for the risk of third-trimester thrombosis in normal pregnant women. Compared to healthy controls, white blood cell, fibrinogen, D-dimer levels increased, while platelet levels decreased (P 0.05). Platelet aggregation and surface activation marker expression significantly increased with the increase of shear rate under flow conditions (P < 0.05). The expression of platelet surface activation markers elevated.So we believe that using microfluidic chip technology to evaluate platelet aggregation and thrombosis in the third-trimester under arterial flow conditions combined with platelet activation can help predict thrombotic diseases. And the results may provide effective clinical application data and a theoretical basis for the diagnosis and prevention of platelet dysfunction and thrombotic diseases during pregnancy. Shear rate platelet adhesion and aggregation late pregnancy thrombosis predictive value Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pregnancy is a unique physiological period in women. Pregnant women are in a hypercoagulable state due to changes in hormone levels, elevated fibrinogen levels, and other factors. These changes can provide preventive hemostatic protection against bleeding of the placental detachment surface and laceration after delivery and convenient conditions for the formation of arteriovenous thrombosis [1,2] . Studies have emphasized that the change of hormone levels and coagulation status during pregnancy changes with the week of pregnancy, the further increase of coagulation factor II, Ⅴ, Ⅶ, and Ⅹ, and other levels in the third trimester of pregnancy, the further decrease of antithrombin activity and fiber activity, and the increase of hypercoagulability, which is conducive to placental stripping during childbirth and reduce postpartum massive bleeding, but can increase the risk of thrombosis in pregnant women. The risk begins during early gestation and increases with increasing gestation time and is the highest in the early postpartum period. Compared with age-appropriate pregnant women, the risk of venous thromboembolism (VTE) increases by 15–35 times, and some women have an 84 times increased risk of thrombosis in the first 6 weeks of the puerperal period [3–5] . In the past, postpartum hemorrhage was the leading cause of death in perinatal women; however, recent studies have shown that the mortality rate of this disease has decreased with increasing attention paid to postpartum hemorrhage in many countries. VTE has become the leading cause of maternal death in some developed countries. Therefore, the early diagnosis of thrombotic diseases during pregnancy is of great significance. Approximately 20% of thrombosis in pregnancy cases include arterial thrombosis and the remaining 80% are venous thrombosis. Venous thrombosis is common in the lower limbs and pelvis thrombosis. The diagnosis of the disease is still based on color Doppler ultrasound and other imaging examinations; however, imaging examination has a certain lag, which is not conducive to the early diagnosis and treatment of the disease [6,7] . Therefore, the prevention of arteriovenous thrombosis (AVT) is critical. AVT often occurs after surgery and childbirth and can lead to death in severe cases. In a normal pregnancy, in the second and third trimesters, physiological hypercoagulability and a continuous increase in blood pressure leads to vasospasm and induce vascular ischemia, hypoxia, and endothelial injuries. During pregnancy, women are up to five times more likely to develop VTE than non-pregnant women of the same age [8] . Studies have reported that platelet aggregation is significantly enhanced in women during early, middle, and late pregnancy and during platelet activation, which may be related to the synthesis and release of a large amount of platelet-activating factor (PAF) [9] , and damage to vascular endothelial cells [10] . In addition, Edwards et al. found that three platelet activity indicators, PAF, CD62p, and CD63, significantly increased during the third trimester and postpartum [11,12] . Their expression levels were positively correlated with the platelet aggregation rate, and platelet aggregation activity also increased in patients with venous thrombosis. Researchers have found significant increases in intracellular Ca 2+ and plasmaβ-thromboglobulin levels [13,14] . Therefore, platelet adhesion, aggregation, and activation in vivo are important components of thrombosis [15] . In addition to agonist induction, another method of platelet activation involves interactions with the cell and extracellular matrix components of the blood vessel wall at different shear rates, thereby generating mechanical forces that cause platelet aggregation and thrombosis. Shear-induced platelet aggregation is a major cause of pathological thrombosis and is rarely observed in normal hemostatic mechanisms. Researchers have long focused on the formation, prevention, and treatment of pathological thrombosis during pregnancy, and few studies have been conducted on the assessed platelet adhesion, aggregation function, and thrombosis ability of women in normal pregnancy and postpartum high-risk periods. Moreover, the platelet aggregation function was measured under static conditions without considering the instantaneous interaction between platelets and increased shear force. The ability to test the efficacy of platelet adhesion and aggregation in hemodynamic environments experienced by platelets in vivo is limited, and thrombosis-related events cannot be observed in real time. Therefore, in this study, a microfluidic chip was used to accurately regulate microfluidic dynamics by simulating the size and structural characteristics of microvessels in vivo to study platelet functions. The microfluidic chip used in this study can simulate in vitro microchannel structures similar to the microvessel size and structure in vivo and can precisely regulate fluid dynamic behavior; therefore, it can be used as an ideal microvessel model in vitro to explore the physiological and pathological mechanisms related to coagulation and platelets, such as platelet aggregation under shear force regulation. We used microfluidic chip technology to simulate the in vitro shear rates of the venous, arteriolar, and arterial walls of moderate stenosis at approximately 500; 1,500; and 4,000 s -1 , respectively, under pathological conditions to study the adhesion and aggregation behaviors of platelets and the process of thrombosis. We found that platelet aggregation function was enhanced in the third trimester and postpartum period. Platelet adhesion increased under the low shear rate of slow blood flow, and aggregates with more and larger thrombi were easily washed away. However, with increasing shear rate, aggregates with fewer thrombi were small and not easily routed. We believe that platelet aggregation is enhanced in the third trimester of pregnancy; however, thrombus formation is weakened under normal blood flow conditions, which increases the risk of thrombus formation if the patient has other pregnancy complications. The purpose of this study was to analyze the value of different platelet function assessment methods and other thrombosis markers in patients with thrombotic diseases in the third trimester of pregnancy to provide new ideas and a theoretical basis for the early diagnosis of thrombotic diseases in pregnancy. Materials and methods 2.1 Materials Sylgard 184 polydimethylsiloxane (Dow Corning, USA), calcein AM (Invitrogen, USA), arachidonic acid(AA)(Jiangsu Inartis), adenosine diphosphate༈ADP༉ (Jiangsu Inartis), collagen༈COL༉ (Jiangsu Inartis), calf serum albumin (Shandong TeliKangxin Medical Technology Co., LTD.), CD61 (Integrin beta3), monoclonal antibody (VI-PL2), PerCP-eFluor710/CD62P (P-Selectin), monoclonal antibody (AK-4), PE/PAC-1 monoclonal antibody (PAC-1),FITC, PBS buffer (Gibco, USA), paraformaldehyde (Biyuntian Biotechnology, USA), sodium citrate venous blood vacuum collection tube (Weigao, Shandong), RSP01-CS bidirectional push-pull precision injection pump (Jiaxan Ruichuang), IX71 inverted fluorescence microscope (Olympus, Japan), plasma cleaner (PDG-32 G-2,Harrick, Germany), and Aggrestar (PL-12) multiparameter platelet function analyzer (Jiangsu Inovartis). 