Proteomics and Metabolomics Profiling of Platelets and Plasma Mediators of Thrombo-Inflammation in Gestational Hypertension and Preeclampsia 妊娠高血压和子痫前期血小板和血浆炎症介质的蛋白质组学和代谢组学分析
Luiz Gustavo N. de Almeida ^(1,2){ }^{1,2}, Daniel Young ^(1){ }^{1}, Lorraine Chow ^(3){ }^{3}, Joshua Nicholas ^(3){ }^{3}, Adrienne Lee ^(4,5){ }^{4,5}, Man-Chiu Poon ^(6){ }^{6} (D) Antoine Dufour ^(1,2,7){ }^{1,2,7} (D) and Ejaife O. Agbani ^(4,7,**(" (D) "){ }^{4,7, *(\text { (D) }} Luiz Gustavo N. de Almeida ^(1,2){ }^{1,2} ， Daniel Young ^(1){ }^{1} ， Lorraine Chow ^(3){ }^{3} ， Joshua Nicholas ^(3){ }^{3} ， Adrienne Lee ^(4,5){ }^{4,5} ， Man-Chiu Poon ^(6){ }^{6} （D） Antoine Dufour ^(1,2,7){ }^{1,2,7} （D） 和 Ejaife O. Agbani ^(4,7,**(" (D) "){ }^{4,7, *(\text { (D) }}1 McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB T2N 1N4, Canada; luizgustavo.almeida@ucalgary.ca (L.G.N.d.A.); daniel.young1@ucalgary.ca (D.Y.); antoine.dufour@ucalgary.ca (A.D.) 1 卡尔加里大学麦凯格骨与关节健康研究所，卡尔加里，AB T2N 1N4，加拿大;luizgustavo.almeida@ucalgary.ca （LGNDA）;daniel.young1@ucalgary.ca （D.Y.）;antoine.dufour@ucalgary.ca （公元）2 Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada 2 卡尔加里大学卡明医学院生物化学与分子生物学系，加拿大卡尔加里 AB T2N 1N43 Department of Anaesthesiology, Perioperative and Pain Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; lorraine.chow121@gmail.com (L.C.); jnichola@ucalgary.ca (J.N.) 3 卡尔加里大学卡明医学院麻醉学、围手术期和疼痛医学系，加拿大卡尔加里 AB T2N 1N4;lorraine.chow121@gmail.com （LC）;jnichola@ucalgary.ca （J.N.）4 Libin Cardiovascular Institute, Calgary, AB T2N 1N4, Canada; adrienne.lee@bccancer.bc.ca 4 利宾心血管研究所，卡尔加里，AB T2N 1N4，加拿大;adrienne.lee@bccancer.bc.ca5 Division of Hematology, Department of Medicine/Medical Oncology, University of British Columbia, Island Health, Victoria, BC V6T 1Z4, Canada 5 英属哥伦比亚大学医学/肿瘤内科血液学系，Island Health，维多利亚，BC V6T 1Z4，加拿大6 Division of Hematology & Hematological Malignancies, Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; mcpoon@ucalgary.ca 6 卡尔加里大学卡明医学院医学系血液学和血液恶性肿瘤部门，AB T2N 1N4，加拿大;mcpoon@ucalgary.ca7 Department of Physiology & Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada 7 卡尔加里大学卡明医学院生理学与药理学系，AB T2N 1N4，加拿大* Correspondence: ejaife.agbani@ucalgary.ca * 对应关系：ejaife.agbani@ucalgary.ca
Citation: de Almeida, L.G.N.; Young, D.; Chow, L.; Nicholas, J.; Lee, A.; Poon, M.-C.; Dufour, A.; Agbani, E.O Proteomics and Metabolomics Profiling of Platelets and Plasma Mediators of Thrombo-Inflammation in Gestational Hypertension and Preeclampsia. Cells 2022, 11, 1256. https://doi.org/10.3390/ cells11081256 引自：de Almeida， L.G.N.;杨，D.;周， L.;尼古拉斯 J.;李，A.;潘，MC;杜福尔，A.;Agbani， E.O 蛋白质组学和代谢组学 妊娠高血压和子痫前期血栓炎症的血浆介质分析。细胞 2022， 11， 1256。https://doi.org/10.3390/ 细胞11081256
Received: 25 February 2022 收稿日期： 2022-02-25
Accepted: 5 April 2022 录用日期： 2022-04-05
