Recombinant VWF fragments and pVWF were denatured with guanidine-HCl [30] as following. Purified pVWF or various VWF fragments (10 mM) were incubated with 1.5 M guanidine-HCl at 37uC for 2 h. Then, guanidine-HCl treated VWF fragments and pVWF were diluted serially with 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl. The binding affinity of denatured VWF and SZ34 was determined by an ELISA. Because the initial concentrations of pVWF and VWF fragments were high, the final concentration of guanidine-HCl in solution was ,20 mM, which did not interfere with the binding activity of SZ34 (data not shown), consistent with what was reported by Tsai [30] in which he showed that guanidine-HCl at 24 mM did not interfere with the platelet aggregation.