Exploring the Dual Role of Extracellular Vesicles in Coagulation and Immune Modulation in Glioblastoma

Background: Glioblastoma (GBM) is often complicated by venous thromboembolism (VTE), primarily driven by tissue factor (TF) and podoplanin (PDPN). These factors promote local hypercoagulation and microthrombosis, thereby contributing to tumor progression by enhancing migration, invasion, and inflammation. Both TF and PDPN can be released via extracellular vesicles (EVs), which carry procoagulant and immunomodulatory cargo.Methods: We developed a translational workflow combining biobanked tumor samples, clinical data, ex vivo GBM cultures, and coagulation assays to investigate mechanisms of hypercoagulation. Intraoperative blood coagulation was profiled using ClotPro. Gene expression of coagulation-related markers was analyzed in tumor tissues and cell lines, complemented by RNAseq-based profiling of coagulation-inflammation links. Functional coagulation assays included clotting time, platelet aggregation, and EV-based analysis of prothrombotic and immunomodulatory activity.Results: Peripheral coagulation in GBM patients was largely unaltered. However, tumor tissues consistently showed high TF and PDPN expression and markedly low tissue factor pathway inhibitor (TFPI) levels (p < 0.001), indicating a shift toward a procoagulant phenotype. Patient-derived GBM cell lines showed variable TF and PDPN expression, which correlated with clotting potential. Distinct procoagulant mechanisms were observed, with some cells engaging both TF-mediated thrombin generation and PDPN-driven platelet activation. EVs isolated from GBM patient plasma and culture media showed similar procoagulant characteristics, with activity proportional to TF expression and immune-modulating effects. Notably, GBM-derived EVs modulated microglial behavior, induced senescence, and triggered immune polarization in a cell line-dependent manner, likely contributing to tumor microenvironment remodeling.Conclusion: GBM-associated hypercoagulability is shaped by heterogeneous tumor-intrinsic pathways and EV-mediated mechanisms. The dual role of EVs in promoting coagulation and modulating immune responses highlights their potential as biomarkers and therapeutic targets for individualized thromboprophylaxis in GBM patients.

背景：
胶质母细胞瘤（Glioblastoma, GBM）常并发静脉血栓栓塞症（venous thromboembolism, VTE），该并发症主要由组织因子（tissue factor, TF）与podoplanin（PDPN）介导。上述因子可促进局部高凝状态与微血栓形成，进而通过增强肿瘤细胞迁移、侵袭与炎症反应，加速肿瘤进展。组织因子与podoplanin均可通过携带促凝与免疫调节载荷的细胞外囊泡（extracellular vesicles, EVs）释放。

方法：
本研究构建了一套转化研究流程，整合生物库储存的肿瘤样本、临床数据、离体胶质母细胞瘤培养体系与凝血功能检测实验，以探究高凝状态的发生机制。采用ClotPro平台对术中凝血状态进行表征分析；对肿瘤组织与细胞系中凝血相关标志物的基因表达水平开展检测，并辅以基于RNA测序的凝血-炎症关联通路分析。功能性凝血检测涵盖凝血时间、血小板聚集实验，以及基于细胞外囊泡的促血栓与免疫调节活性分析。

结果：
胶质母细胞瘤患者的外周凝血功能整体无明显改变。但肿瘤组织始终呈现高表达组织因子与podoplanin的特征，且组织因子途径抑制物（tissue factor pathway inhibitor, TFPI）水平显著降低（p < 0.001），提示肿瘤微环境向促凝表型转变。患者来源的胶质母细胞瘤细胞系的组织因子与podoplanin表达水平存在异质性，且与凝血潜能呈正相关。研究观察到多种不同的促凝机制：部分细胞同时依赖组织因子介导的凝血酶生成与podoplanin驱动的血小板活化。从胶质母细胞瘤患者血浆与细胞培养上清中分离的细胞外囊泡呈现相似的促凝特性，其活性与组织因子表达水平正相关，且具备免疫调节功能。值得注意的是，胶质母细胞瘤来源的细胞外囊泡可调控小胶质细胞行为、诱导细胞衰老，并以细胞系依赖的方式触发免疫极化，这一过程可能参与肿瘤微环境的重塑。

结论：
胶质母细胞瘤相关的高凝状态由异质性的肿瘤内在通路与细胞外囊泡介导的机制共同塑造。细胞外囊泡在促进凝血与调节免疫应答中的双重作用，凸显了其作为胶质母细胞瘤患者个体化血栓预防的生物标志物与治疗靶点的潜在价值。