Published: 7 April 2022 已发布： 7 四月 2022
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Platelets may be pivotal mediators of the thrombotic and coagulopathic complications of preeclampsia (PE), linking inflammation and thrombosis with endothelial and vascular dysfunction. Both PE and gestational hypertension (GH) fall within the spectrum of hypertensive complications of pregnancy, with GH being a risk factor for preeclampsia. However, it is unclear what biomarkers distinguish PE from GH. Using a discovery size cohort, we aimed to characterize specific plasma and platelet thrombo-inflammatory drivers indicative of PE and differentiate PE from GH. We performed multiplex immunoassays, platelet and plasma quantitative proteomics and metabolomics of PE patients, comparing with non-pregnant (NP), healthy pregnant controls (PC) and GH participants. The expression pattern of plasma proteins and metabolites in PE/GH platelets was distinct from that of NP and PC. Whilst procoagulation in PC may be fibrinogen driven, inter-alpha-trypsin inhibitors ITIH2 and ITIH3 are likely mediators of thrombo-inflammation in GH and PE, and fibronectin and S100A8/9 may be major procoagulant agonists in PE only. Also enriched in PE were CCL1 and CCL27 plasma cytokines, and the platelet leucine-rich repeat-containing protein 27 and 42 (LRRC27/42), whose effects on platelets were explored using STRING analysis. Through protein-protein interactions analysis, we generated a new hypothesis for platelets’ contribution to the thrombo-inflammatory states of preeclampsia. 血小板可能是子痫前期 （PE） 血栓形成和凝血功能障碍的关键介质，将炎症和血栓形成与内皮和血管功能障碍联系起来。PE 和妊娠高血压 （GH） 都属于妊娠高血压并发症的范围，其中 GH 是子痫前期的危险因素。然而，目前尚不清楚哪些生物标志物将 PE 与 GH 区分开来。使用发现规模队列，我们旨在表征指示 PE 的特定血浆和血小板血栓炎症驱动因素，并区分 PE 和 GH。我们对 PE 患者进行了多重免疫测定、血小板和血浆定量蛋白质组学以及代谢组学，并与非妊娠 （NP） 、健康妊娠对照 （PC） 和 GH 参与者进行比较。PE/GH 血小板中血浆蛋白和代谢物的表达模式与 NP 和 PC 不同。虽然 PC 中的凝血可能是纤维蛋白原驱动的，但 α 胰蛋白酶间抑制剂 ITIH2 和 ITIH3 可能是 GH 和 PE 中血栓炎症的介质，而纤连蛋白和 S100A8/9 可能仅是 PE 中的主要促凝激动剂。PE 中还富含 CCL1 和 CCL27 浆细胞因子，以及富含血小板亮氨酸重复序列的蛋白 27 和 42 （LRRC27/42），使用 STRING 分析探讨了它们对血小板的影响。通过蛋白质-蛋白质相互作用分析，我们提出了血小板对子痫前期血栓炎症状态的贡献的新假设。
Preeclampsia (PE) complicates up to 8%8 \% of pregnancies; it is defined as elevated blood pressure ( > 140//90mmHg>140 / 90 \mathrm{mmHg} on at least two occasions and > 6h>6 \mathrm{~h} apart) at or beyond 20 weeks gestation, and proteinuria ( >= 300mg//24h\geq 300 \mathrm{mg} / 24 \mathrm{~h} ) or adverse conditions, such as elevated liver enzymes, which are known to increase the risk of severe complications [1,2]. Gestational hypertension (GH), considered a risk factor for PE [3-5], is characterized by an elevated 子痫前期 （PE） 使 8%8 \% 怀孕复杂化;其定义为妊娠 20 周或之后血压升高（ > 140//90mmHg>140 / 90 \mathrm{mmHg} 至少间隔 2 次） > 6h>6 \mathrm{~h} 和蛋白尿 （ >= 300mg//24h\geq 300 \mathrm{mg} / 24 \mathrm{~h} ） 或肝酶升高等不良情况，已知这些情况会增加严重并发症的风险 [1,2]。妊娠高血压（gestational hypertension， GH）被认为是肺栓塞的危险因素[3-5]，其特征是
