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.

Female mammals are endowed with a finite number of oocytes at birth, each enclosed by a single layer of somatic (granulosa) cells in a primordial follicle. The fate of most follicles is atretic degeneration, a process that culminates in near exhaustion of the oocyte reserve at approximately the fifth decade of life in women, leading to menopause. Apoptosis has a fundamental role in follicular atresia, and recent studies have shown that Bax, which is expressed in both granulosa cells and oocytes, may be central to ovarian cell death. Here we show that young adult female Bax-/- mice possess threefold more primordial follicles in their ovarian reserve than their wild-type sisters, and this surfeit of follicles is maintained in advanced chronological age, such that 20-22-month-old female Bax-/- mice possess hundreds of follicles at all developmental stages and exhibit ovarian steroid-driven uterine hypertrophy. These observations contrast with the ovarian and uterine atrophy seen in aged wild-type female mice. Aged female Bax-/- mice fail to become pregnant when housed with young adult males; however, metaphase II oocytes can be retrieved from, and corpora lutea form in, ovaries of aged Bax-/- females following superovulation with exogenous gonadotropins, and some oocytes are competent for in vitro fertilization and early embryogenesis. Therefore, ovarian lifespan can be extended by selectively disrupting Bax function, but other aspects of normal reproductive performance remain defective in aged Bax-/- female mice.