2.2 Collection and processing of blood samples From January 2021 to December 2022, blood samples were collected from pregnant women registered with the Obstetrics Department of Yongchuan Hospital of Chongqing Medical University and from women who gave birth naturally. Eligible non-pregnant women were 20 healthy volunteers recruited by the Physical Examination Center of Yongchuan Hospital of Chongqing Medical University. The inclusion criteria were as follows: pregnant woman with no pregnancy-related complications, no history of thrombosis, and normal platelet count. Non-pregnant volunteers were eligible if they had not taken the following drugs in the last 2 weeks: clopidogrel, aspirin, other antiplatelet drugs, statins, and lipid-lowering drugs and had normal platelet counts. This study was approved by the Ethics Committee of the Yongchuan Hospital of Chongqing Medical University (approval no. 2021016), and all participants provided written informed consent. Venous blood samples were collected in vacuum, 3.2% sodium citrate anticoagulant at 1:9 (v/v), and EDTA-K2 was added and used within 2 h. Calcein AM fluorescent dye was used to fluorescently label platelets in blood samples at a concentration of 1 mmol/L and was added to the blood sample at 1:500 (v/v), gently shaken, and incubated at 37°C for 10 min without light. All study methods were carried out in accordance with the Ethics Committee of Yongchuan Hospital of Chongqing Medical University. 2.3 Basic data detection Anticoagulant whole blood without additives (5 mL) and anticoagulant whole blood with sodium citrate (3 mL) were collected simultaneously from the pregnant and non-pregnant groups, and progesterone, estradiol, fibrinogen, and D-dimer levels were detected by the instrument. White blood cell count, hemoglobin level, and platelet count were measured using an automatic blood cell analyzer. Differences in laboratory indices between the two groups were compared. 2.4 Continuous multiparameter method for detecting platelet adhesion and aggregation functions Platelet aggregation induced by AA, ADP, and COL was detected by continuous multiparameter method. The test program was set in Aggrestar (PL-12), a 30 µL inducer was added corresponding to the program settings to the specified position. Sodium citrate anticoagulant was added to whole blood 300 µL to the sample test position 1. The instrument begins automatic detection by clicking on the start of the test button. The maximum platelet adhesion and aggregation rates induced by different inducers were recorded. 2.5 Hydrodynamic analysis of microchannel The microchannels were subjected to a hydrophilic treatment before analysis using microfluidic chips. After treatment, the chip was placed on an inverted fluorescence microscope platform. The chip outlet was connected to the injection pump with a polytetrafluoroethylene tube (inner diameter 1.0 mm, outer diameter, 1.5 mm), and the flow speed of the blood sample in the microchannel was controlled in the pull-back mode. When the blood began to flow in the microchannel, Streampix 5.0 software was used to control the camera to record images of the adhesion and aggregation of fluorescent-labeled platelets on the narrow surface and narrow downstream surface after flowing through the microchannel at a frame rate of 1 frame/s (objective lens, ×20). A total of 180 sequential fluorescence images were obtained after 3 min of recording. A schematic and photographs of the experimental equipment are shown in Fig. 1 . The flow rate in the chip through channel was set to 14, 52, and 40 µlL/min in the narrow channel. According to the finite element analysis, the shear rates formed by the three flow rates were 1,000; 1,500; and 4,000 s − 1 , respectively. These three flow rates simulated normal venous, small-artery, and moderate-stenosis flow rates. Anticoagulant whole blood samples of sodium citrate labeled with AM fluorescent dye were incubated at 37℃ for 100 min under light protection, and then added to the sample pool successively to observe platelet adhesion and aggregation behavior and thrombosis in real time. 2.6 Platelet activation analysis by flow cytometry Women with thrombosis were incubated at 37℃ for 30 min, then perfused into the microchannel sealed by bovine serum albumin, and the blood samples were collected at the volume flow rate of 52 µL/min and 100 µL/min, respectively. Five microliters of each sample were incubated with anti-human CD61, CD62 P, and PAC-1 antibodies for 20 min at room temperature in the dark, and one blood sample was labeled as a negative control. The labeled samples were mixed with 1% paraformaldehyde (1 mL) and fixed for 10 min. The expression of CD62P and PAC-1 was analyzed by flow cytometry. Data were analyzed. 2.7 Statistical analysis Measurement data were expressed as mean ± standard deviation. A randomized block design analysis of variance was used to compare the means of multiple groups, least significant difference test was used for multiple comparisons, paired t-test was used to compare the means of the two groups, P < 0.05 was considered statistically significant. Results 3.1 During the third trimester and postpartum period, hormone levels in women increase, and the number of red blood cells and platelets decrease. The number of white blood cells slightly increased. Protein C activity, D-dimer levels, and fibrinogen levelsincreased (Table 1 ). Table 1 Basic data of research object (n = 76) Item Control Third trimester of pregnancy P Estradiol (ng/mL) 25.14 ± 3.19 Progesterone (ng/mL) 215.12 ± 30.58 Erythrocytes (*10^12/L) 3.77 ± 0.35 3.26 ± 0.29 0.516 Leukocytes (*10^9/L) 6.41 ± 1.95 9.53 ± 3.31 0.006 Platelets (*10^9/L) 239.4 ± 50.05 196.4 ± 48.22 0.015 D-dimer (µg/mL) 1.58 ± 0.21 2.74 ± 1.51 0.021 FiB (g/L) 2.76 ± 0.35 5.95 ± 0.72 0.008 P < 0.05 is significant. FiB, fibrinogen 3.2 Platelet adhesion and aggregation in third trimester women under static conditions After blood was added to the inducers, AA, ADP, COL, and PL-12 levels were used to evaluate the platelet reactivity to the inducers under static conditions. No significant difference in platelet aggregation function (AA 47.52 ± 5.43, ADP 61.08 ± 5.09, COL 52.97 ± 4.78) between pregnant women and control group (P > 0.05) was noted, as shown in Fig. 2 . 3.3 Platelet adhesion and aggregation enhancement and increased thrombosis in shear rate environment Previous studies have shown that shear-induced platelet aggregation is the major cause of thrombosis [16] This study investigated the differences in platelet aggregation in the blood of different women in the third trimester under different shear force conditions. Figure 3 (a) and (b) show the image sequence of platelets flowing for 200 s under different shear rate gradients and the images after binarization. When whole blood flowed at 1,000 s − 1 input shear rate, platelets were “small and scattered” in the microchannel. When the shear rate was increased to 1,500 s − 1 , the platelet aggregation density increased. When the wall shear rate reached 4,000 s − 1 , the degree of platelet aggregation was further enhanced and a dense, highlighted state was observed. Figure 3 c, d (c,d) show the binarization images of platelet aggregation. A gradual increase in the wall shear rate was observed. The total area and coverage of platelet aggregates significantly increased (P < 0. 