blood pressure (sBP >= 140mmHg\geq 140 \mathrm{mmHg} and/or dBP >= 90mmHg\mathrm{dBP} \geq 90 \mathrm{mmHg} ) at or beyond week 20 of pregnancy but without proteinuria or adverse conditions [1,2]. In this study, we used the definitions of hypertensive disorders of pregnancy according to the Society of Obstetricians and Gynecologists of Canada guidelines [1,6], to distinguish between preeclampsia and gestational hypertension. Both GH and PE are considered hypertensive disorders of pregnancy [7-9], with PE being more severe and resulting in more clinical complications than GH [7-9]. Preeclampsia can lead to significant adverse outcomes, including fetal and intrauterine growth restriction, severe maternal hypertension, and end-organ dysfunction that may result in preterm delivery, placental abruption, and maternal and perinatal death [1,10-14]. Its hematological manifestations include thrombocytopenia, thrombosis, and postpartum hemorrhage [1,6]. The cause of preeclampsia is still unclear, but there is evidence that platelets may be pivotal mediators of its complications, linking inflammation and thrombosis with endothelial and vascular dysfunction [15-19]. 妊娠 20 周或之后的血压（sBP >= 140mmHg\geq 140 \mathrm{mmHg} 和/或 dBP >= 90mmHg\mathrm{dBP} \geq 90 \mathrm{mmHg} ），但没有蛋白尿或不良情况 [1,2]。在这项研究中，我们根据加拿大妇产科医师协会指南[1,6]使用妊娠期高血压疾病的定义来区分子痫前期和妊娠高血压。GH 和 PE 都被认为是妊娠期高血压疾病 [7-9]，其中 PE 比 GH 更严重，导致的临床并发症更多 [7-9]。子痫前期可导致显著的不良结局，包括胎儿和宫内生长受限、重度母体高血压和终末器官功能障碍，这些都可能导致早产、胎盘早剥以及孕产妇和围产期死亡[1,10-14]。其血液学表现包括血小板减少、血栓形成和产后出血[1,6]。子痫前期的病因尚不清楚，但有证据表明血小板可能是其并发症的关键介质，将炎症和血栓形成与内皮和血管功能障碍联系起来[15-19]。
Early in pregnancy, platelets undergo differential granular secretions, releasing vasoactive agents important to the formation and maintenance of blood and lymphatic vessels, as well as to the maintenance of endothelial function and vascular integrity [17,20-23]. Procoagulant platelet factors are increased in patients with preeclampsia, such as plasma levels of transforming growth factor- beta\beta (TGF- beta\beta ) and platelet factor-4 (PF4 or CXCL4) [18,22]. Additionally, several studies report on the role of micro- and nanoparticles present in the circulation in preeclampsia. When isolated and injected into pregnant mouse models, these particles can induce a preeclampsia state via direct effects on the endothelium to reduce proliferation and cause dysfunction [19,24]. The micro- and nanoparticles appear to originate mostly from activated platelets ( 97-99%97-99 \% ), but they are also released from syncytiotrophoblasts [25]. It is well established that platelets are abnormally activated in preeclampsia [26,27], yet the precise agonist, or combination of agonists, at play in preeclampsia is unknown. Such a degree of procoagulation is not present in a healthy pregnancy. Taken together, the platelet activation process itself may contribute to the progression, if not the pathogenesis of preeclampsia. 