Treatment with NH₄Cl diverts VWF and AgII into the constitutive secretory pathway. (A) PhosphorImager data of VWF (left) and AgII (right) immunoprecipitated from cell lysates (C) and medium (M) of HUVECs metabolically labeled for 30 minutes with [³⁵S]-cysteine/methionine and chased in the presence (NH₄Cl) or absence (Control) of 25 mM NH₄Cl for 24 hours. (B) The time courses for appearance of proVWF (□, ■) and mature VWF (▵, ▴) in the media of HUVECs metabolically labeled as in panel A and chased for the times indicated in the presence (■, ▴) or absence (□, ▵) of 25 mM NH₄Cl. Data are expressed as a percentage of the maximum value for each protein species secreted. Data shown are the means (± range) of 2 observations. The experiment was repeated with similar results. (C) A representative time course for the appearance of VWF immunoreactivity, determined by ELISA, in fresh growth medium (□) and growth medium containing 25 mM NH₄Cl (▴) or 30 μM histamine (●). Data points represent means (± range) of duplicate observations. The experiment was repeated on at least 3 occasions with similar results. The amount of VWF immunoreactivity secreted is expressed as IU/mL after calibration against a known standard (see “Measurement of VWF secretion by enzyme-linked immunosorbent assay”).