von Willebrand factor (vWF) in the circulation is subjected to proteolysis. In a recent study, we reported that normal plasma contains a protease activity that cleaves vWF in a shear-dependent manner, causing a decrease in its multimer size while generating dimers of the 140-kD and the 176-kD fragments indistinguishable from those found in normal plasma. In this study, the plasma protease has been partially purified and characterized and the role of vWF conformation in its cleavage by the protease has been further investigated. Guanidine HCl caused unfolding of vWF in a concentration-dependent manner, resulting in a shift in its fluorescence emission maxima to longer wavelengths. A dramatic increase in its proteolytic susceptibility was seen at 1.1 to 1.2 mol/L guanidine HCl, a concentration causing only a 3- to 4-nm shift in vWF emission maxima. Although vWF molecules refolded as guanidine HCl was removed by dialysis, the refolding was accompanied only by a partial recovery of the proteolytic resistance. The plasma protease, partially purified by approximately 900 folds by Sephacryl S-300 HR gel filtration, Matrex gel orange A dye affinity chromatography, and Q Sepharose anion exchange, had a molecular mass of approximately 200 kD and was inhibited by EDTA, EGTA, or 1,10-phenanthroline. The inhibition by EGTA or EDTA could be reversed by Ca2+ but not by mg2+. It was not inhibited by a panel of synthetic and natural protease inhibitors or adsorbed by gelatin-agarose, and it was present in plasmas deficient in proteins involved in coagulation and anticoagulation. The vWF fragments generated by the protease, as mapped by peptide-specific antibodies VP-1 and LJ-7745, were in distinguishable from the natural fragments but distinct from those produced by plasmin. High molecular weight endothelial vWF, after exposure to guanidine HCLI or high shear stress, was cleaved by the protease to smaller forms. These results support the model that endothelial secreted vWF is converted to multimers by a novel plasma metalloproteinase. Although native vWF exists in a conformation relatively resistant to cleavage, an alteration in the conformation by shear stress can lead to enhanced proteolytic susceptibility. This model may explain the decrease in vWF multimer sizes in various clinical conditions.