Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

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.

SummaryPatients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with non-familial TTP.

In patients with thrombotic thrombocytopenic purpura (TTP), excessive intravascular platelet aggregation has been associated with appearance in plasma of unusually large von Willebrand factor (vWF ) multimers. These extremely adhesive vWF multimers may arise due to deficiency of a “depolymerase” cleaving vWF to smaller molecular forms, either by reducing the interdimeric disulfide bridges or by proteolytic degradation. We studied the activity of a recently described vWF-cleaving protease in four patients with chronic relapsing TTP. Diluted plasma samples of TTP patients were incubated with purified normal human vWF in the presence of a serine protease inhibitor, at low ionic strength, and in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. Four patients, that included two brothers, with chronic relapsing TTP displayed either substantially reduced levels or a complete absence of vWF-cleaving protease activity. In none of these patient plasmas was an inhibitor of or an antibody against the vWF-cleaving protease established. Our data suggest that the unusually large vWF multimers found in TTP patients may be caused by deficient vWF-cleaving protease activity. Deficiency of this protease may be inherited in an autosomal recessive manner and seems to predispose to chronic relapsing TTP. The assay of the vWF-cleaving protease activity may be used as a sensitive diagnostic tool for identification of subjects with a latent TTP tendency.

von Willebrand factor-cleaving protease (vWF-cp) is responsible for the continuous 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 useful 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 chromatography on the IgG fraction from a patient with autoantibodies to vWF-cp and by a series of further chromatographic procedures, including affinity chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. Four single-chain protein bands, separated by sodium dodecyl sulfatepolyacrylamide 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 acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence (Ala-Ala-Gly-Gly-Ile-Leu-His-LeuGlu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel filtration as a highmolecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by the use of concentrated chaotropic salts. vWF-cp in normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37°C; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was even a temporary increase in protease activity during the first 3 days of incubation. (Blood. 2001;98:1654-1661)