Journal: TH Open: Companion Journal to Thrombosis and Haemostasis
Article Title: The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells
doi: 10.1055/s-0039-1688934
Figure Lengend Snippet: Analysis of the TF and fVIIa content of microvesicles. Five cell lines (HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7) were propagated in 25 cm 2 flasks, washed with phosphate-buffered saline (PBS) pH 7.4 and adapted to respective serum-free medium, for 1 hour. Microvesicles were prepared from the conditioned media by ultracentrifugation and the functional density determined using the Zymuphen MP-assay kit. ( A ) The TF antigen concentration of the microvesicles was measured using the Quantikine TF-ELISA kit and ( B ) the associated TF activities were measured using the fXa-generation assay ( n = 5). ( C ) The presence of fVII/fVIIa antigen in the cells was detected by western blot analysis. The samples were separated by 12% (w/v) SDS-PAGE, transferred onto nitrocellulose membranes and then blocked with TBST (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20). The membranes were then probed with a rabbit polyclonal anti-fVII antibody diluted 1:2000 (v/v) in TBST and developed with an alkaline phosphatase-conjugated goat antirabbit antibody, diluted 1:4000 (v/v). The bands were visualized using the Western Blue stabilized alkaline phosphatase-substrate and ( D ) quantified by ImageJ program. (The micrographs are representative of three separate experiments). ( E ) Microvesicle-associated factor VII/VIIa antigen levels were quantified using the Assaymax FVII-ELISA kit ( n = 5). ( F ) Microvesicle-associated fVIIa activity was also measured by fXa-generation assay but in the absence of exogenous fVIIa and presence of TF (1 U/mL) ( n = 5). ( G ) The molar ratio of fVIIa:TF in microvesicles derived from the cells lines was calculated using the data generated above. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TF, tissue factor.
Article Snippet: The TF antigen associated with the microvesicles was measured using the Quantikine TF-ELISA kit (R&D Systems) and the factor VII/VIIa antigen levels were determined using the Assaymax FVII-ELISA kit (Assaypro/Universal Biologicals Ltd., Cambridge, United Kingdom) according to the manufacturers' instructions.
Techniques: Functional Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, SDS Page, Activity Assay, Derivative Assay, Generated, Polyacrylamide Gel Electrophoresis
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