Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis

Abstract

Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximately 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximately 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. vWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, of calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N-ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe-CHN2. Activation by divalent metal ions was found to increase in the following order: Zn2+ approximately Cu2+ approximately CD2+ approximately Ni2+ approximately Co2+ <Mn2+ <Mg2+ <Ca2+ <Sr2+ <Ba2+. The observed properties of the vWF- degrading enzyme differ from those of all other hitherto described proteases. Purified vWF was incubated with the protease, and the degraded material subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after disulfide reduction. The size, amino acid composition, and amino terminal sequence of the reduced fragments confirmed that the peptide bond 842Tyr-843Met had been cleaved, ie, the same bond that has been proposed to be cleaved in vivo.

Diagnosis of thrombotic thrombocytopenic purpura based on modulation by patient plasma of normal platelet adhesion under flow condition

“…The VWF multimers found in the normal circulation are synthesized by megakaryocytes and endothelial cells (Moake, 1997). Both cell types consist of unusually large multimers that are normally degraded by a specific metalloprotease cleaving the peptide bond Tyr842-Met843 (Dent et al, 1990;Furlan et al, 1996;Tsai, 1996). In vitro, the protease generates fragments of VWF that are similar to those found in normal plasma.…”

“…Different methods for VWF-cleaving protease activity have been developed recently. These methods are based on a change in the multimeric pattern of VWF (Furlan et al, 1996;Tsai, 1996;Obert et al, 1999). In another method, the protease activity was measured using a collagen-coated surface, where a decrease in collagen binding reflected degraded VWF (Gerritsen et al, 1999).…”

Pediatric thrombotic thrombocytopenic purpura

“…Von Willebrand factor-cleaving protease was purified in 1996, 10,11 cloned [12][13][14] and identified as the 13th member of the family of metalloprotease ADAMTS in 2001. 15,16 ADAMTS13 is synthetized in the liver (hepatic stellate cells) and vascular endothelial cells.…”

Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia

“…Regulation of VWF size and function (large multimeric forms are most haemostatically effective) is based on proteolytic modifications, which occur soon after secretion or release into the bloodstream. This is mediated by a recently identified specific VWF cleaving protease (ADAMTS13) (Furlan et al, 1996;Tsai, 1996;Levy et al, 2001). In vivo the cleavage site for ADAMTS13 in the intact VWF subunit is normally protected but becomes susceptible to proteolysis under conditions affecting the VWF protein confirmation, such as high shear in the microcirculation.…”