背景

血小板来源的外泌体（platelet-derived exosomes，PEXOs）是由血小板分泌的异质性的双层膜分子，参与细胞通讯和物质转运，调节体内多种生物过程，例如：凝血、组织修复等。PEXOs不仅具有和血小板相似的分子负载和功能，同时它便于运输和储存；PEXOs具有纳米级尺寸、低免疫原性和局部释放能力，能够跨越组织屏障，实现更广泛的功能调节，因此在部分医疗领域不仅被认为是血小板和富血小板血浆（platelet-rich plasma，PRP）的理想替代品，也在药物载体方面具有广阔的应用前景。血小板的激活条件影响PEXOs的生物学特征和功能，由于缺乏统一的制备标准，其研究和临床应用仍存在局限性，PEXOs在疾病治疗中具体效应分子和机制并不明确。以往关于PEXOs的研究多采用化学方式激活血小板，并未关注物理激活条件对PEXOs的影响，尤其是蛋白组学的影响，而化学激活条件在一定程度上增加了工程化生产外泌体的成本和难度。外泌体在形成和分泌过程中可以差异化包裹来自母细胞的生物分子，但是PEXOs和血小板之间的蛋白质组成差异并未得到充分的探讨和验证。因此，本研究探讨多种不同激活条件下PEXOs的生物学特征差异，并进一步分析反复冻融与其他化学激活条件诱导的PEXOs之间，以及血小板与PEXOs之间的蛋白组学特征差异，分析PEXOs的蛋白组成和外泌体差异化包裹物质的机制，为标准化制备PEXOs提供理论依据，并初步探讨PEXOs在凝血与抗凝、组织修复等方面应用的潜在效应分子和机制。

方法

从新鲜全血中离心分离血小板悬液，采用五种不同的激活条件（包括对照、二磷酸腺苷、凝血酶、钙离子载体和反复冻融）分别激活血小板悬液，经超速离心法分离富集PEXOs；经纳米流式分析仪、透射电镜和免疫印迹法等对PEXOs进行鉴定；检测血小板悬液和五组PEXOs样本中的蛋白质浓度与含量；利用数据非依赖性扫描模式 (Data-independent acquisition，DIA)的定量蛋白组学技术分析血小板悬液和五组PEXOs的蛋白组学特征，基于平行反应监视（parallel reaction monitoring，PRM）靶向蛋白组学技术进一步验证目的蛋白在血小板悬液、二磷酸腺苷组PEXOs、凝血酶组PEXOs和反复冻融组PEXOs中的表达情况。

结果

1. PEXOs的鉴定

纳米流式结果显示：所有PEXOs尺寸分布主峰在75-85nm之间，85%-95%的外泌体＜100nm；PEXOs在透射电镜下呈外泌体经典的杯状结构；蛋白质免疫印迹实验结果显示：所有外泌体均表达特异性标志物CD9、CD81和TSG101，且血小板来源标志物CD41阳性，均不表达外泌体阴性标志物Calnexin；以上结果提示研究所提取的囊泡为PEXOs。

2. 不同激活条件下PEXOs的生物学特征差异

反复冻融组PEXOs的浓度最高，与其他各组间的差异均具有统计学意义（P＜0.05）。反复冻融组PEXOs蛋白质浓度最高（1.11±0.51）μg/μL，大于对照组PEXOs（0.32±0.39）μg/μL、二磷酸腺苷组PEXOs（0.41±0.31）μg/μL和凝血酶组PEXOs（0.38±0.37）μg/μL，差异均具有统计学意义（P＜0.05），反复冻融组PEXOs蛋白质浓度大于钙离子载体组PEXOs（0.51±0.41）μg/μL和血小板组（0.79±0.52）μg/μL，差异均无统计学意义（P＞0.05）；反复冻融组PEXOs的蛋白质含量最高（125.40±58.32）μg，大于对照组PEXOs（25.53±25.96）μg和其他三个实验组PEXOs（二磷酸腺苷组：37.24±15.73、凝血酶组：36.28±24.18、钙离子载体组：47.09±23.29）μg，差异均具有统计学意义（P＜0.05），反复冻融组PEXOs的蛋白质含量与血小板组（77.78±32.95）μg的差异无统计学意义（P＞0.05）。PEXOs的蛋白质浓度和含量与血小板激活条件相关，不同激活条件基于诱导PEXOs浓度、蛋白质浓度或含量高低的排序为：反复冻融＞钙离子载体＞二磷酸腺苷≈凝血酶＞对照。

