Regulated secretion of soluble P-selectin from HUVECs. (A) HUVECs were stimulated with thrombin (10 nM), PAR1-AP (100 μM), or PAR2-AP (200 μM) for 20 minutes after which the conditioned medium was collected and assayed for soluble P-selectin. Data represent the mean ± SEM of 3 independent experiments conducted in triplicate. There was no statistical difference between the agonists (P > .05 as determined by 2-tailed t test). (B) Membrane-bound P-selectin predominates over soluble P-selectin in HUVECs. Shown are agarose gel electrophoresis of PCR-amplified P-selectin and soluble P-selectin fragments from HUVECs. Lane M represents PCR amplification of P-selectin constructed into pECFP, whereas lane S represents PCR amplification of soluble P-selectin constructed into pECFP. Lanes 1 to 10 represent PCR amplification using cDNA obtained from HUVECs (10 different cord preparations) as templates. (C) Protein synthesis inhibition does not decrease forskolin or PAR2-AP stimulation of VWF release. HUVECs were preincubated with cycloheximide (10 μM) or vehicle for 30 minutes prior to stimulation with vehicle, PAR1-AP (100 μM), PAR2-AP (200 μM), or forskolin (20 μM with 100μM IBMX). Medium was collected after 30 minutes and assayed for VWF. Values represent the mean ± SEM of an experiment conducted 3 times.