Mass spectrometry analysis of cleavage site on FRETS-VWF73 by membrane-bound proteases on neutrophils. A commercial FRETS-VWF73 peptide cleaved by (A) normal pooled plasma (NPP), (B) PMA-activated neutrophils in assay buffer, (C) activated neutrophils combined with NPP, (D) or activated neutrophils combined with ADAMTS13-deficient TTP plasma were analyzed by MALDI-TOF mass spectrometry. The doublet peaks (shown by asterisks above brackets) observed when NPP or TTP plasmas were present differed by 157 Da, and are consistent with the removal of a carboxyl terminal arginine of the FRETS-VWF73 substrate by plasma carboxypeptidase B. NPP alone generated a doublet peak consistent with cleavage at the Y¹⁶⁰⁵-M¹⁶⁰⁶ peptide bond (A). Activated neutrophils combined with NPP or TTP plasma generated a major peak of 6649 Da, consistent with cleavage at the V¹⁶⁰⁷-T¹⁶⁰⁸ bond and the removal of the carboxyl terminal arginine (C-D).