VWF multimer analysis following incubation with TSP-1 proteins. (A) 100 nM of TSP-1 in HEPES buffer (50 mM HEPES, 0.125 M NaCl, and 0.1 mM or 2 mM CaCl2) was incubated with TTP plasma for 1 hour at 37°C and then directly visualized by gel electrophoresis. The percentage of high molecular weight multimers was not affected by the addition of TSP-1 (P = .50) as measured by densitometry. (B-C) Recombinant VWF (0.4 U/mL) was incubated with full-length TSP-1 or N700S recombinant protein (10 nM or 100 nM) for 1 hour at 37°C. Controls were incubated with HEPES buffer alone. No significant reduction was observed in VWF multimer size by gel electrophoresis or as measured by collagen-binding activity relative to VWF antigenic concentrations (ANOVA 1-way analysis, P = .08). Error bars represent standard deviations. (D) rVWF (0.4 U/mL) was incubated overnight with recombinant ADAMTS13 (10 μg/mL) or platelet TSP-1 (100 nM) at 37°C in Tris (tris[hydroxymethyl]aminomethane) buffer (10 mM Tris, 1.5 mM urea, 3 mM BaCl2, 0.1 mM CaCl2).