Exocyst depletion impairs VWF multimerization but augments WPB exocytosis. (A) Immunoblot showing total VWF antigen levels in media of EXOC4-depleted and control HUVECs at 1, 2, 5, 10, 15, and 30 minutes after stimulation with 1 U/mL thrombin resolved by SDS-PAGE and immunoblotted with anti-VWF antibody. (B) Densitometry of 3 individual thrombin-stimulation experiments as described in panel A showing significant augmentation of VWF exocytosis on EXOC4 depletion at 30 minutes (***P < .001). (C) Resting conditioned media was collected following 8-hour incubation with control or EXOC4-depleted HUVECs. VWF antigen levels were measured using VWF ELISA. (D) EXOC4-depleted and control HUVECs were stimulated with 1 U/mL thrombin. Harvested media concentrated in centrifugal concentrators and fractionated by nonreducing 1.4% agarose gel electrophoresis was immunoblotted using anti-VWF antibodies. Blot shows reduced HMW multimers in EXOC4-depleted cells. (E) Densitometry analysis of VWF multimers in panel D quantified as the ratio of HMW multimer to the LMW oligomer. (n = 2; **P < .01). (F) Control and BLOC-2–, EXOC4–, and combined BLOC-2– and EXOC4–depleted cells were stimulated with 1 U/mL thrombin, and total VWF antigen in media at 30 minutes was measured using ELISA. The bar graph shows that differing from BLOC-2 depletion, EXOC4 depletion augments VWF exocytosis. EXOC4 depletion augments VWF exocytosis in BLOC-2–depleted cells compared with BLOC-2 depletion alone (**P < .001 from 2 individual experiments each with triplicates).