Quantification of VWF 176-kD and 140-kD fragments in plasma-derived pVWF. (A) VWF 140-kD fragments (gray) and 176-kD fragments (black) in size-fractionated pVWF were detected with Mab LJ140 and Mab LJ176 by immunoassay, and expressed as percentages of VWF fragments per total quantity of VWF antigen. Results are means ± SD. (B) VWF multimer immunoblot (1% agarose) showing multimer size ranges in pVWF fractions. (C) pVWF (multimer sizes similar to 82) was reduced and separated by SDS-5% PAGE. The immunoblot using polyclonal goat anti–VWF displays intact VWF monomer (225 kD) and faint bands for the 140-kD and 176-kD fragments. (D) Nonreduced samples separated by SDS-0.8% agarose demonstrate the larger plasma VWF multimers present in pVWF and the smaller multimers in CS. (E) Reduced samples of CS and pVWF were separated by SDS-5% PAGE and immunoblotted with Mab LJ140. The VWF 140-kD fragment was undetectable in pVWF samples that contained 1000-fold more VWF antigen (8 μg) than CS samples (8.4 ng). Immunoblots represent similar results obtained in 4 to 5 experiments. (F) Target epitopes of Mab LJ140 and Mab LJ176 that react specifically with either fragment 140-kD or 176-kD flanking the ADAMTS-13 cleavage site within the A2 domain of the VWF monomeric subunit. Cleavage of VWF at peptide bond M¹⁶⁰⁵-Y¹⁶⁰⁶ by ADAMTS-13 results in an increased proportion of 140-kD and 176-kD fragments relative to total VWF protein.