Sheared VWF interacts with PS in plasma and interferes with free PS measurement. Streptavidin beads–immobilized biotinylated VWF was exposed to pooled human plasma or single donor plasma, under static conditions or under shear (vortexing), and bound proteins were analyzed with nanoLC-MS/MS, and probed for PS and C4BP-α by immunoblot, with SA used as a control. (A) Mass spectrometry analysis of VWF pull down in pooled normal plasmas (pooled plasma 1 from Innovative Research; pooled plasma 2 from Precision Biologics) and single donor. (B-C) Western blot of VWF pull down probed for PS (B) and reprobed for C4BP-α (C). Arrows indicate the bands used for densitometric analysis. LC-MS, liquid chromatography-mass spectrometry, SA, serum albumin.