05), as shown in Fig. 4 . 3.4 Platelet activation was enhanced in late pregnancy and postpartum women Flow cytometry was used to assess the expression of the platelet surface activation markers (P-selectin and PAC-1) flowing through the closed microchannels (Fig. 5 ). Platelets were identified in the whole blood using a CD61 monoclonal antibody, and the adhered parts of the platelets were excluded. The expression levels of P-selectin and activated GDPⅡb/Ⅲa in single platelets were analyzed. The results showed that the expression levels of P-selectin and GPⅡb/Ⅲas on the platelet surface of pregnant women increased after high-shear induction, the expression levels in postpartum thrombus patients had significantly increased, and platelet aggregation had occurred. 3.5 ROC curve analysis According to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) of dynamic shear rate, static platelet function detection, thromboembolic marker (D-dimer), and platelet activation marker (CD62P) detection for predicting the risk of thrombosis in women in late pregnancy were 0.707, 0.628, 0.823, and 0.878, respectively, (see Table 2 ,6 Fig. 6 ). Table 2 ROC curve analysis results. Item AUC 95% CI P Sensitivity(%) Specificity(%) Static platelet function test 0.628 0.533–0.723 0.008 65.2 54.1 D dimer 0.707 0.621–0.792 < 0.001 75.3 69.2 Dynamic platelet function test 0.823 0.762–0.892 < 0.001 80.3 65.6 CD62P 0.878 0.825–0.931 < 0.001 79.6 80.3 Discussion In healthy people, the fibrinolytic and coagulation systems are in a state of long-term dynamic balance through which coagulation and hemostasis are achieved [17] .Thrombosis can be caused when blood stasis, blood in a hypercoagulable state, or vascular endothelial injury occurs. In addition to these factors, the maternal body can be in a hypercoagulable state to meet childbirth needs. In addition, the sudden increase in fetal weight in late pregnancy aggravates the suppression of the uterus in maternal lower limb circulation and causes the lower limb venous blood flow speed to drop sharply. At the same time, the pregnant women were less active than usual. Under the combined effect of these factors, the risk of thrombotic disease in late pregnancy significantly increased [18,19] . Thromboembolic diseases have a rapid onset and can cause sudden death if not treated in a timely and effective manner. Therefore, predicting thromboembolic diseases during pregnancy is important. Although imaging is the gold standard for the diagnosis of thromboembolic disease in pregnancy, it has a certain lag and limited predictive value [20,21 ] . Platelet function is being increasingly evaluated under physiological flow conditions to simulate the flow shear rate environment in vivo . In this study, we measured the optical aggregation of platelets under different morphological conditions and used microfluidic chip technology to monitor platelet thrombosis in whole blood, a method used to simulate human hemostasis and thrombosis in vitro . Therefore, detection of platelet aggregation under physiological conditions is important. During pregnancy, uterine vessels undergo significant vascular remodeling mediated by vascular endothelial and placental growth factors, leading to potential platelet-collagen matrix interactions and changes in blood flow rheology. The results of this study showed that platelet adhesion and aggregation effects in pregnant women in the third trimester of pregnancy were not significantly different from those in non-pregnant women under static conditions. The expression of platelet activation markers in pregnant women and significantly increased in platelet aggregation was observed, and the platelets showed aggregation. Platelet adhesion and aggregation were enhanced under arterial shear conditions; however, stable platelet aggregation did not occur, indicating that the small blood aggregation function was enhanced at high shear rates in the third trimester of pregnancy, but adhesion stability was decreased. This may reduce the risk of arterial thrombosis to a certain extent and may also be related to the partial deaggregation of platelets induced by the pathological shear rate in vitro over a certain period; however, the specific mechanism requires further study. It can also be seen from Fig. 6 assessing the platelet aggregation function in a dynamic environment to predict the risk of thrombosis is the most valuable method for the current diagnosis of thrombosis. Therefore, our results suggest that the detection of platelet aggregation effects under arterial flow conditions may provide valuable research data for the diagnosis of potential thrombosis or assessment of bleeding risk during pregnancy. The current clinical methods used to evaluate the platelet function have significant limitations. These are usually measured as changes in absorbance caused by platelet aggregation under the stimulation of thrombin, collagen, adenosine diphosphate, and other chemical activators, which do not reflect the shear force of platelets flowing through narrow blood vessels. Shear stress is a key factor in platelet activation through the regulation of mechanical action. Moreover, in arterial thrombotic diseases, blood vessel stenosis can cause blood flow to be subjected to a shear force much higher than the physiological levels, and such a high shear force can directly induce rapid platelet activation/aggregation in vitro [22–24] . It affects membrane receptor aggregation, membrane tension, integrin expression, cytoskeletal modification, and signal transduction. Therefore, the microfluidic chip technology used in this study provides a controllable shear rate for detecting platelet reactivity and thrombosis under simulated arterial conditions in vitro. PAC-1 is the fibrinogen binding site exposed after the activation of glycoprotein Ⅱb/Ⅲa, namely activated glycoprotein Ⅱb/Ⅲa. CD62P is a protein component of the granulosa membrane of the resting platelets. The degranulosa membrane rapidly fuses with platelets during platelet activation, and P-selectin is transferred to the surface of the platelets or the degranulosa becomes soluble in P-selectin. Previous studies have shown that CD62P is a reliable marker of split-dependent platelet activation [25] . In this study, flow cytometry was used to detect the expression of activation markers in platelets subjected to shear rate, indicating that an increase in the shear rate activates platelets and leads to increased platelet activation. However, no significant platelet aggregation was found, which may be related to the changes in blood flow status and hormone levels in the third trimester [24] . Strengths:In our study, microfluidic chip technology was used to simulate bionic blood vessels in vitro, so that the functional detection of platelets was exposed to physiological flow environment, and the results may be more reliable. Limitations:The sample size is small, and the corresponding mechanism of platelet function enhancement in late pregnancy remains to be studied Conclusion In summary, compared to healthy non-pregnant women in the third trimester, the platelet aggregation effect was significantly enhanced and activation was increased; however, the stability of the formed platelet aggregates was not significantly increased, and the specific mechanism remains to be further studied. Our study preliminatively proves that the microfluidic chip system can be used to evaluate the clip-induced platelet aggregation and activation