Partial amino acid sequence of purified von Willebrand factor-cleaving protease

von Willebrand factor-cleaving protease (vWF-cp) is responsible for the con tinuous degradation of plasma vWF multimers released from endothelial cells . It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in plasma. Purified vWF-cp may be usefu l for replacement in these patients, who are. now treated by plasma therapy . In this study, vWF-cp was purified from normal human plasma by affinity c hromatography on the IgG fraction from a patient with autoantibodies to vWF -cp and by a series of further chromatographic procedures, including affini ty chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. F our single-chain protein bands, separated by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis under nonreducing conditions, showed M-r of 150 , 140, 130, and 110 kd and were found to share the same N-terminal amino ac id sequence, suggesting that they were derived from the same polypeptide ch ain that had been partially degraded at the carboxy-terminal end. A hydroph obic sequence (Ala-Ala-Gly-Gly-lie-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel fil tration as a high-molecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by th e use of concentrated chaotropic salts. vWF-cp in normal human plasma or se rum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity o f vWF-cp is unusually stable during incubation at 37 degreesC; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer tha n 1 week. There was even a temporary increase in protease activity during t he first 3 days of incubation. (C) 2001 by The American Society of Hematolo gy.