3. DIA蛋白组学结果

在六组样本中共检出3216种蛋白质，血小板及不同激活条件下PEXOs的蛋白质数量存在差异，血小板组（3046）＞反复冻融组PEXOs（2944）＞钙离子载体组PEXOs（2462）＞凝血酶组PEXOs（1877）＞二磷酸腺苷组PEXOs（1700）＞对照组PEXOs（1269）；其中有1090个蛋白质在六组样本间均表达，这些共有蛋白主要参与信号传导、免疫系统和癌症等相关信号通路；有26个蛋白质在五组PEXOs中均表达，但未在血小板中检出；各组样本中均含有仅在该组中表达的蛋白质即“独有蛋白”（对照组PEXOs：1个、二磷酸腺苷组PEXOs：1个、凝血酶组PEXOs：3个、钙离子载体组PEXOs：4个、反复冻融组PEXOs：52个、血小板组：145个）。对差异蛋白进行生物信息学分析显示：与血小板相比，二磷酸腺苷组PEXOs、凝血酶组PEXOs和钙离子载体组PEXOs的上调蛋白均在补体与凝血级联通路显著富集；蛋白质PDGFB和PDGFD在凝血酶组PEXOs表达上调，该组中的上调蛋白还在细胞外基质相互作用的信号通路显著富集。

4. PRM靶向蛋白组学结果

基于DIA结果，采用PRM靶向蛋白组学方法验证30个目的蛋白在三组外泌体和血小板中的表达情况，PRM结果显示：凝血酶组PEXOs和反复冻融组PEXOs表达相同的15种目的蛋白，二磷酸腺苷组PEXOs表达其中14种目的蛋白，不表达EGF，血小板组则表达16种目的蛋白；四组间均表达12个蛋白质（SERPINC1、SERPINA1、A2M、PDGFB、PDGFD、APOA1、FN1、SPARC、THBS1、PF4、PDCD6、TMED10）；基于DIA结果，从26个各组PEXOs均表达但血小板不表达的蛋白质中筛选4个目的蛋白进行PRM验证，结果显示：蛋白质EVA1B在三组PEXOs均表达但未在血小板中表达，其余三个蛋白质在各组中均不表达。基于DIA结果筛选二磷酸腺苷组PEXOs（蛋白质PLA2G2A）、凝血酶组PEXOs（蛋白质RPS15）和反复冻融组PEXOs（蛋白质EXOC5和蛋白质COMMD3）的“独有蛋白”进行验证，未在PRM实验中检出对应的蛋白质；与血小板相比，凝血酶组PEXOs存在1个下调蛋白（TMED10），和9个上调蛋白（THBS1、PF4、SPARC、PDGFB、PDGFD、A2M、SERPINA1、SERPINC1、APOA1），除蛋白质SERPINA1，其余蛋白质调节方向均与DIA结果一致。

结论

PEXOs是血小板的重要效应分子，它在形成和释放过程中差异化包裹来自母细胞的蛋白质，从而影响其生物学功能；PEXO的蛋白组学特征和血小板激活条件的分子途径与强度密切相关，不同的激活条件可能诱导外泌体形成或包裹特定的蛋白质，并且PEXOs中存在非血小板来源的蛋白质，其来源可能与外泌体的形成与功能相关。反复冻融法可以诱导血小板产生高浓度的外泌体，其蛋白组分和血小板高度相似，反复冻融可能是一种理想的PEXOs的研究和制备条件，而凝血酶组 PEXOs中部分生长因子相关蛋白表达升高，并且可能通过细胞外基质的相互作用在组织修复中发挥调节功能。

Background

Platelet-derived exosomes (PEXOs) are heterogeneous and bilayer membrane molecules secreted by platelets, which not only serve as important mediators of intercellular communication and cargo transport, but also can regulate many biological processes such as hemostasis, tissue repair and immune response. PEXOs have nanoscale size, low immunogenicity and local release capacity, can cross the tissue barrier, achieve a wider range of regulation. Due to its stable preservation and transport, PEXOs have become ideal substitute for platelet-rich plasma (PRP), and promising drug carrier. The characterization of PEXOs are affected by many factors, especially the activation conditions of platelets. Previous studies did not pay attention to physical activation methods, while chemically activating methods increases costs to a certain extent. The clinical use of PEXOs is still limited due to its main drawbacks that are the lack of standard preparation methods, the variability among separation and activation methods. Exosomes can differentially package cargos from the mother cell during formation and secretion, but differences between PEXOs and platelets have not been fully investigated. Therefore, this study aim to analyze the proteomic characterization of PEXOs induced by different activation conditions, including repeated freezing-thawing. We aim to investigate the differences in proteins between PEXOs and platelets and to explore the potential taregets of PEXOs for therapeutic intervention in tissue repair and anticoagulation.