in the third trimester of pregnancy when the blood flow changes significantly and the hormone levels are high and can analyze the differences in platelet reactivity under different shear rates. The combined detection of platelet activation markers is helpful for predicting thrombotic diseases in the third trimester of pregnancy. This may provide a new theoretical basis for the early diagnosis of thrombotic diseases during pregnancy. Declarations Financial support : This work was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission [grant number KGQN202100442]; the Natural Science Foundation of Yongchuan District [grant numbers 2022yc-jckx20052, 2023yc-jckx20027]. Conflicts of Interest : The authors declare that there are no conflicts of interest. Data availability : Data is provided within the manuscript. Author contributions ： H.C. conceived the experiments and wrote the article, M.H.D contributed to the experimental design and data analysis, H.X.J.andG.X.M. contributed to the experimental operation, L.Y. contributed to specimen collection, and D.S.R. contributed to the experimental guidance and review of the article.All authors reviewed the manuscript. References Wang C, Yang HX. New recommendations for thromboembolism in pregnancy by the American College of Obstetricians and Gynecologists in 2018 [J]. Chinese Journal of Perinatal Medicine 2019;22:139–40. SUN M, LIU C, ZHAO N, et al. Predictive value of platelet aggregation rate in postpartum deep venous thrombosis and its possible mechanism [J].Exp Ther Med 2018;15:5215–20. E.R. Pomp, A.M. Lenselink, F.R. Rosendaal, C.J. Doggen, Pregnancy, the postpartum period and prothrombotic defects: risk of venous thrombosis in the MEGA study, J Thromb Haemost. 6 (2008) 632–637 E. 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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-4203479","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292006435,"identity":"1feb0af4-bab5-4c2c-b9de-4d761b66af8a","order_by":0,"name":"cui he","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACNv7GhsM/fvy3sz/efIA4LXwShxsfM/YwJzOcOZZAnBY5hvRmYwY2ZsaGGz4GRDqM4WCbdAEPUM8Mno833jDYyek2ENLC3NgmPcOCh49Zunez5RyGZGOzA0TYIsHDI8HMJnN2mzQPw4HEbYS1JAK1sBkw9kjkPCNaS7MxD1sC4wyJHDYitUgcbHw4s+dAsgHPMWPLOQZE+EW+v/3BgQ8/DtgZsDc/vPGmwk6OoBYUIMFDZNQgayFVxygYBaNgFIwIAADRA0DRMmJbNwAAAABJRU5ErkJggg==","orcid":"","institution":"Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"cui","middleName":"","lastName":"he","suffix":""},{"id":292006436,"identity":"1189f0b2-1a2a-4613-b656-1be13db2c1e6","order_by":1,"name":"haidong ma","email":"","orcid":"","institution":"Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"haidong","middleName":"","lastName":"ma","suffix":""},{"id":292006437,"identity":"b674f4e2-e4b3-46db-9830-9cbf96cba528","order_by":2,"name":"xuemei gao","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"xuemei","middleName":"","lastName":"gao","suffix":""},{"id":292006438,"identity":"d5314323-d0ac-4c56-b34e-814c2059bce7","order_by":3,"name":"xiaojing huang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"xiaojing","middleName":"","lastName":"huang","suffix":""},{"id":292006439,"identity":"5b05ad7a-c9ef-4000-a1bf-199f9a8833a1","order_by":4,"name":"surong deng","email":"","orcid":"","institution":"Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"surong","middleName":"","lastName":"deng","suffix":""},{"id":292006440,"identity":"d3749595-4856-402b-b4ae-7190ecd4d286","order_by":5,"name":"yu liu","email":"","orcid":"","institution":"Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"yu","middleName":"","lastName":"liu","suffix":""}],"badges":[],"createdAt":"2024-04-02 03:14:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4203479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4203479/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54827076,"identity":"c8d2c4cd-74ea-42b9-87b7-5ca4abd5cef5","added_by":"auto","created_at":"2024-04-17 10:15:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":207961,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the microfluidic chip structure. Physical diagram of the microfluidic chip, with orange dye indicating the sample pool, microchannels, and outlets of the microfluidic chip. A schematic diagram of the operation principle of the analysis system.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/9b2cdfdf667e106a4284a76d.png"},{"id":54827075,"identity":"cf0b3937-a8a9-4322-a8c3-73bcd5715ab9","added_by":"auto","created_at":"2024-04-17 10:15:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23482,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Platelet Adhesion Function Induced by Inducer, (b) Platelet Aggregation Function under the Action of Inducer\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/a973df1dce7b730ea33b1017.png"},{"id":54827080,"identity":"593db6aa-1356-4533-9246-58785728f824","added_by":"auto","created_at":"2024-04-17 10:15:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":475593,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of platelets with shear rate of 1,000, 1,500 and 4,000 s-1 and blood flow of 200 s ; Binarization image of platelet aggregation during blood flow for 200 s with shear rate of 1,000, 1,500, and 4,000 s-1 and blood flow of 200 s\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/4a4066c28ce36fea76ee9092.png"},{"id":54827078,"identity":"25b16990-c7de-4614-bfe6-a2afc86d6234","added_by":"auto","created_at":"2024-04-17 10:15:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15649,"visible":true,"origin":"","legend":"\u003cp\u003ePlatelet aggregation coverage, with the increase of shear rate, the aggregation coverage ratio increases\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/949cb597ff3d3022b52190b1.png"},{"id":54827079,"identity":"e132441a-b3c7-4fd5-a58f-e87582dd23e5","added_by":"auto","created_at":"2024-04-17 10:15:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":150300,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of shear rate on platelet aggregation and activation under flow condition\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/2a208d8679c09ec2038196b9.png"},{"id":54827077,"identity":"09936390-1ba4-454c-85fc-8e7a569b080d","added_by":"auto","created_at":"2024-04-17 10:15:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45242,"visible":true,"origin":"","legend":"\u003cp\u003eROC curve analysis results\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/ca814c34570eee5057406a18.png"},{"id":59390514,"identity":"a96a1e0c-bf4a-4fce-9bcd-d48aa872dbad","added_by":"auto","created_at":"2024-07-01 07:59:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1468661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4203479/v1/15ef7031-31a0-4c38-b9d2-591fada22d30.