第一部分

背景与目的：血小板减少症是抗磷脂综合征（Antiphospholipid syndrome，APS）常见的标准外临床表现，与APS患者预后的相关性尚不明确。本研究拟描述原发性APS患者中血小板减少症患者的临床特征，并进一步阐述血小板减少症与原发性APS患者新发血栓事件、病理妊娠及严重分类标准外表现的相关性。

材料与方法：本研究为基于北京协和医院中国风湿病数据中心（Chinese Rheumatism Data Center，CRDC）开展的APS单中心前瞻性队列研究，序贯纳入符合2006年悉尼分类标准的原发性APS患者，收集基线人口学资料、临床特征、既往血栓事件、病理妊娠等，根据患者病情每3-6个月安排随访，记录随访过程中新发事件及治疗反应等。结局定义为血栓事件、病理妊娠、严重诊断标准外事件（除网状青斑、浅静脉血栓的标准外事件），及出血和死亡。

结果：本研究纳入91例（31.3%）合并血小板减少症及200例（68.7%）血小板持续正常的原发性APS患者。对两组患者人口学特征、基线临床特征进行对比，合并血小板减少的APS患者基线发生过静脉血栓事件的比例显著高于血小板持续正常患者（62.6% vs 46.5%，p=0.015），其中腹腔静脉血栓最为显著（13.2% vs 4.5%，p=0.016）；分类标准外临床表现患者的比例在血小板减少组显著更高（25.3% vs 9%，p<0.001）；各类动脉血栓事件、病理妊娠均未见明显差异。IgG亚型的抗心磷脂抗体（Anticardiolipin，aCL）、抗β2 糖蛋白I（Glycoprotein I，GP I）抗体及狼疮抗凝物（Lupus anticoagulant，LA），和三抗体阳性的比例在合并血小板减少症的APS患者中均显著高于血小板正常患者（p<0.001）。中位随访时间为48（28,65.5）个月。对随访过程中新发血栓事件、病理妊娠及严重标准外表现进行生存分析，均提示合并血小板减少症APS患者生存率显著低于血小板正常组（p=0.0097，p=0.049， p<0.001）。血小板减少症患者中共85（93.4%）人对治疗有反应。血小板减少程度、血小板减少症治疗反应对终点事件发生未见显著影响。在多因素Cox回归分析中，血小板减少症对新发血栓事件（HR 2.4，95%CI 1.1-5.4, p=0.03）、病理妊娠（HR 3.3，95%CI 1.01-10.9，p=0.047）和严重标准外表现（HR 3.4，95%CI 1.5-7.8，p=0.003）均为独立危险因素。

结论：根据是否合并血小板减少症可以识别发生血栓事件、病理妊娠和严重标准外事件高风险的原发性APS患者。

第二部分

背景与目的：抗磷脂综合征（APS）是一种非炎症性自身免疫性疾病。发病机制尚未充分发掘，其中“二次打击”学说较为广泛认可。循环中存在的抗磷脂抗体（aPL）被认为是“第一次打击”，血小板激活可能参与“第二次打击”。为进一步挖掘血小板活性功能状态在APS疾病中的作用，拟从蛋白表达层面，通过蛋白质组学探究APS血小板分子学特征。

材料与方法：本研究纳入原发性APS患者及性别、年龄匹配的健康对照（Healthy controls，HC），留取外周血样本并分离血小板，通过气相色谱-质谱串联分析的方法进行蛋白质测定，通过生物信息学分析方法筛选差异表达蛋白（Differentially expressed protein，DEP），进行通路富集分析，通过Western blot、ELISA等方法针对差异蛋白进行验证。

结果：研究共纳入原发性APS患者36例，性别、年龄匹配HC15例。数据库检索完成后得到不同蛋白在不同样本中的强度值，对比在组内、组间分散程度在同一水平线，质控样本质量良好。主成分分析（Principle component analysis，PCA）发现两组样本区分度良好。以差异倍数绝对值＞1.3，且p值<0.05为阈值筛选DEP，得到178个上调DEP和52和下调DEP。GO富集上调通路主要包括蛋白翻译过程调控、蛋白定位等生物过程，下调通路包含氧化磷酸化负调控等，KEGG富集提示血小板活化改变。分析富集通路中主要DEP，筛选JAK-STAT通路重要分子STAT1、STAT2及PI3K-AKT通路中的蛋白激酶Akt3。通过WGCNA构建蛋白共表达网络，联合临床特征进行关联性分析，将不同基因表达蛋白分为12个模块，提取与动脉血栓相关性最大模块进行GO通路富集，提示APS血小板可能存在凋亡通路异常，BCL2L1参与相关通路改变。根据生信分析结果及临床意义，筛选STAT2、Akt3、BCL2L1进行Western blot验证，ELISA法验证CCL5在血浆中水平，发现STAT2在APS血小板中表达显著上升，CCL5在APS患者血浆中水平下降。

