FIGURE 4.

Activation of PAR2 on HTECs induces increased secretion of tissue factor. (A–D) 2F (2 μM) induced increased secretion of TF into cell culture medium as measured by Western blots (A–C), and a tissue factor activity assay (D). (A) Cell associated and secreted TF are the same molecular weight. TF secreted by 2F-treated cells is retained by a spin column membrane with a 3 kDa and 100 kDa cut-off membrane and in panels (B,C) TF is sedimented by centrifugation at 20k × g and 200k × g. Before analysis, medium was concentrated 10-fold. (C) The exosome marker, ALIX, is sedimented along with TF by centrifugation of conditioned medium at 200k × g. ALIX is equally present in both control and 2F condition medium pelleted from 200k × g centrifugation step, but TF is predominantly present in the 2F conditioned medium pellet. (D) 2F induced secretion of increased levels of active tissue factor into the cell culture medium from HTECs as measured by enzymatic production of Xa in the presence of factor VIIa. In panel (C) equal volumes of conditioned medium was sequentially centrifuged at 300 × g, 2k × g, 20k × g, and 200k × g. Analysis was by an unpaired two-sided Student’s t-test. Mean ± SEM, * indicates a significant (p < 0.05) difference when compared to control vehicle treatment; All the data in (A–D), n ≥ 3. Representative blots are shown.