在妊娠早期，血小板会经历不同的颗粒状分泌，释放出对血液和淋巴管的形成和维持以及维持内皮功能和血管完整性很重要的血管活性物质[17,20-23]。子痫前期患者的促凝血小板因子升高，例如血浆转化生长因子 （ beta\beta TGF- beta\beta ） 和血小板因子 4 （PF4 或 CXCL4） 水平 [18,22]。此外，几项研究报告了子痫前期循环中存在的微粒和纳米颗粒的作用。当分离并注射到怀孕的小鼠模型中时，这些颗粒可以通过直接作用于内皮细胞来诱导子痫前期状态，从而减少增殖并导致功能障碍[19,24]。微粒和纳米颗粒似乎主要来源于活化的血小板 （ 97-99%97-99 \% ），但它们也从合体滋养层细胞中释放出来 [25]。已明确指出，血小板在子痫前期中异常激活[26,27]，但在子痫前期中起作用的确切激动剂或激动剂联合作用尚不清楚。这种程度的促凝血在健康妊娠中不存在。综上所述，血小板活化过程本身可能有助于子痫前期的进展，如果不是子痫前期的发病机制的话。
Understanding the role of platelets in the development and or perpetuation of preeclampsia may enable the early identification of patients at risk of preeclampsia and the development of more targeted therapies. Besides having distinct epidemiology and hemodynamic characteristics, PE also differs from GH in pathology and pathogenesis [3,28-32]. In this study, we analyzed proteomics and metabolomics changes in plasma and platelets derived from PE and GH patients, compared with non-pregnant (NP) and healthy pregnant (PC) participants, to identify potential drivers of thrombo-inflammation in PE and GH. Next, we addressed whether PE can be distinguished from GH at the initial finding of hypertension by assessing biomarkers at time of diagnosis. Unique to PE was the overexpression in platelets of leucine-rich repeat-containing proteins 27/42 (LRRC27/42) [33,34], two proteins with limited characterization that could play biological roles in cell adhesion, signal transduction and apoptosis, and S100A9, a Ca^(2+)\mathrm{Ca}^{2+} and Zn^(2+)\mathrm{Zn}^{2+} protein regulating inflammation and immune responses [35,36], which we functionally validated. Lastly, we utilized pathway analysis to generate a new hypothesis for platelets’ contribution to the thrombo-inflammatory state of preeclampsia. 了解血小板在子痫前期发展和/或持续发展中的作用可能有助于早期识别有子痫前期风险的患者并开发更有针对性的治疗方法。除了具有不同的流行病学和血流动力学特征外，PE 在病理学和发病机制上也与 GH 不同 [3,28-32]。在这项研究中，我们分析了 PE 和 GH 患者血浆和血小板的蛋白质组学和代谢组学变化，与非怀孕 （NP） 和健康怀孕 （PC） 参与者相比，以确定 PE 和 GH 中血栓炎症的潜在驱动因素。接下来，我们通过评估诊断时的生物标志物，解决了是否可以在最初发现高血压时区分 PE 和 GH。PE 的独特之处在于在血小板中过表达富含亮氨酸重复序列的蛋白质 27/42 （LRRC27/42） [33,34]，这两种蛋白质的特性有限，可以在细胞粘附、信号转导和细胞凋亡中发挥生物学作用，以及 S100A9，一种 Ca^(2+)\mathrm{Ca}^{2+} 调节 Zn^(2+)\mathrm{Zn}^{2+} 炎症和免疫反应的蛋白质 [35,36]，我们对此进行了功能验证。最后，我们利用通路分析为血小板对子痫前期血栓炎症状态的贡献提出了新的假设。
2. Methods 2. 方法
2.1. Methods Statement 2.1. methods 语句
