Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles
Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles
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DOI：
10.3402/jev.v2i0.19728
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发表时间：
2013-01-01
影响因子：
16
通讯作者：
Mullier, Francois
中科院分区：
文献类型：
--
作者：
Gheldof, Damien;Hardij, Julie;Mullier, Francois
癌症患者发生静脉血栓栓塞的风险增加7至10倍。循环微泡可能是癌症静脉血栓栓塞的有用预测生物标志物。验证和标准化的技术，可用于确定完整的微泡表型是必需的。这些措施有两个方面：a）通过流式细胞术（FCM）、透射电子显微镜（TEM）和凝血酶生成测定（TGA）来检测由培养的乳腺癌细胞MDA-MB-231释放的携带组织因子（TF）的微泡;和B）验证TGA作为研究微泡的促凝血活性（PCA）的有用方法的灵敏度和测定内/测定间的可变性。将培养的乳腺癌细胞MDA-MB-231在378 ℃下孵育45分钟。然后将样品在4，500 g下离心或不离心15分钟，并将细胞和MV或含MV的上清液用于TEM、FCM和TGA。在活性测定中，将微泡（即细胞耗尽的上清液）与抗TF抗体或膜联蛋白V孵育，以评估TF和磷脂对PCA的贡献。或者，将上清液通过0.1、0.22、0.45或0.65 mm膜过滤并进行TGA。大多数PCA与小于0.1 mm的微泡相关，并且在10，000 g离心后通过TEM估计的平均微泡尺寸为12 +/- 54 nm，大多数囊泡在100 nm和200 nm之间。来源于5，000 MDA-MB-231细胞/ml的微泡足以显著增加正常混合血浆的凝血酶生成。TEM，FCM和过滤耦合TGA代表一个有用的组合来研究的PCA TF轴承微泡，无论其大小。在病人身上实施这些技术将是有趣的。
Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required.Objectives. These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/ interassay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles.Methods. Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 378C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 mm membranes and subjected to TGA.Results. The majority of the PCA was associated with microvesicles smaller than 0.1 mm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 12 +/- 54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma.Conclusions. TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients.