PAK2 signaling regulates VWF release from endothelial cells. (A) Confocal analysis localizes PAK2 (green) to the cytoplasm and at the end of dorsal actin stress fibers (magenta) in HUVECs. (B) PAK2 (green) is not present on the actin ring (magenta) that forms at exocytosis (inset). Scale bars, 10 μm (full size) and 1 μm (inset); brightness and contrast increased for clarity. (C-D) The effect of siRNA depletion of the 6 PAK isoforms on (C) PMA-stimulated and (D) HAI-stimulated VWF secretion. (E) PAK2 depletion reduced PMA stimulated VWF release in a dose-dependent fashion. HUVECs were independently transfected with 4 different siRNA oligonucleotides targeting PAK2. VWF secretion was determined by fluorescent dot blot and PAK2 protein abundance determined by western blotting. (F) Western blotting of HUVEC lysate detected as single band at the estimated size of 61 kDa. (G) Schematic representation of PAK2 structure and targets for pharmacological inhibition. (H) Pharmacological inhibition of the autoregulatory domain of PAK2 with 25 to 50 μM IPA-3 prevents VWF release. (I) Targeting the catalytic kinase domain of PAK2 with FRAX486 inhibits VWF release (0.78-12.5 μM). (J) The specific PAK1 inhibitor NVS PAK1.1 has no effect on VWF release at 12.5 μM. ∗P < .05, ∗∗P < .01, ∗∗∗P < .005 (one-way ANOVA with Dunnett multiple comparison). (K-L) Effect of FRAX486 administration on adrenaline-stimulated VWF secretion in vivo. C57 black WT mice were administered FRAX486 or an equivalent volume of DMSO. Tail vein bleeds were performed to assess basal VWF levels. Intraperitoneal injection of adrenaline (0.5 mg/kg) was used to stimulate VWF secretion from the murine vasculature (DMSO, n = 5; FRAX486, n = 4). After 30 minutes, mice were sacrificed and the plasma was isolated. Plasma VWF levels and multimer composition were assessed by near-infrared dot blot (K) and multimer gel (L). MW, molecular weight. ∗P < .05.