Written informed consent was obtained in accordance with the Declaration of Helsinki. Blood samples were obtained from participants meeting study inclusion criteria (see Section 3) under the University of Calgary, Research Ethics Board approval (REB18-1545). All methods were performed in accordance with the Alberta Health Services and The University of Calgary research guidelines and regulations. 根据赫尔辛基宣言获得书面知情同意书。从符合卡尔加里大学研究伦理委员会批准 （REB18-1545） 的研究纳入标准 （见第 3 节） 的参与者那里获得血样。所有方法均按照阿尔伯塔省卫生服务和卡尔加里大学的研究指南和规定进行。
2.2. Human Platelet-Rich Plasma Preparation 2.2. 人富血小板血浆制备
Blood was collected into blue-capped sodium citrate tubes ( 0.109M//3.2%0.109 \mathrm{M} / 3.2 \% ) and centrifuged at 180 xx g180 \times g for 17 min to prepare platelet-rich plasma (PRP). All blood samples were processed within 1-2 h of collection. 将血液收集到蓝色帽柠檬酸钠管 （ 0.109M//3.2%0.109 \mathrm{M} / 3.2 \% ） 中，离心 180 xx g180 \times g 17 分钟以制备富血小板血浆 （PRP）。所有血样在采集后 1-2 小时内处理完毕。
2.3. Plasma and Platelet Lysates Preparation 2.3. 血浆和血小板裂解物制备
PRP was centrifuged at 650 xx g650 \times g to obtain the platelet-poor plasma (supernatant) for storage ( -80^(@)C-80^{\circ} \mathrm{C} ) until used. Plasma and platelets were lysed with buffer containing 1%1 \% SDS, 200 mM HEPES ( pH 8.0 ), 100 mM ammonium bicarbonate, 10 mM EDTA and protease inhibitor cOmplete ^(TM){ }^{\mathrm{TM}} tablets (Roche, Mississauga, ON, Canada). Disulfide bonds were reduced with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific, Mississauga, ON, Canada) at 55^(@)C55^{\circ} \mathrm{C} for 1 h and cysteines were alkylated with 15 mM iodoacetamide (VWR, Mississauga, ON, Canada) for 25 min in the dark at room temperature. Protein precipitation was performed with 600 muL600 \mu \mathrm{~L} of ice-cold acetone, incubated at -20^(@)C-20^{\circ} \mathrm{C} overnight, and followed with centrifugation at 8000 xx g8000 \times g for 10 min . 将 PRP 离心 650 xx g650 \times g 以获得贫血小板血浆（上清液）用于储存 （ -80^(@)C-80^{\circ} \mathrm{C} ） 直至使用。用含有 1%1 \% SDS、200 mM HEPES （pH 8.0）、100 mM 碳酸氢铵、10 mM EDTA 和蛋白酶抑制剂 cOmplete ^(TM){ }^{\mathrm{TM}} 片剂的缓冲液（Roche，Mississauga，ON，Canada）裂解血浆和血小板。用 10 mM Tris（2-羧乙基）膦盐酸盐（Thermo Fisher Scientific，Mississauga，ON，Canada） 55^(@)C55^{\circ} \mathrm{C} 还原二硫键 1 小时，用 15 mM 碘乙酰胺（VWR，Mississauga，ON，Canada）在室温下避光烷基化半胱氨酸 25 分钟。用 600 muL600 \mu \mathrm{~L} 冰冷的丙酮进行蛋白质沉淀，孵育 -20^(@)C-20^{\circ} \mathrm{C} 过夜，然后离心 8000 xx g8000 \times g 10 分钟。
2.4. Quantitative Proteomics 2.4. 定量蛋白质组学
The four groups of patients’ plasma and platelets were subjected to quantitative shotgun proteomics analysis, as previously described (Table 1) [37]. Plasma and platelet analysis were performed on non-pregnant (NP), healthy pregnant controls (PC), gestational hypertension (GH), and preeclampsia (PE) participants. A total of 100 mug100 \mu \mathrm{~g} per samples was analyzed. Proteins were resuspended in 100 muL100 \mu \mathrm{~L} of 50 mM triethyl ammonium bicarbonate