HUVEC exposed to PCLP for 16 hours exhibit a VWF trafficking defect. HUVEC were exposed to dimethyl sulfoxide (DMSO) or a range of concentrations of PCLP and incubated overnight (16 hours). (A) Immunoblotting of the resulting lysates displayed changes in pro-VWF and mature VWF in relation to tubulin. Densitometry indicated a decrease in total VWF (B) and mature-VWF levels (C) alongside a dose-dependent increase in the ratio of pro/mature VWF (D). n = 4 Ratio paired t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005 (E) 16-hour incubation with PCLP resulted in inhibition of regulated secretion of VWF in response to thrombin and (F) HAI. (G) HUVEC preincubated with DMSO or PCLP for 16 hours were stimulated with HAI for 10 minutes before application of 5 dyne/cm² shear stress. VWF strings were visualized by immunofluorescence and confocal microscopy. (H-I) The number (H) and length (I) of VWF strings secreted under flow in response to HAI in the presence or absence of DMSO or PCLP (n = 3). (J) IF analyses using anti-LAMP1 (green) and anti-VWF (blue) antibodies indicated numerous VWF positive lysosomes in PCLP-treated cells. (K) IF analyses using anti-TGN46 (yellow) and anti-VWF (blue) antibodies indicated a gross defect in TGN morphology (fragmented and swollen) in PCLP-treated cells. Scale bars, 10 μm. Inset scale bars, 1 μm.