VIIa-induces association of TF with β1 integrin in HaCaT nonmalignant human keratinocytes. (A) Time course of coimmunoprecipitation of TF with β1 integrin after addition of 10 nmol/L VIIa to HaCaT cells. Immunoprecipitations were from cells lysed with Brij35 buffers containing either 1 mmol/L CaCl2 and MgCl2, or 1 mmol/L EDTA. Western blots for β1 integrin and TF in the immunoprecipitates and in the cleared cell lysate (CL) before immunoprecipitation are shown. (B) Dose-dependence of VIIa-induced association of TF with β1 integrin after 1 hour of incubation. (C) Coprecipitation of VIIa in the β1 integrin-TF complex detected after 10 nmol/L VIIa addition for 1 hour. (D) VIIa-induced β1-associated TF is coagulant inactive. Cells were treated with VIIa for 60 minutes, and β1 was precipitated from the lysate. β1 and TF precipitation were assessed on Western blots, and coagulant activity of the immunoprecipitate was assessed by adding immunoprecipitates to an Xa generation assay; mean and standard deviations, n = 3. (E) TF associates with integrin on the cell surface. After addition of 10 nmol/L VIIa for the times indicated, cells were surface biotin-labeled. Biotinylated proteins are detected by horseradish peroxidase (HRP)-streptavidin (SA-HRP) relative to total immunoprecipitated TF and β1. Densitometry from 3 separate experiments showed similar ∼3-fold up-regulation in total and cell surface TF upon VIIa addition as depicted in the graph. (F) VIIa-induced TF-integrin interaction is independent of PAR2 activation and activity of VIIa. PAR2 activation was blocked with 100 μg/mL polyclonal anti-PAR2 in cells stimulated with 10 nmol/L VIIa or active site-inhibited VIIa (VIIai) was added for 1 hour.