and trypsinized (Thermo Fisher Scientific, Mississauga, ON, Canada) overnight at a 1:10 enzyme-to-substrate ratio. TMTsixplex ^(TM){ }^{\mathrm{TM}} (Thermo Fisher Scientific, Mississauga, ON, Canada) labeling was performed according to the manufacturer’s manual (Table S1). In summary, 0.8 mg of TMT reagent was resuspended in 41 muL41 \mu \mathrm{~L} of acetonitrile (ACN), samples were spun down at 4000 xx g4000 \times g for 10 s and incubated at room temperature for 1 h . The reaction was quenched with 8muL8 \mu \mathrm{~L} of 5%5 \% hydroxylamine and incubated for 15 min at 25^(@)C25^{\circ} \mathrm{C}. Peptides with different labels were combined followed by 100%100 \% formic acid (FA) addition to each sample to reach a volumetric concentration of 1%1 \% FA. Samples were spun at 5000 rpm for 10 min before desalting using Sep-Pak C18 columns (Waters, 130 mg WAT023501) conditioned with 1xx3mL90%1 \times 3 \mathrm{~mL} 90 \% methanol/0.1% TFA, 1xx2mL0.1%1 \times 2 \mathrm{~mL} 0.1 \% formic acid. Each sample was loaded onto a column, washed with 1xx3mL0.1%TFA//5%1 \times 3 \mathrm{~mL} 0.1 \% \mathrm{TFA} / 5 \% methanol, and the peptides were eluted off the column with 1xx1mL50%ACN//0.1%1 \times 1 \mathrm{~mL} 50 \% \mathrm{ACN} / 0.1 \% FA. Peptides were lyophilized prior to resuspension in 1% FA and a colorimetric peptide assay (Thermo Fisher Scientific, Mississauga, ON, Canada) was used to determine the concentration in each sample. The resulting tryptic peptide samples were dried down and stored at -80^(@)C-80^{\circ} \mathrm{C}. 如前所述，对四组患者的血浆和血小板进行定量鸟枪法蛋白质组学分析（表 1）[37]。对非妊娠 （NP） 、健康妊娠对照 （PC） 、妊娠高血压 （GH） 和子痫前期 （PE） 参与者进行血浆和血小板分析。分析了每个样本的总数 100 mug100 \mu \mathrm{~g} 。将蛋白质重悬于 100 muL100 \mu \mathrm{~L} 50 mM 碳酸氢三乙基铵中，并以 1：10 的酶与底物比例（Thermo Fisher Scientific，Mississauga，ON，Canada）过夜。根据制造商手册（表 S1）进行 TMTsixplex ^(TM){ }^{\mathrm{TM}} （Thermo Fisher Scientific，Mississauga，ON，Canada）标记。综上所述，将 0.8 mg TMT 试剂重悬于乙腈 （ACN） 中 41 muL41 \mu \mathrm{~L} ，将样品离心 4000 xx g4000 \times g 10 秒并在室温下孵育 1 小时。用 8muL8 \mu \mathrm{~L}5%5 \% 羟胺淬灭反应，并在 中孵育 25^(@)C25^{\circ} \mathrm{C} 15 分钟。合并具有不同标记的肽，然后向每个样品中加入 100%100 \% 甲酸 （FA），以达到 FA 的 1%1 \% 体积浓度。样品以 5000 rpm 离心 10 分钟，然后使用 Sep-Pak C18 色谱柱（沃特世，130 mg WAT023501）脱盐，用 1xx3mL90%1 \times 3 \mathrm{~mL} 90 \% 甲醇/0.1% TFA、 1xx2mL0.1%1 \times 2 \mathrm{~mL} 0.1 \% 甲酸活化。将每个样品上样到色谱柱上，用 1xx3mL0.1%TFA//5%1 \times 3 \mathrm{~mL} 0.1 \% \mathrm{TFA} / 5 \% 甲醇洗涤，然后用 1xx1mL50%ACN//0.1%1 \times 1 \mathrm{~mL} 50 \% \mathrm{ACN} / 0.1 \% FA 将肽从色谱柱上洗脱。将肽冻干，然后用 1% FA 重悬，并使用比色肽分析法（Thermo Fisher Scientific，Mississauga，ON，Canada）测定每个样品中的浓度。将所得胰蛋白酶样品干燥并储存在 -80^(@)C-80^{\circ} \mathrm{C} 。
Table 1. Demographics, Anthropometrics and Platelet Indices in Study Participants. 表 1.研究参与者的人口统计学、人体测量学和血小板指数。