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thrombosis and Platelet Adhesion and Aggregation in the Third Trimester of Pregnancy under Arterial Shear Strip","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePregnancy is a unique physiological period in women. Pregnant women are in a hypercoagulable state due to changes in hormone levels, elevated fibrinogen levels, and other factors. These changes can provide preventive hemostatic protection against bleeding of the placental detachment surface and laceration after delivery and convenient conditions for the formation of arteriovenous thrombosis \u003csup\u003e[1,2]\u003c/sup\u003e. Studies have emphasized that the change of hormone levels and coagulation status during pregnancy changes with the week of pregnancy, the further increase of coagulation factor II, Ⅴ, Ⅶ, and Ⅹ, and other levels in the third trimester of pregnancy, the further decrease of antithrombin activity and fiber activity, and the increase of hypercoagulability, which is conducive to placental stripping during childbirth and reduce postpartum massive bleeding, but can increase the risk of thrombosis in pregnant women. The risk begins during early gestation and increases with increasing gestation time and is the highest in the early postpartum period. Compared with age-appropriate pregnant women, the risk of venous thromboembolism (VTE) increases by 15\u0026ndash;35 times, and some women have an 84 times increased risk of thrombosis in the first 6 weeks of the puerperal period \u003csup\u003e[3\u0026ndash;5]\u003c/sup\u003e. In the past, postpartum hemorrhage was the leading cause of death in perinatal women; however, recent studies have shown that the mortality rate of this disease has decreased with increasing attention paid to postpartum hemorrhage in many countries. VTE has become the leading cause of maternal death in some developed countries. Therefore, the early diagnosis of thrombotic diseases during pregnancy is of great significance. Approximately 20% of thrombosis in pregnancy cases include arterial thrombosis and the remaining 80% are venous thrombosis. Venous thrombosis is common in the lower limbs and pelvis thrombosis. The diagnosis of the disease is still based on color Doppler ultrasound and other imaging examinations; however, imaging examination has a certain lag, which is not conducive to the early diagnosis and treatment of the disease \u003csup\u003e[6,7]\u003c/sup\u003e. Therefore, the prevention of arteriovenous thrombosis (AVT) is critical. AVT often occurs after surgery and childbirth and can lead to death in severe cases. In a normal pregnancy, in the second and third trimesters, physiological hypercoagulability and a continuous increase in blood pressure leads to vasospasm and induce vascular ischemia, hypoxia, and endothelial injuries. During pregnancy, women are up to five times more likely to develop VTE than non-pregnant women of the same age \u003csup\u003e[8]\u003c/sup\u003e. Studies have reported that platelet aggregation is significantly enhanced in women during early, middle, and late pregnancy and during platelet activation, which may be related to the synthesis and release of a large amount of platelet-activating factor (PAF) \u003csup\u003e[9]\u003c/sup\u003e, and damage to vascular endothelial cells \u003csup\u003e[10]\u003c/sup\u003e. In addition, Edwards et al. found that three platelet activity indicators, PAF, CD62p, and CD63, significantly increased during the third trimester and postpartum \u003csup\u003e[11,12]\u003c/sup\u003e. Their expression levels were positively correlated with the platelet aggregation rate, and platelet aggregation activity also increased in patients with venous thrombosis. Researchers have found significant increases in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and plasmaβ-thromboglobulin levels \u003csup\u003e[13,14]\u003c/sup\u003e. Therefore, platelet adhesion, aggregation, and activation \u003cem\u003ein vivo\u003c/em\u003e are important components of thrombosis \u003csup\u003e[15]\u003c/sup\u003e. In addition to agonist induction, another method of platelet activation involves interactions with the cell and extracellular matrix components of the blood vessel wall at different shear rates, thereby generating mechanical forces that cause platelet aggregation and thrombosis. Shear-induced platelet aggregation is a major cause of pathological thrombosis and is rarely observed in normal hemostatic mechanisms. Researchers have long focused on the formation, prevention, and treatment of pathological thrombosis during pregnancy, and few studies have been conducted on the assessed platelet adhesion, aggregation function, and thrombosis ability of women in normal pregnancy and postpartum high-risk periods. Moreover, the platelet aggregation function was measured under static conditions without considering the instantaneous interaction between platelets and increased shear force. The ability to test the efficacy of platelet adhesion and aggregation in hemodynamic environments experienced by platelets \u003cem\u003ein vivo\u003c/em\u003e is limited, and thrombosis-related events cannot be observed in real time. Therefore, in this study, a microfluidic chip was used to accurately regulate microfluidic dynamics by simulating the size and structural characteristics of microvessels \u003cem\u003ein vivo\u003c/em\u003e to study platelet functions.\u003c/p\u003e \u003cp\u003eThe microfluidic chip used in this study can simulate \u003cem\u003ein vitro\u003c/em\u003e microchannel structures similar to the microvessel size and structure \u003cem\u003ein vivo\u003c/em\u003e and can precisely regulate fluid dynamic behavior; therefore, it can be used as an ideal microvessel model \u003cem\u003ein vitro\u003c/em\u003e to explore the physiological and pathological mechanisms related to coagulation and platelets, such as platelet aggregation under shear force regulation. We used microfluidic chip technology to simulate the \u003cem\u003ein vitro\u003c/em\u003e shear rates of the venous, arteriolar, and arterial walls of moderate stenosis at approximately 500; 1,500; and 4,000 s\u003csup\u003e-1\u003c/sup\u003e, respectively, under pathological conditions to study the adhesion and aggregation behaviors of platelets and the process of thrombosis. We found that platelet aggregation function was enhanced in the third trimester and postpartum period. Platelet adhesion increased under the low shear rate of slow blood flow, and aggregates with more and larger thrombi were easily washed away. However, with increasing shear rate, aggregates with fewer thrombi were small and not easily routed. We believe that platelet aggregation is enhanced in the third trimester of pregnancy; however, thrombus formation is weakened under normal blood flow conditions, which increases the risk of thrombus formation if the patient has other pregnancy complications. The purpose of this study was to analyze the value of different platelet function assessment methods and other thrombosis markers in patients with thrombotic diseases in the third trimester of pregnancy to provide new ideas and a theoretical basis for the early diagnosis of thrombotic diseases in pregnancy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eSylgard 184 polydimethylsiloxane (Dow Corning, USA), calcein AM (Invitrogen, USA), arachidonic acid(AA)(Jiangsu Inartis), adenosine diphosphate༈ADP༉ (Jiangsu Inartis), collagen༈COL༉ (Jiangsu Inartis), calf serum albumin (Shandong TeliKangxin Medical Technology Co., LTD.), CD61 (Integrin beta3), monoclonal antibody (VI-PL2), PerCP-eFluor710/CD62P (P-Selectin), monoclonal antibody (AK-4), PE/PAC-1 monoclonal antibody (PAC-1),FITC, PBS buffer (Gibco, USA), paraformaldehyde (Biyuntian Biotechnology, USA), sodium citrate venous blood vacuum collection tube (Weigao, Shandong), RSP01-CS bidirectional push-pull precision injection pump (Jiaxan Ruichuang), IX71 inverted fluorescence microscope (Olympus, Japan), plasma cleaner (PDG-32 G-2,Harrick, Germany), and Aggrestar (PL-12) multiparameter platelet function analyzer (Jiangsu Inovartis).