Abstract：
Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have obvious advantages over MSC therapy. But the strong procoagulant properties of MSC-EVs pose a potential risk of thromboembolism, an issue that remains insufficiently explored. In this study, we systematically investigated the procoagulant activity of large EVs derived from human umbilical cord MSCs (UC-EVs) both in vitro and in vivo. UC-EVs were isolated from cell culture supernatants. Mice were injected with UC-EVs (0.125, 0.25, 0.5, 1, 2, 4 μg/g body weight) in 100 μL PBS via the tail vein. Behavior and mortality were monitored for 30 min after injection. We showed that these UC-EVs activated coagulation in a dose- and tissue factor-dependent manner. UC-EVs-induced coagulation in vitro could be inhibited by addition of tissue factor pathway inhibitor. Notably, intravenous administration of high doses of the UC-EVs (1 μg/g body weight or higher) led to rapid mortality due to multiple thrombus formations in lung tissue, platelets, and fibrinogen depletion, and prolonged prothrombin and activated partial thromboplastin times. Importantly, we demonstrated that pulmonary thromboembolism induced by the UC-EVs could be prevented by either reducing the infusion rate or by pre-injection of heparin, a known anticoagulant. In conclusion, this study elucidates the procoagulant characteristics and mechanisms of large UC-EVs, details the associated coagulation risk during intravenous delivery, sets a safe upper limit for intravenous dose, and offers effective strategies to prevent such mortal risks when high doses of large UC-EVs are needed for optimal therapeutic effects, with implications for the development and application of large UC-EV-based as well as other MSC-EV-based therapies.
摘要：
间充质干细胞来源的细胞外囊泡（MSC-EV）相对于MSC治疗具有明显的优势。但MSC-EV的强促凝特性构成了血栓栓塞的潜在风险,一个仍然没有充分探索的问题。在这项研究中,我们系统地研究了体外和体内源自人脐带MSCs（UC-EVs）的大型EVs的促凝血活性。从细胞培养上清液中分离UC-EV。通过尾静脉注射100μLPBS中的UC-EV(0.125、0.25、0.5、1、2、4μg/g体重)。注射后30分钟监测行为和死亡率。我们发现这些UC-EV以剂量和组织因子依赖性方式激活凝血。添加组织因子途径抑制剂可以抑制UC-EV体外诱导的凝血。值得注意的是,静脉内给予高剂量的UC-EV（1μg/g体重或更高）导致由于肺组织中的多个血栓形成而导致的快速死亡,血小板,和纤维蛋白原消耗，和延长凝血酶原和活化部分凝血活酶时间。重要的是,我们证明，UC-EV引起的肺血栓栓塞可以通过降低输注速率或预注射肝素来预防，一种已知的抗凝剂。总之,本研究阐明了大型UC-EV的促凝血特性和机制，详细说明静脉注射期间相关的凝血风险，为静脉注射剂量设定安全上限，当需要高剂量的大型UC-EV以获得最佳治疗效果时，提供有效的策略来预防此类致命风险，对基于大型UC-EV以及其他基于MSC-EV的疗法的开发和应用具有重要意义。
