Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures.
Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures.
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DOI：
10.3402/jev.v3.23592
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发表时间：
2014
影响因子：
16
通讯作者：
Ettelaie R
中科院分区：
文献类型：
--
作者：
Ettelaie C;Collier ME;Maraveyas A;Ettelaie R
在一定范围的相对离心力下，从3株细胞系（MDA-MB-231、AsPC-1和A375）的条件培养基中分离出微囊泡，比较组织因子（TF）蛋白和活性、微囊泡数量、大小分布和相对密度。同时，通过在细胞中表达tgf - tgfp并分离微泡，确定了含tgf微泡的相对密度。纳米颗粒跟踪分析（NTA）表明，直径较大的微泡（> ~ 200nm）主要在100,000g时沉积，具有tf依赖性凝血酶和Xa因子生成的潜力，而在没有因子VII的情况下，所有微泡都具有一定的凝血酶生成能力。含tf微泡的免疫沉淀和NTA也表明这些微泡的范围在200-400 nm之间。梯度密度离心分析发现，10万g回收的样品中主要存在低密度（<1.1 g/ml）的微囊泡，且微囊泡与TF抗原和活性有关。NTA分析证实，这些组分主要由较大直径的微囊泡组成。对健康或患者血浆中微囊泡的类似分析支持从条件培养基中获得的结果，表明TF活性主要与低密度微囊泡相关。此外，对健康血浆进行离心，补充含tf - tgfp的微泡，100,000g时荧光微泡回收率为67%，20,000g时回收率仅为26%。条件介质或等离子体在10,000g时进行预离心，通过随后的20,000g或100,000g离心，提高了回收含tf微囊泡的速度和产量。总之，TF似乎与低密度（1.03-1.08 g/ml）、大直径（200-350 nm）微泡有关。
Microvesicles were isolated from the conditioned media of 3 cell lines (MDA-MB-231, AsPC-1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF-tGFP in cells and isolating the microvesicles, the relative density of TF-containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger-diameter microvesicles (>200 nm) were primarily sedimented at 100,000g and possessed TF-dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immuno-precipitation of TF-containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200–400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower-density (<1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger-diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower-density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TF-tGFP-containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre-centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF-containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low-density (1.03–1.08 g/ml), larger-diameter (200–350 nm) microvesicles.