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Collection and processing of blood samples\u003c/h2\u003e \u003cp\u003eFrom January 2021 to December 2022, blood samples were collected from pregnant women registered with the Obstetrics Department of Yongchuan Hospital of Chongqing Medical University and from women who gave birth naturally. Eligible non-pregnant women were 20 healthy volunteers recruited by the Physical Examination Center of Yongchuan Hospital of Chongqing Medical University. The inclusion criteria were as follows: pregnant woman with no pregnancy-related complications, no history of thrombosis, and normal platelet count. Non-pregnant volunteers were eligible if they had not taken the following drugs in the last 2 weeks: clopidogrel, aspirin, other antiplatelet drugs, statins, and lipid-lowering drugs and had normal platelet counts. This study was approved by the Ethics Committee of the Yongchuan Hospital of Chongqing Medical University (approval no. 2021016), and all participants provided written informed consent. Venous blood samples were collected in vacuum, 3.2% sodium citrate anticoagulant at 1:9 (v/v), and EDTA-K2 was added and used within 2 h. Calcein AM fluorescent dye was used to fluorescently label platelets in blood samples at a concentration of 1 mmol/L and was added to the blood sample at 1:500 (v/v), gently shaken, and incubated at 37\u0026deg;C for 10 min without light.\u003c/p\u003e \u003cp\u003e All study methods were carried out in accordance with the Ethics Committee of Yongchuan Hospital of Chongqing Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Basic data detection\u003c/h2\u003e \u003cp\u003eAnticoagulant whole blood without additives (5 mL) and anticoagulant whole blood with sodium citrate (3 mL) were collected simultaneously from the pregnant and non-pregnant groups, and progesterone, estradiol, fibrinogen, and D-dimer levels were detected by the instrument. White blood cell count, hemoglobin level, and platelet count were measured using an automatic blood cell analyzer. Differences in laboratory indices between the two groups were compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Continuous multiparameter method for detecting platelet adhesion and aggregation functions\u003c/h2\u003e \u003cp\u003ePlatelet aggregation induced by AA, ADP, and COL was detected by continuous multiparameter method. The test program was set in Aggrestar (PL-12), a 30 \u0026micro;L inducer was added corresponding to the program settings to the specified position. Sodium citrate anticoagulant was added to whole blood 300 \u0026micro;L to the sample test position 1. The instrument begins automatic detection by clicking on the start of the test button. The maximum platelet adhesion and aggregation rates induced by different inducers were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Hydrodynamic analysis of microchannel\u003c/h2\u003e \u003cp\u003eThe microchannels were subjected to a hydrophilic treatment before analysis using microfluidic chips. After treatment, the chip was placed on an inverted fluorescence microscope platform. The chip outlet was connected to the injection pump with a polytetrafluoroethylene tube (inner diameter 1.0 mm, outer diameter, 1.5 mm), and the flow speed of the blood sample in the microchannel was controlled in the pull-back mode. When the blood began to flow in the microchannel, Streampix 5.0 software was used to control the camera to record images of the adhesion and aggregation of fluorescent-labeled platelets on the narrow surface and narrow downstream surface after flowing through the microchannel at a frame rate of 1 frame/s (objective lens, \u0026times;20). A total of 180 sequential fluorescence images were obtained after 3 min of recording. A schematic and photographs of the experimental equipment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The flow rate in the chip through channel was set to 14, 52, and 40 \u0026micro;lL/min in the narrow channel. According to the finite element analysis, the shear rates formed by the three flow rates were 1,000; 1,500; and 4,000 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. These three flow rates simulated normal venous, small-artery, and moderate-stenosis flow rates. Anticoagulant whole blood samples of sodium citrate labeled with AM fluorescent dye were incubated at 37℃ for 100 min under light protection, and then added to the sample pool successively to observe platelet adhesion and aggregation behavior and thrombosis in real time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Platelet activation analysis by flow cytometry\u003c/h2\u003e \u003cp\u003eWomen with thrombosis were incubated at 37℃ for 30 min, then perfused into the microchannel sealed by bovine serum albumin, and the blood samples were collected at the volume flow rate of 52 \u0026micro;L/min and 100 \u0026micro;L/min, respectively. Five microliters of each sample were incubated with anti-human CD61, CD62 P, and PAC-1 antibodies for 20 min at room temperature in the dark, and one blood sample was labeled as a negative control. The labeled samples were mixed with 1% paraformaldehyde (1 mL) and fixed for 10 min. The expression of CD62P and PAC-1 was analyzed by flow cytometry. Data were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eMeasurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A randomized block design analysis of variance was used to compare the means of multiple groups, least significant difference test was used for multiple comparisons, paired t-test was used to compare the means of the two groups, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e3.1 During the third trimester and postpartum period, hormone levels in women increase, and the number of red blood cells and platelets decrease. The number of white blood cells slightly increased. Protein C activity, D-dimer levels, and fibrinogen levelsincreased (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBasic data of research object (n\u0026thinsp;=\u0026thinsp;76)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThird trimester of pregnancy\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEstradiol (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProgesterone (ng/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e215.12\u0026thinsp;\u0026plusmn;\u0026thinsp;30.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eErythrocytes (*10^12/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.516\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeukocytes (*10^9/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlatelets (*10^9/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e239.4\u0026thinsp;\u0026plusmn;\u0026thinsp;50.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e196.4\u0026thinsp;\u0026plusmn;\u0026thinsp;48.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD-dimer (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFiB (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is significant. FiB, fibrinogen\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.2 Platelet adhesion and aggregation in third trimester women under static conditions\u003c/h2\u003e\n \u003cp\u003eAfter blood was added to the inducers, AA, ADP, COL, and PL-12 levels were used to evaluate the platelet reactivity to the inducers under static conditions. No significant difference in platelet aggregation function (AA 47.52\u0026thinsp;\u0026plusmn;\u0026thinsp;5.43, ADP 61.08\u0026thinsp;\u0026plusmn;\u0026thinsp;5.09, COL 52.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78) between pregnant women and control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was noted, as shown in Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.3 Platelet adhesion and aggregation enhancement and increased thrombosis in shear rate environment\u003c/h2\u003e\n \u003cp\u003ePrevious studies have shown that shear-induced platelet aggregation is the major cause of thrombosis \u003csup\u003e[16]\u003c/sup\u003e This study investigated the differences in platelet aggregation in the blood of different women in the third trimester under different shear force conditions. Figure\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e(a) and (b) show the image sequence of platelets flowing for 200 s under different shear rate gradients and the images after binarization. When whole blood flowed at 1,000 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e input shear rate, platelets were \u0026ldquo;small and scattered\u0026rdquo; in the microchannel. When the shear rate was increased to 1,500 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the platelet aggregation density increased. When the wall shear rate reached 4,000 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the degree of platelet aggregation was further enhanced and a dense, highlighted state was observed. Figure\u0026nbsp;\u003cspan\u003e3\u003c/span\u003ec, d (c,d) show the binarization images of platelet aggregation. A gradual increase in the wall shear rate was observed. The total area and coverage of platelet aggregates significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0. 05), as shown in Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.4 Platelet activation was enhanced in late pregnancy and postpartum women\u003c/h2\u003e\n \u003cp\u003eFlow cytometry was used to assess the expression of the platelet surface activation markers (P-selectin and PAC-1) flowing through the closed microchannels (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e). Platelets were identified in the whole blood using a CD61 monoclonal antibody, and the adhered parts of the platelets were excluded. The expression levels of P-selectin and activated GDPⅡb/Ⅲa in single platelets were analyzed. The results showed that the expression levels of P-selectin and GPⅡb/Ⅲas on the platelet surface of pregnant women increased after high-shear induction, the expression levels in postpartum thrombus patients had significantly increased, and platelet aggregation had occurred.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.5 ROC curve analysis\u003c/h2\u003e\n \u003cp\u003eAccording to the receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) of dynamic shear rate, static platelet function detection, thromboembolic marker (D-dimer), and platelet activation marker (CD62P) detection for predicting the risk of thrombosis in women in late pregnancy were 0.707, 0.628, 0.823, and 0.878, respectively, (see Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e,6 Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eROC curve analysis results.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAUC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSensitivity(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecificity(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStatic platelet function test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.533\u0026ndash;0.723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD dimer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.621\u0026ndash;0.792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDynamic platelet function test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.762\u0026ndash;0.892\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD62P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.878\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.825\u0026ndash;0.931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn healthy people, the fibrinolytic and coagulation systems are in a state of long-term dynamic balance through which coagulation and hemostasis are achieved \u003csup\u003e[17]\u003c/sup\u003e.Thrombosis can be caused when blood stasis, blood in a hypercoagulable state, or vascular endothelial injury occurs. In addition to these factors, the maternal body can be in a hypercoagulable state to meet childbirth needs. In addition, the sudden increase in fetal weight in late pregnancy aggravates the suppression of the uterus in maternal lower limb circulation and causes the lower limb venous blood flow speed to drop sharply. At the same time, the pregnant women were less active than usual. Under the combined effect of these factors, the risk of thrombotic disease in late pregnancy significantly increased \u003csup\u003e[18,19]\u003c/sup\u003e. Thromboembolic diseases have a rapid onset and can cause sudden death if not treated in a timely and effective manner. Therefore, predicting thromboembolic diseases during pregnancy is important. Although imaging is the gold standard for the diagnosis of thromboembolic disease in pregnancy, it has a certain lag and limited predictive value \u003csup\u003e[20,21 ]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlatelet function is being increasingly evaluated under physiological flow conditions to simulate the flow shear rate environment \u003cem\u003ein vivo\u003c/em\u003e. In this study, we measured the optical aggregation of platelets under different morphological conditions and used microfluidic chip technology to monitor platelet thrombosis in whole blood, a method used to simulate human hemostasis and thrombosis \u003cem\u003ein vitro\u003c/em\u003e. Therefore, detection of platelet aggregation under physiological conditions is important. During pregnancy, uterine vessels undergo significant vascular remodeling mediated by vascular endothelial and placental growth factors, leading to potential platelet-collagen matrix interactions and changes in blood flow rheology. The results of this study showed that platelet adhesion and aggregation effects in pregnant women in the third trimester of pregnancy were not significantly different from those in non-pregnant women under static conditions. The expression of platelet activation markers in pregnant women and significantly increased in platelet aggregation was observed, and the platelets showed aggregation. Platelet adhesion and aggregation were enhanced under arterial shear conditions; however, stable platelet aggregation did not occur, indicating that the small blood aggregation function was enhanced at high shear rates in the third trimester of pregnancy, but adhesion stability was decreased. This may reduce the risk of arterial thrombosis to a certain extent and may also be related to the partial deaggregation of platelets induced by the pathological shear rate \u003cem\u003ein vitro\u003c/em\u003e over a certain period; however, the specific mechanism requires further study. It can also be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e assessing the platelet aggregation function in a dynamic environment to predict the risk of thrombosis is the most valuable method for the current diagnosis of thrombosis. Therefore, our results suggest that the detection of platelet aggregation effects under arterial flow conditions may provide valuable research data for the diagnosis of potential thrombosis or assessment of bleeding risk during pregnancy.