Figure 2.
FVIII aggregation is reversible upon restoration of Glc metabolism. (A) FVIII aggregates disassociate and refold into functional secreted FVIII. (i-iii) Kinetics of FVIII aggregation and disaggregation upon altered Glc metabolism was characterized by simultaneous membrane filtration, pulse-chase analysis, and measurement of secreted FVIII activity. (i-iii) The corresponding lane numbers are indicated below the image. 10A1 cells were pulse-labeled for 20 minutes with [35S]-Met/Cys and then chased for 20 minutes with media containing excess-unlabeled Met/Cys to compete synthesis of nascent chains (lane 1). Radiolabeled cells were then fed with complete media for indicated times (ii, lanes 2-3) or Glc-free media containing 20 mM 2DG and 10 mM NaN3 for 2 hours (i-ii, lanes 4-9). Radiolabeled [35S]-Met/Cys cell lysates and media were harvested after 2 hours (lane 4) or allowed to recover in complete media (5 mM Glc) from 30 minutes to 4 hours (lanes 5-8), or complete media with 10 µg/mL CHX (lane 9). (i) At the indicated times, cell lysates were analyzed by filtration on CA and NC membranes and probed with FVIII and β-actin antibodies. (ii) FVIII was immunoprecipitated from cell lysates and media for analysis by SDS–polyacrylamide gel electrophoresis and autoradiography. FVIII HCs and LCs are indicated. The percentages of radiolabeled intracellular FVIII and secreted FVIII are shown relative to intracellular FVIII in lane 1. (iii) Secreted FVIII is functional by the activated partial thromboplastin time (aPTT) assay. (B) Inhibition of Glc metabolism and not oxidative phosphorylation causes FVIII aggregation. H9 cells treated with SAHA for 19 hours were subsequently treated with a mixture of 1 µM oligomycin A (Oligo) and 1 µM rotenone (Rot) which are complex II– and complex I–specific inhibitors of oxidative phosphorylation or 50 mM 2DG for indicated times (0-60 minutes). Cell lysates were filtered through CA membranes and probed with FVIII antibody. Blue bars represent technical duplicates. (C) FVIII aggregation is due to hydrophobic interactions. H9 cells treated with SAHA for 22 hours were treated with 20 mM 2DG and 10 mM NaN3 for 2 hours. Treatment with tunicamycin (Tm), an ER stress inducer, started at 18 hours after SAHA. The percentage of FVIII retention on CA membranes was normalized to FVIII on NC membranes from technical duplicates. The longer exposure shows ∼3% of the FVIII aggregates are resistant to 1% SDS.

FVIII aggregation is reversible upon restoration of Glc metabolism. (A) FVIII aggregates disassociate and refold into functional secreted FVIII. (i-iii) Kinetics of FVIII aggregation and disaggregation upon altered Glc metabolism was characterized by simultaneous membrane filtration, pulse-chase analysis, and measurement of secreted FVIII activity. (i-iii) The corresponding lane numbers are indicated below the image. 10A1 cells were pulse-labeled for 20 minutes with [35S]-Met/Cys and then chased for 20 minutes with media containing excess-unlabeled Met/Cys to compete synthesis of nascent chains (lane 1). Radiolabeled cells were then fed with complete media for indicated times (ii, lanes 2-3) or Glc-free media containing 20 mM 2DG and 10 mM NaN3 for 2 hours (i-ii, lanes 4-9). Radiolabeled [35S]-Met/Cys cell lysates and media were harvested after 2 hours (lane 4) or allowed to recover in complete media (5 mM Glc) from 30 minutes to 4 hours (lanes 5-8), or complete media with 10 µg/mL CHX (lane 9). (i) At the indicated times, cell lysates were analyzed by filtration on CA and NC membranes and probed with FVIII and β-actin antibodies. (ii) FVIII was immunoprecipitated from cell lysates and media for analysis by SDS–polyacrylamide gel electrophoresis and autoradiography. FVIII HCs and LCs are indicated. The percentages of radiolabeled intracellular FVIII and secreted FVIII are shown relative to intracellular FVIII in lane 1. (iii) Secreted FVIII is functional by the activated partial thromboplastin time (aPTT) assay. (B) Inhibition of Glc metabolism and not oxidative phosphorylation causes FVIII aggregation. H9 cells treated with SAHA for 19 hours were subsequently treated with a mixture of 1 µM oligomycin A (Oligo) and 1 µM rotenone (Rot) which are complex II– and complex I–specific inhibitors of oxidative phosphorylation or 50 mM 2DG for indicated times (0-60 minutes). Cell lysates were filtered through CA membranes and probed with FVIII antibody. Blue bars represent technical duplicates. (C) FVIII aggregation is due to hydrophobic interactions. H9 cells treated with SAHA for 22 hours were treated with 20 mM 2DG and 10 mM NaN3 for 2 hours. Treatment with tunicamycin (Tm), an ER stress inducer, started at 18 hours after SAHA. The percentage of FVIII retention on CA membranes was normalized to FVIII on NC membranes from technical duplicates. The longer exposure shows ∼3% of the FVIII aggregates are resistant to 1% SDS.

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