von Willebrand factor (VWF) is synthesized primarily in vascular endothelial cells and secreted into the plasma as unusually large VWF multimers. Normally, these multimers are quickly degraded into smaller forms by a plasma metalloproteinase, VWF-cleaving protease (VWF-CP). Decreases in the activity of this enzyme result in congenital and acquired thrombotic thrombocytopenic purpura (TTP). The human VWF-CP has recently been purified. Cloning of the corresponding cDNA revealed that the 1,427-aa polypeptide is a member of the ADAMTS gene family, termed ADAMTS13. Twelve rare mutations in this gene have been identified in patients with congenital TTP. Here, we report missense and nonsense mutations in two Japanese families with Upshaw-Schulman syndrome, congenital TTP with neonatal onset and frequent relapses. The comparison of individual ADAMTS13 genotypes and plasma VWF-CP activities indicated that the R268P, Q449stop, and C508Y mutations abrogated activity of the enzyme, whereas the P475S mutant retained low but significant activity. The effects of these mutations were further confirmed by expression analysis in HeLa cells. Recombinant VWF-CP containing either the R268P or C508Y mutations was not secreted from cells. In contrast, Q449stop and P475S mutants were normally secreted but demonstrated minimal activity. Genotype analysis of 364 Japanese subjects revealed that P475S is heterozygous in 9.6% of individuals, suggesting that approximately 10% of the Japanese population possesses reduced VWF-CP activity. We report on a single-nucleotide polymorphism associated with alterations in VWF-CP activity; it will be important to assess this single-nucleotide polymorphism as a risk factor for thrombotic disorders.

Precise analysis of human CD34-negative (CD34 ؊ ) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34 ؊ severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin ؊ ) CD34 ؊ cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4-mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34 ؉ progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34 ؉ cells in vivo. These in vivo-generated CD34 ؉ cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34 ؊ SRCs in nonobese diabetic (

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Summary.  Upshaw–Schulman syndrome (USS) is an extremely rare hereditary deficiency of ADAMTS13 activity, termed congenital TTP. The clinical signs are usually mild during childhood, often with isolated thrombocytopenia. But their symptoms become more evident when patients have infections or get pregnant. We identified 43 USS‐patients in Japan, who ranged in age from early childhood to 79 years of age. Analysing the natural history of these USS patients based on ADAMTS13 gene mutations may help characterise their clinical phenotypes. Severe neonatal jaundice that requires exchange blood transfusion, a hallmark of USS, was found in 18 of 43 patients (42%). During childhood, 25 of 43 patients were correctly diagnosed with USS without gender disparity. These 25 patients were categorised as having ‘the early‐onset phenotype’. Between 15 and 45 years of age, 15 were correctly diagnosed, and, interestingly, they were all female. The remaining three patients were male and were diagnosed when they were older than 45 years of age, suggesting that they were ‘the late‐onset phenotype’. Two of these three males developed sudden overt TTP when they were 55 and 63 years old, respectively. These two men had two different homozygous ADAMTS13 gene mutations, p.R193W/p.R193W and p.C1024R/p.C1024R, respectively. Both of which were not discovered in the US or Western countries. In vitro expression studies showed that these two proteins were consistently secreted into the culture medium but to a lesser extent and with reduced activity compared to the wild‐type protein. Our results indicate that ‘the late‐onset phenotype’ of USS is formed with ethnic specificity.

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Previous studies have demonstrated that a high level of shear stress can produce platelet aggregation without the addition of any agonist. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A coneplate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and the remnant platelets showed the exposure of procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor (vWF) to platelet glycoprotein lb, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C (PKC) promoted shear-dependent microparticle formation. Epinephrine did not influence microparticle formation, although it enhanced platelet aggregation by high shear stress. These findings suggest the possibility that local generation of microparticles in atherosclerotic arteries, the site that pathologically high shear stress could occur, may contribute to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

A severe lack of von Willebrand factorcleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity. IntroductionVon Willebrand factor (VWF) functions as a molecular glue through its anchoring of platelets at sites of injured vessel walls under high shear stress. 1 Mature VWF contains 2050-amino acid residues, has a molecular weight of approximately 250 kDa, and is released from endothelial cells as an "unusually large" multimer (UL-VWFM) with a molecular weight of approximately 30 000 kDa. [2][3][4][5] In healthy individuals, UL-VWFM is converted rapidly in the circulation to smaller forms, a series of multimers with molecular weights ranging from approximately 500 to 20 000 kDa. 6 It is known that the larger multimers have more potent biologic activity. 7,8 Under shear stress conditions in the circulation, VWF becomes more susceptible to proteolysis, 9-12 resulting in the formation of small but consistent proportions of 176-kDa and 140-kDa fragments derived from the approximately 250-kDa VWF subunit. 13 On the basis of these findings, the existence of a specific VWF-cleaving protease (VWF-CP) has been proposed.UL-VWFM has been found in plasma from patients with thrombotic thrombocytopenic purpura (TTP). 14 TTP is characterized by thrombocytopenia, microangiopathic hemolytic anemia, renal failure, neurologic dysfunction, and fever. 15 In 1996, a metalloprotease that cleaves a peptide bond between amino acid residues Tyr842 and Met843 within the VWF A2 domain was partially purified. 16,17 It was also reported that a deficiency in VWF-CP activity was associated with TTP. 18 On the basis of these findings, it was proposed that the UL-VWFM produced as a result of the deficiency in VWF-CP activity promotes microvascular thrombus formation. Congenital or acquired deficiency of VWF-CP activity can cause TTP. Congenital TTP with neonatal onset and frequent relapse...

A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.