HDL/ApoA-I prevents VWF self-association under both static and shear conditions. (A) Boiled plasma and ApoA-I prevent VWF surface adsorption and self-association under static conditions. Purified recombinant Bio-VWF (8 μg/mL) in 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mM CaCl₂ was incubated with buffer or different proteins at 22°C for 4 hours in 1.5-mL microfuge tubes. At the end of incubations, unbound proteins remaining in solution were removed. Proteins adsorbed to the tube surfaces were sequentially eluted first with buffer containing sodium dodecyl sulfate (SDS) (2% SDS, 2 mM CaCl₂, 10 mM HEPES, pH 7.4), and then with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) elution buffer (4% CHAPS, 8 M urea, 5 mM dithiothreitol, 40 mM tris(hydroxymethyl)aminomethane [Tris], pH 9.5). Bio-VWF remaining in the fluid phase and that bound to the surface were both analyzed by western blot after electrophoresis on reducing polyacrylamide gels. (B) VWF self-association is enhanced by shear stress and prevented by HDL. Purified VWF from cryoprecipitate (7 μg/mL in 10 mM HEPES, 25 mM NaCl, 10 mM EDTA, pH 7.5), Bio-VWF in serum-free medium desalted into the buffer above at 10 μg/mL, and 50% citrated plasma in 10 mM EDTA were sheared by vortexing at 2400 rpm in a rotary mixer for 90 minutes at 37°C. VWF remaining in solution was quantified by enzyme-linked immunosorbent assay (ELISA) and expressed as a percentage of the VWF in parallel samples not exposed to shear stress (n = 3). The VWF multimer distribution was analyzed by SDS-agarose gel electrophoresis and western blotting as described in supplemental Data. (C) HDL/ApoA-I dose-dependently inhibits VWF self-association under shear stress. Samples of 50% citrated plasma in 10 mM EDTA were sheared as above in increasing concentrations of HDL and ApoA-I or in single concentrations of alpha-1-acid glycoprotein (AAG) and transthyretin (each at 500 μg/mL). VWF remaining in solution was determined by ELISA. (D) Correlation of VWF remaining in solution after exposure to shear stress with the amount of VWF–ApoA-I complex formation measured by sandwich ELISA is described in supplemental Data. BSA, bovine serum albumin.