Sachetto Ana T A, Archibald Sierra J, Hisada Yohei, Rosell Axel, Havervall Sebastian, van Es Nick, Nieuwland Rienk, Campbell Robert A, Middleton Elizabeth A, Rondina Matthew T, Thålin Charlotte, Mackman Nigel
Department of Medicine, UNC Blood Research Center, Division of Hematology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Department of Clinical Sciences, Division of Internal Medicine, Karolinska Institutet, Danderyd Hospital, Stockholm, Sweden.
Res Pract Thromb Haemost. 2023 Mar;7(3):100124. doi: 10.1016/j.rpth.2023.100124. Epub 2023 Mar 15.
Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g.
In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19.
EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured.
LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs.
We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.
在癌症、脓毒症和新冠肺炎等疾病中，循环促凝血细胞外囊泡（EVs）会增加。EV组织因子（TF）活性与脓毒症中的弥散性血管内凝血以及胰腺癌和新冠肺炎患者的静脉血栓形成有关。EVs通常通过在约20,000g的离心力下离心分离。
在本研究中，我们分析了脓毒症、胰腺癌或新冠肺炎患者中富集的大、小两种EV群体的TF活性。
通过依次在20,000g（大EVs，LEVs）和100,000g（小EVs，SEVs）下离心从血浆中分离出EVs。我们分析了来自有或无脂多糖（LPS）刺激的健康个体全血样本制备的血浆中的EVs，以及来自脓毒症、胰腺癌或新冠肺炎患者血浆样本中的EVs。测量了EVs的TF依赖性（EV-TF活性）和TF非依赖性因子Xa（FXa）生成。
LPS增加了LEVs中的EV-TF活性，但未增加SEVs中的活性。同样，在2名脓毒症患者中，其EV-TF活性高于检测背景，我们在LEVs中观察到了EV-TF活性，但在SEVs中未观察到。胰腺癌或新冠肺炎患者的LEVs和SEVs中均有循环EV-TF活性。
我们建议通过在100,000g而不是20,000g的离心力下从患者血浆中分离EVs，以更准确地测量循环EV-TF活性水平。
