Characterization of physical properties of tissue factor–containing microvesicles and a comparison of ultracentrifuge-based recovery procedures

摘要

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 reco...