von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelial cells as a very large multimer, but circulates in plasma as a group of multimers ranging from 500 to 10 000 kd. An important mechanism for depolymerization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleaving protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first described as a Ca ؉؉ -dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and characterized. In this study, the purification of human vWF-cleaving protease from a commercial preparation of factor VIII/vWF concentrate by means of several column chromatographic steps, including 2 steps of heparin-Sepharose column, is reported. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the anion exchange and gel filtration column fractions showed that the vWF-cleaving protease activity corresponded to a protein band of 150 kd. Introductionvon Willebrand factor (vWF) plays an essential role in platelet adhesion to damaged blood vessels by forming a bridge between platelet surface glycoproteins and damaged subendothelium. vWF also plays an important role in hemostasis by binding and stabilizing coagulation factor VIII, protecting it from proteolysis. 1,2 vWF is composed of subunits of 2050 amino acid residues with the following repeated motifs or domains: DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. 3,4 vWF precursor is synthesized as a very large protein in endothelial cells 5 and megakaryocytes 6 and undergoes several posttranslational events; these include removal of a signal peptide and a large propeptide, the formation of intrachain and interchain disulfide bonds, and glycosylation. Dimers are formed in the endoplasmic reticulum through the formation of C-terminal interchain disulfide bonds, 7 and these dimers form multimers in the Golgi apparatus by the formation of interdimer disulfide bonds at the N-terminal region of the protein. This structure has been referred to as a head-to-head and tail-to-tail mode. 8 The mature vWF is synthesized and stored in endothelial cells 9,10 and then slowly released into the circulating blood. Cultured human umbilical vein endothelial cells release only the fully polymerized form of vWF to the condition medium. 11 Also, normal platelets contain exclusively the intact form of vWF in their granules. 12 In plasma, however, vWF exists as a mixture of disulfide-bonded multimers, with sizes ranging from dimers (500 kd) to highly polymerized forms as large as 10 000 kd. 1,2 A size distribution of multimers is important for normal hemostasis in that the larger multimers have a higher thrombotic activity than smaller polymers. 13 An excess of the very large vWF multimers in circulation, however, can result in platelet clumping, thrombosis, and thrombocytopenia. 14 The large polymerized forms of vWF un...

Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".

T.Kawakami and T.Chiba contributed equally to this workNEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modi®er that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modi®cation of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our ®ndings indicate that the NEDD8-modifying system accelerates the formation of the E2±E3 complex, which stimulates protein polyubiquitylation.

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