American Society of Hematology

Figure 5.

$BLOC-2 depletion impairs endothelial WPB exocytosis and VWF multimerization. (A) Immunoblot showing total VWF antigen levels in the media at 1, 2, 5, 10, 15, and 30 minutes after 1 U/mL thrombin stimulation of BLOC-2–depleted and control HUVECs resolved by SDS-PAGE and immunoblotted with anti-VWF antibody. (B) VWF antigen released from HPS6-depleted and control HUVECs 30 minutes after 1 U/mL thrombin stimulation determined by ELISA. (***P < .001; n = 3). (C) Whole cell lysates of BLOC-2–depleted and control cells were resolved by SDS-PAGE and immunoblotted with antibodies to VWF and HPS6. Total endothelial cell VWF content is unaltered on BLOC-2 depletion. (D) Resting conditioned media was collected after 8-hour incubation with control or BLOC-2–depleted HUVECs. VWF antigen levels were measured using VWF ELISA (**P < .01; n = 3). (E) BLOC-2–depleted and control HUVECs were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes. Total VWF antigen (Ag) and activity (Act) were measured using a VWF ELISA and a type 3 collagen binding assay, respectively. The bar graph shows the Act to Ag ratios (**P < .01; n = 3). (F) Qualitative analysis of exocytosed VWF multimers was performed using agarose gel electrophoresis. BLOC-2–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 with anti-VWF antibodies. Blot shows reduced HMW multimers in BLOC-2–depleted cells. (G) Densitometry analysis of VWF multimers in e quantified as the ratio of HMW multimer to the lowest-molecular-weight (LMW) oligomer, as shown. (n = 3; ***P < .001). (H) WT and HPS6−/− mice were treated subcutaneously with epinephrine. VWF antigen levels in plasma obtained before and 15 minutes after epinephrine treatment were determined using VWF ELISA (n = 3; **P < .01). (I) Plasma from an individual with a BLOC-2 mutation and a healthy control was resolved by nonreducing 1.4% agarose gel electrophoresis and immunoblotted with VWF antibody showing reduced HMW multimers.$

BLOC-2 depletion impairs endothelial WPB exocytosis and VWF multimerization. (A) Immunoblot showing total VWF antigen levels in the media at 1, 2, 5, 10, 15, and 30 minutes after 1 U/mL thrombin stimulation of BLOC-2–depleted and control HUVECs resolved by SDS-PAGE and immunoblotted with anti-VWF antibody. (B) VWF antigen released from HPS6-depleted and control HUVECs 30 minutes after 1 U/mL thrombin stimulation determined by ELISA. (***P < .001; n = 3). (C) Whole cell lysates of BLOC-2–depleted and control cells were resolved by SDS-PAGE and immunoblotted with antibodies to VWF and HPS6. Total endothelial cell VWF content is unaltered on BLOC-2 depletion. (D) Resting conditioned media was collected after 8-hour incubation with control or BLOC-2–depleted HUVECs. VWF antigen levels were measured using VWF ELISA (**P < .01; n = 3). (E) BLOC-2–depleted and control HUVECs were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes. Total VWF antigen (Ag) and activity (Act) were measured using a VWF ELISA and a type 3 collagen binding assay, respectively. The bar graph shows the Act to Ag ratios (**P < .01; n = 3). (F) Qualitative analysis of exocytosed VWF multimers was performed using agarose gel electrophoresis. BLOC-2–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 with anti-VWF antibodies. Blot shows reduced HMW multimers in BLOC-2–depleted cells. (G) Densitometry analysis of VWF multimers in e quantified as the ratio of HMW multimer to the lowest-molecular-weight (LMW) oligomer, as shown. (n = 3; ***P < .001). (H) WT and HPS6^−/− mice were treated subcutaneously with epinephrine. VWF antigen levels in plasma obtained before and 15 minutes after epinephrine treatment were determined using VWF ELISA (n = 3; **P < .01). (I) Plasma from an individual with a BLOC-2 mutation and a healthy control was resolved by nonreducing 1.4% agarose gel electrophoresis and immunoblotted with VWF antibody showing reduced HMW multimers.

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