Gheldof Damien, Mullier François, Chatelain Bernard, Dogné Jean-Michel, Chatelain Christian
Hematology Laboratory, NARILIS, Namur Thrombosis and Hemostasis Center, CHU Mont-Godinne, Université Catholique de Louvain, Leuven, Belgium.
Blood Coagul Fibrinolysis. 2013 Jul;24(5):567-72. doi: 10.1097/MBC.0b013e328360a56e.
Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.
癌症患者发生静脉血栓栓塞（VTE）的风险增加7至10倍。循环微泡可能是癌症患者VTE的一种预测生物标志物。凝血酶生成试验（TGA）是检测微泡促凝活性的一种有用技术。然而，由于血浆中存在组织因子途径抑制剂（TFPI），TGA缺乏敏感性。本研究的目的是通过限制TFPI的干扰来提高TGA对组织因子的敏感性。将MDA-MB231细胞系列稀释液在37°C孵育45分钟以生成微泡。然后将样品离心，含有微泡的上清液用于TGA。将正常混合血浆与TFPI抑制剂孵育或稀释两次以降低血浆TFPI水平。将滞后时间用作TGA的替代标志物以检测微泡的促凝活性。抑制TFPI可使显著缩短滞后时间所需的细胞浓度降低两倍，并使测定内变异性降低2.4倍。当由MDA-MB-231衍生的微泡触发TGA时，血浆稀释对TGA敏感性没有影响。凝血酶生成是研究携带组织因子的微泡促凝活性的一种非常敏感的方法。通过用针对其Kunitz结构域I的特异性单克隆抗体抑制TFPI可以提高敏感性。两倍血浆稀释是通过TGA研究微泡促凝活性的一种有趣的更便宜的替代方法，具有良好的敏感性，特别是当血浆量较少时。