结论：通过血小板蛋白质组学分析发现APS患者血小板在蛋白质翻译、转运等通路上较HC存在差异，生信分析方法筛选DEP包含STAT1、STAT2、Akt3、MPO、BCL2L1、CCL5等蛋白，提示APS患者血小板可能存在活化、凋亡和氧化磷酸化异常。验证发现STAT2在APS血小板中表达增高，CCL5在APS患者血浆中水平降低。

第三部分

背景与目的：血小板在抗磷脂综合征（APS）不止参与血栓形成过程，还可能存在活性及功能的异常，并作为免疫细胞参与调节免疫系统。本研究拟多方位描述原发性APS患者血小板活性与功能变化。

对象与方法：纳入符合2006年悉尼分类标准的原发性APS患者，及性别、年龄匹配的HC，留取外周血全血样本。密度梯度离心法分离血小板，利用流式细胞术检测血小板表面活性标志物。对血小板进行全转录组测序分析，利用生物信息学筛选差异表达基因，利用通路富集分析等方法解读血小板参与的关键通路，并通过实时荧光定量PCR、流式细胞术、Western blot等方法验证关键分子。

结果：血小板活性功能研究共纳入原发性APS患者38例，性别、年龄匹配的HC 23例。与HC相比，APS患者血小板流式细胞术检测CD62p平均荧光强度显著更高（p=0.0028），提示APS血小板活性增高，活性氧（Reactive oxygen species，ROS）强度较HC明显增高（p=0.0037），经凝血酶或ADP刺激后两者无差异。两组血小板线粒体数量无显著差异（p=0.93）。Seahorse能量代谢分析提示APS患者血小板线粒体呼吸氧耗量更大，尤其是最大呼吸能力（p=0.048）和备用呼吸能力（p =0.042）。转录组测序分析中，PCA分析提示APS患者异质性较大。将显著水平设置在padj<0.05&| log2(FoldChange) |> =1，鉴定得到上调差异基因1860个，下调174个，表达差异以上调改变为主。通路富集分析发现多条与淋巴细胞活性、分化相关通路，以T细胞为主。分析各通路中差异基因，发现II类MHC分子均被富集在相关通路中，以HLA-DR最为显著，通过多种方式验证APS患者血小板表面表达HLA-DR。

结论：原发性APS患者血小板较健康人显著活化，活性氧产生增加，线粒体压力增加。转录组测序富集分析提示APS患者血小板与Th细胞分化相关，异常表达II类MHC分子。

Part I

Background and purpose: Thrombocytopenia, a frequent clinical manifestation in patients with antiphospholipid syndrome (APS), its association with recurrent thrombotic, obstetric and severe extra-criteria events is not completely clear. This study aims to further elucidate the characteristics of APS patients with thrombocytopenia, and the association between thrombocytopenia and the occurrence of endpoint events in APS patients.

Materials and Methods: This study is based on the single-center prospective cohort study conducted by Peking Union Medical College Hospital, and consecutively included primary APS that met the 2006 Sydney classification criteria. For patients, baseline demographic information, clinical characteristics, past thrombotic events, pregnancy morbidity were collected. Follow-up visits were arranged every 3-6 months according to the patient's condition, and new events and treatments during follow-up were recorded. Outcomes were defined as thrombotic events, pregnancy morbidity, severe extra-criteria events (extra-criteria manifestations except for livedo reticularis and superficial venous thrombosis), bleeding and death.

Results: This study included 91 (31.3%) primary APS patients with thrombocytopenia and 200 (68.7%) with persistently normal platelet count. We compared the baseline demographic and clinical characteristics of the two groups and found the proportion of APS patients with thrombocytopenia who had experienced venous thrombotic events at baseline was significantly higher than that of patients with persistently normal platelets (62.6% vs 46.5%, p = 0.015), among which the celiac vein Thrombosis was most significant (13.2% vs 4.5%, p = 0.016). The proportion of patients with extra-criteria criteria manifestations was significantly higher in the thrombocytopenic group (25.3% vs 9%, p < 0.001). No significant differences were found in arterial thrombosis and pregnancy morbidity. In terms of antiphospholipid antibodies (aPL), the proportions of positive LA, IgG subtype of aCL and anti-β2 GPI antibody, and triple positivity were significantly bigger than APS patients with normal platelet count (p < 0.001). Survival analysis of newly developed thrombotic events, pregnancy morbidity, and severe extra-criteria manifestations during follow-up showed that the survival rate of APS patients with thrombocytopenia was significantly lower than that of the normal platelet group (p = 0.0097, p = 0.049, p < 0.001). The degree of thrombocytopenia and thrombocytopenia treatment response had no significant impact on the occurrence of endpoint events. In multivariate Cox regression analysis, thrombocytopenia was an independent risk factor for all endpoint events including thrombotic events (HR 2.4, 95%CI 1.1-5.4, p = 0.03), pregnancy morbidity (HR 3.3, 95%CI 1.01-10.9, p = 0.047), and severe extra-criteria manifestations (HR 3.4, 95% CI 1.5-7.8, p = 0.003).

Conclusion: Patients with primary APS at high risk for thrombotic events, pathological pregnancy, and severe extra-criteria manifestations could be identified based on the presence of thrombocytopenia.

Part II

Background and purpose: Antiphospholipid syndrome (APS) is a non-inflammatory, systemic autoimmune disease of which the pathogenesis is not fully understood. The second-hit theory is rather widely accepted. Antiphospholipid antibodies (aPL) are considered the first hit, and platelet activation might be involved in the second hit. In order to further explore the characteristics of platelet function and status of platelet activation, we seek to investigate the molecular characteristics of APS platelets from protein expression level through proteomics.

Materials and methods: This study included primary APS patients and gender, age-matched healthy controls (HC). Peripheral blood samples were collected and platelets were separated. Proteomics was measured by liquid chromatography- mass spectrometry tandem analysis. Bioinformatics analysis was used to screen differentially expressed proteins (DEP). We also enriched pathways using GO and KEGG databases. Screened DEPs were verified using Western blot and ELISA.

Results: A total of 36 patients with primary APS and 15 HC were included in the proteomics. After database search, the intensity of proteins in each sample are obtained. The degree of dispersion within and between groups is at the same level, indicating that the samples qualified. Principal component analysis (PCA) found that the two groups were well differentiated. DEP were screened using the threshold of |FoldChange|>1.3 and p value <0.05, and 178 up regulated DEPs, 52 down regulated DEPs were obtained. The up regulated biological process of GO enrichment mainly falls into the category of protein translation and localization. The down regulated pathways include negative regulation of oxidative phosphorylation. KEGG enrichment indicated changes in platelet activation. After analyzing main DEPs in the enriched pathways, we focused on several important molecules including STAT1, STAT2 in the JAK/STAT pathway and a protein kinase Akt3 in the PI3K/AKT pathway. A protein co-expression network was constructed using WGCNA, and correlation analysis between gene expression and clinical traits was performed. Different gene-expressed proteins were divided into 12 modules. Arterial thrombosis was most correlated with the yellow module, and the related proteins were extracted for GO pathway enrichment, suggesting that APS platelets might be associated with apoptosis pathways, and BCL2L1 was involved. Based on the bioinformatics analysis and clinical significance, STAT2, Akt3, BCL2L1 and CCL5 were chosen for subsequent verification. Western blot results showed the level of STAT2 in APS platelets increased significantly, while the level of CCL5 in plasma of APS patients decreased compared with HC.

Conclusion: Through platelet proteomics analysis, it was found that APS platelet was different from HC in protein translation, protein transport and other related pathways. Several DEPs were screened including STAT1, STAT2, Akt3, MPO, BCL2L1, and CCL5, which suggested that APS platelet might have abnormal activation, apoptosis and oxidative phosphorylation. STAT2 was validated to be increased in platelet and CCL5 decreased in plasma.

Part III

Background and purpose: Platelets play more than the part of thrombosis in antiphospholipid syndrome (APS), but also exhibit abnormal activity and function. Platelets may serve as immune cells to participate in the regulation of the immune system. This study intends to describe the changes in platelet activity and function among patients with primary APS.

Materials and methods: Patients diagnosed with primary APS according to the 2006 Sydney classification criteria. Gender and age-matched healthy controls (HC) were recruited. Peripheral blood samples were collected. Density gradient centrifugation was used to separate platelets, and flow cytometry was used to detect platelet activity markers. RNA-sequencing analysis of platelets from APS patients and HC was performed. Bioinformatic was used to screen differentially expressed genes. Pathway enrichment analysis was used to interpret the key pathways involved in platelets. To validate the screened genes, real-time quantitative PCR, flow cytometry and Western blot were performed.

Results: This study included 38 patients with primary APS and 23 age/gender-matched HC. Compared with HC, the mean fluorescence intensity (MFI) of CD62p detected by flow cytometry of platelets in APS patients was significantly higher (p=0.0028), indicating that APS platelet activity was increased. The production of reactive oxygen species (ROS) was significantly higher than that of HC (p=0.0037). However, no difference was found after the stimulation of thrombin and ADP. Mitotracker indicated that there was no difference in the number of mitochondria in platelets between the two groups (p=0.93). Seahorse analysis showed that patients with primary APS had greater respiratory oxygen consumption in platelet mitochondria, especially the maximum respiratory capacity (p=0.048) and spare respiratory capacity (p=0.042). In RNA-sequencing, principal component analysis (PCA) showed that APS patients were highly heterogeneous. The significance level was set at adjusted p-value (padj) <0.05 & |log2FoldChange|>=1. A total of 1860 up-regulated differentially expressed genes (DEG) and 174 down-regulated DEG were identified. The difference was mainly up-regulation. Pathway enrichment analysis found multiple pathways related to lymphocyte activity and differentiation, T cells particularly. After browsing the DEGs in enriched pathways, major histocompatibility complex (MHC) class II molecules were key DEG in relevant pathways, with HLA-DR being the most significant. The expression of HLA-DR on platelet surface of APS patients was validated by various methods.

Conclusion: Platelets in primary APS were significantly activated compared with HC, and ROS production was significantly increased. The mitochondrial stress was increased in APS platelets. Enrichment analysis of RNA-sequencing showed that APS platelets were related to T helper cell differentiation and abnormal expression of MHC class II molecules.