\u003c/p\u003e\u003cp\u003eThe current clinical methods used to evaluate the platelet function have significant limitations. These are usually measured as changes in absorbance caused by platelet aggregation under the stimulation of thrombin, collagen, adenosine diphosphate, and other chemical activators, which do not reflect the shear force of platelets flowing through narrow blood vessels. Shear stress is a key factor in platelet activation through the regulation of mechanical action. Moreover, in arterial thrombotic diseases, blood vessel stenosis can cause blood flow to be subjected to a shear force much higher than the physiological levels, and such a high shear force can directly induce rapid platelet activation/aggregation \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e[22–24]\u003c/sup\u003e. It affects membrane receptor aggregation, membrane tension, integrin expression, cytoskeletal modification, and signal transduction. Therefore, the microfluidic chip technology used in this study provides a controllable shear rate for detecting platelet reactivity and thrombosis under simulated arterial conditions in vitro. PAC-1 is the fibrinogen binding site exposed after the activation of glycoprotein Ⅱb/Ⅲa, namely activated glycoprotein Ⅱb/Ⅲa. CD62P is a protein component of the granulosa membrane of the resting platelets. The degranulosa membrane rapidly fuses with platelets during platelet activation, and P-selectin is transferred to the surface of the platelets or the degranulosa becomes soluble in P-selectin. Previous studies have shown that CD62P is a reliable marker of split-dependent platelet activation \u003csup\u003e[25]\u003c/sup\u003e. In this study, flow cytometry was used to detect the expression of activation markers in platelets subjected to shear rate, indicating that an increase in the shear rate activates platelets and leads to increased platelet activation. However, no significant platelet aggregation was found, which may be related to the changes in blood flow status and hormone levels in the third trimester\u003csup\u003e[24]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStrengths:In our study, microfluidic chip technology was used to simulate bionic blood vessels in vitro, so that the functional detection of platelets was exposed to physiological flow environment, and the results may be more reliable.\u003c/p\u003e\n\u003cp\u003eLimitations:The sample size is small, and the corresponding mechanism of platelet function enhancement in late pregnancy remains to be studied\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, compared to healthy non-pregnant women in the third trimester, the platelet aggregation effect was significantly enhanced and activation was increased; however, the stability of the formed platelet aggregates was not significantly increased, and the specific mechanism remains to be further studied. Our study preliminatively proves that the microfluidic chip system can be used to evaluate the clip-induced platelet aggregation and activation in the third trimester of pregnancy when the blood flow changes significantly and the hormone levels are high and can analyze the differences in platelet reactivity under different shear rates. The combined detection of platelet activation markers is helpful for predicting thrombotic diseases in the third trimester of pregnancy. This may provide a new theoretical basis for the early diagnosis of thrombotic diseases during pregnancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial support :\u003c/strong\u003eThis work was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission [grant number KGQN202100442]; the Natural Science Foundation of Yongchuan District [grant numbers 2022yc-jckx20052, 2023yc-jckx20027].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflicts of Interest :\u003c/strong\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability :\u003c/strong\u003eData is provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eH.C. conceived the experiments and wrote the article, M.H.D contributed to the experimental design and data analysis, H.X.J.andG.X.M. contributed to the experimental operation, L.Y. contributed to specimen collection, and D.S.R. contributed to the experimental guidance and review of the article.All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang C, Yang HX. New recommendations for thromboembolism in pregnancy by the American College of Obstetricians and Gynecologists in 2018 [J]. Chinese Journal of Perinatal Medicine 2019;22:139\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eSUN M, LIU C, ZHAO N, et al. Predictive value of platelet aggregation rate in postpartum deep venous thrombosis and its possible mechanism [J].Exp Ther Med 2018;15:5215\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eE.R. Pomp, A.M. Lenselink, F.R. Rosendaal, C.J. Doggen, Pregnancy, the postpartum period and prothrombotic defects: risk of venous thrombosis in the MEGA study, J Thromb Haemost. 6 (2008) 632\u0026ndash;637\u003c/li\u003e\n\u003cli\u003eE. Jackson, K.M. Curtis, M.E. Gaffield, Risk of venous thromboembolism during the postpartum period: a systematic review, ObstetGynecol. 117 (2011) 691-703\u003c/li\u003e\n\u003cli\u003eA. Sultan, J. West, L.J. Tata, K.M. Fleming, C. Nelson-Piercy, M.J. 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Tovar-Lopez, et al., A shear gradient-dependent platelet aggregation mechanism drives thrombus formation, Nat Med. 15 (2009) 665-673\u003c/li\u003e\n\u003cli\u003eKamada H, Imai Y, Nakamura M, et al. Shear-induced platelet aggregation and distribution of thrombogenesis at stenotic vessels. Micnocirculation 2017;24.\u003c/li\u003e\n\u003cli\u003eMcEver RP, Seleclins. Initiators of leucocyte adhesion and signalling at the vascular wall, Cardiovasc Res 2015;107:331\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eVal\u0026eacute;ra MC, Parant O, Cenac C, et al. Platelet adhesion and thrombus formation in whole blood at arterial shear rate at the end of pregnancy. Am J Reprod Immunol 2015;74:533\u0026ndash;41. doi: 10.1111/aji.12433. Epub 2015 Oct 4. PMID: 26435170.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Shear rate, platelet, adhesion and aggregation, late pregnancy thrombosis, predictive value","lastPublishedDoi":"10.21203/rs.3.rs-4203479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4203479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlatelet adhesion and aggregation effect increases in third-trimester wemen, and the risk of thrombosis increases, so how to achieve early diagnosis is particularly important.In this study, microfluidic chip technology was used to study the adhesion and aggregation behavior of platelets in third-trimester under different arterial shear rates (1000s-1, 1500s-1, 4000s-1). Flow cytometry was used to analyze platelet surface activation markers (PAC-1 and P-selectin CD62P), and to explore the diagnostic value of different platelet function assessment methods for the risk of third-trimester thrombosis in normal pregnant women. Compared to healthy controls, white blood cell, fibrinogen, D-dimer levels increased, while platelet levels decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant difference observed in platelet reactivity to agonist induction under static conditions ( P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Platelet aggregation and surface activation marker expression significantly increased with the increase of shear rate under flow conditions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression of platelet surface activation markers elevated.So we believe that using microfluidic chip technology to evaluate platelet aggregation and thrombosis in the third-trimester under arterial flow conditions combined with platelet activation can help predict thrombotic diseases. And the results may provide effective clinical application data and a theoretical basis for the diagnosis and prevention of platelet dysfunction and thrombotic diseases during pregnancy.\u003c/p\u003e","manuscriptTitle":"Thrombosis and Platelet Adhesion and Aggregation in the Third Trimester of Pregnancy under Arterial Shear Strip","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 10:15:37","doi":"10.21203/rs.3.rs-4203479/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4eba3543-8470-41d1-9a32-428e9919be3f","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T07:51:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-17 10:15:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4203479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4203479","identity":"rs-4203479","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}