Methods

Platelets were isolated by differential centrifugation from freshly drawn，citrate anti-coagulation blood obtained from healthy volunteers. Platelet suspensions were activated using five different activation conditions. The ctrl group added no extra stimulus, and the other four groups were treated with adenosine diphosphate (ADP), thrombin, Ca2+ ionophore and freeze-thaw cycles (FT) to activate platelets. PEXOs were isolated by differential centrifugations, and then were determined by nano-flow cytometry and electron microscopy. The protein markers of PEXOs also were identified by western-blot. The protein concentration and content of PEXOs were also detected. The proteomic characterization of PEXOs and the platelet suspensions were analyzed by data-independent acquisition mass spectrometry (DIA-MS) and then validated by parallel reaction monitoring (PRM) mass spectrometry.

Results

1.Identification of PEXOs

Flow Nano Analyzer showed that the main size peak of all groups of PEXOs is between 75-85nm, 85%-95% of PEXOs were＜100nm. PEXOs were cup-holder-like bilayer membrane vesicles under transmission electron microscope. WB showed that CD9, CD81, TSG101 and CD41 proteins were positive, while calnexin is negative. These results suggested that these vesicles were PEXOs.

2. The biological characterization of PEXOs under different activation pathways

The concentration of PEXOs in the FT group was the highest, higher than that in the control group and other groups (P<0.05).The protein concentration of PEXOs in the FT group (1.11±0.51) μg/μL was higher than that in the control group (0.32±0.39) μg/μL, ADP group (0.41±0.31) μg/μL and thrombin group (0.38±0.37) μg/μL, the differences are statistically significant(p＜0.05). The protein concentration of FT group was higer than the Ca2+ ionophore group (0.51±0.41) μg/μL,（p＞0.05) and platelets (0.79±0.52) μg/μL,(p＞0.05). The total protein of FT group (125.40±58.32) μg was higher than that in other groups (the ctrl group 25.53±25.96 vs ADP group 37.24±15.73 vs thrombin group 36.28±24.18 vs Ca2+ ionophore group 47.09±23.29 vs platelets 77.78±32.95) μg, the differences are statistically significant(p＜0.05). The activation pathway regulates the quality of PEXOs. Regarding the concentration or protein concentration or protein content of PEXOs, the order in our study is FT group＞Ca2+ ionophore group＞ADP group≈ thrombin group＞ ctrl group.

3. DIA-MS analysis

MS detected 3216 proteins, 1090 proteins were expressed in all groups, and these proteins were mainly involved in signal transduction, immune system and cancer-related signaling pathway. 26 proteins were expressed in all groups of PEXOs but not in platelets. Each group of PEXOs expressed unique proteins which had not expressesd in other groups. Compared with platelets, the up-regulated proteins in the adenosine diphosphate group, thrombin group and calcium ionophore group were significantly enriched in the complement and coagulation cascade. PDGFB, PDGFD and a group of proteins involved in extracellular matrix interacting increased in the thrombin group.

4. PRM validation study

30 proteins were selected based on the analysis of DIA-MS and then were validated by PRM study. The thrombin group and FT group expressed the same 15 target proteins, while the ADP group expressed 14 target proteins except EGF, platelets expressed 16 target proteins. The platelets and PEXOs have 12 common proteins, such as A2M, PDGFB, PDGFD, FN1, SPARC, THBS1 and PF4. EVA1B was expressed in all three PEXOs groups but not in platelets. We tried to detect the unique proteins of ADP group (PLA2G2A), thrombin group (RPS15) and FT group PEXOs (EXOC5 and COMMD3) based on DIA-MS analysis, failed in PRM. TMED10 decreased in thrombin group, 9 proteins(THBS1,PF4,SPARC,PDGFB,PDGFD,A2M,SERPINA1,SERPINC1,APOA1) increasd, compared with platelets. Except SERPINA1, the regulation type were all consistent with that in DIA-MS.

Conclusion

The protein differentially packaged process of PEXOs is closely related to the activation pathways of platelets, which can not only regulate their proteomic characterization, but also determine their function. The greater stimulus intensity, the more protein contents and types. There are non-mother-cell derived proteins in PEXOs, which may be related to the formation process and roles of exosomes. Each group of PEXOs has unique proteins which only expressd in one group. Freeze-thaw cycles can induce a large amount protein-rich PEXOs which has little difference in protein profiling compared with platelets, it may be an ideal condition for the use of study and preparation of PEXOs. Compared with platelets, PDGFB and PFGFD increased in PEXOs induced by thrombin, and they may have therapeutic effects in tissue repair through interaction of extracellular matrix.