Direct measurement of the PS/VWF complex in plasma. (A) PS/VWF complex was measured by ELISA, using polyclonal antibodies to capture VWF and detect bound PS. Experiments were performed with purified proteins (200 nM PS, 10 μg/mL VWF, and 5 mM CaCl₂, as indicated, in HEPES-buffered saline, with albumin) or HNP, with or without shear or 1 μM hirudin, 5 mM Gly-Pro-Arg-Pro peptide, and 5 mM CaCl₂ supplementation. Samples were incubated on the plate for the indicated time. (B) Biotinylated PS (150 nM) was added to PS-immunodepleted plasma, and PS/VWF complex was detected as in panel A, except using streptavidin-HRP to detect. Experiments were performed in the presence or absence of saturating concentrations of an anti-PS polyclonal antibody, TFPIα, APC, or MerTK. (C) Free PS was measured, as in Figure 1, using purified PS (200 nM), with or without purified VWF (10 μg/mL) and ristocetin (2 mg/mL). (D-E) Washed platelets (2.5 × 10⁸/mL) were aggregated in the presence or absence of VWF (10 μg/mL), PS (150 nM), and ristocetin (2 mg/mL). Experiments were performed in the absence (D) or presence (E) of vortexing to unfold VWF. In panel E, VWF was vortexed for 30 seconds, 5 minutes, or 1 hour. (F) VWF multimer blot of 0.25 μL HNP with additional 150 nM purified VWF, with or without 50 nM recombinant ADAMTS13, 200 nM PS, or 1 μM hirudin, 5 mM Gly-Pro-Arg-Pro peptide, and 5 mM CaCl_2, supplementation with or without shear (∼2500 rpm for 60 minutes). Every data point is the average of 3 replicates (mean ± SD). P values for panels B-C are according to Kruskal-Wallis with Dunn multiple comparison test; ∗P < .05; ∗∗P < .01. For panels D-E, experiments were performed using platelets from 3 different donors. Shown are the average aggregation curves. HRP, horseradish peroxidase; rpm, revolutions per minute.