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.

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.

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...

Congenital thrombotic thrombocytopenic purpura is an autosomal recessive inherited disease with a clinically heterogeneous course and an incompletely understood genotype-phenotype correlation. In 2006, the Hereditary TTP Registry started recruitment for a study which aimed to improve the understanding of this ultra-rare disease. The objective of this study is to present characteristics of the cohort until the end of 2017 and to explore the relationship between overt disease onset and ADAMTS13 activity with emphasis on the recurring ADAMTS13 c.4143_4144dupA mutation. Diagnosis of congenital thrombotic thrombocytopenic purpura was confirmed by severely deficient ADAMTS13 activity (≤10% of normal) in the absence of a functional inhibitor and the presence of ADAMTS13 mutations on both alleles. By the end of 2017, 123 confirmed patients had been enrolled from Europe (n=55), Asia (n=52, 90% from Japan), the Americas (n=14), and Africa (n=2). First recognized disease manifestation occurred from around birth up to the age of 70 years. Of the 98 different ADAMTS13 mutations detected, c.4143_4144dupA (exon 29; p.Glu1382Argfs*6) was the most frequent mutation, present on 60 of 246 alleles. We found a larger proportion of compound heterozygous than homozygous carriers of ADAMTS13 c.4143_4144dupA with overt disease onset at < 3 months of age (50% vs. 37%), despite the fact that ADAMTS13 activity was <1% in 18 of 20 homozygous, but in only 8 of 14 compound heterozygous carriers. An evaluation of overt disease onset in all patients with an available sensitive ADAMTS13 activity assay (n=97) shows that residual ADAMTS13 activity is not the only determinant of age at first disease manifestation. Registered at clinicaltrials.gov identifier NCT01257269.

Assays of vWF-CPase activity and its inhibitor may be useful for predicting the response to therapy and the outcome of patients with TTP. In some patients, nonfamilial TTP with a poor prognosis may not be caused by a constitutional or acquired deficiency of vWF-CPase with its inhibitor. Although PE and immunosuppressive therapy are effective in patients with nonfamilial TTP and a vWF-CPase inhibitor, other therapeutic modalities may be needed for nonfamilial TTP with unknown etiology.

We report here 7 new mutations in the ADAMTS13 gene responsible for UpshawSchulman syndrome (USS), a catastrophic phenotype of congenital thrombotic thrombocytopenic purpura, by analyzing 5 Japanese families. There were 3 mutations that occurred at exon-intron boundaries: 414؉1G>A at intron 4, 686؉1G>A at intron 6, and 1244؉2T>G at intron 10 (numbered from the A of the initiation Met codon), and we confirmed that 2 of these mutations produced aberrantly spliced messenger RNAs (mRNAs). The remaining 4 mutations were missense mutations: R193W, I673F, C908Y, and R1123C. In expression experiments using HeLa cells, all mutants showed no or a marginal secretion of ADAMTS13. Taken together with the findings in our recent report we determined the responsible mutations in a total of 7 Japanese patients with USS with a uniform clinical picture of severe neonatal hyperbilirubinemia, and in their family members, based on ADAMTS13 gene analysis. Of these patients, 2 were homozygotes and 5 were compound heterozygotes. The parents of one homozygote were related (cousins), while those of the other were not. Molecular models of the metalloprotease, fifth domain of thrombospondin 1 (Tsp1-5), and Tsp1-8 domains of ADAMTS13 suggest that the missense mutations could cause structural defects in the mutants. IntroductionThrombotic thrombocytopenic purpura (TTP) is a life-threatening generalized disorder, and its diagnosis is made according to the criteria of Moschcowitz's pentad 1 : thrombocytopenia, microangiopathic hemolytic anemia (MAHA), fluctuating neurologic signs, renal failure, and fever. These criteria, however, are almost undistinguishable from those of hemolytic-uremic syndrome (HUS) with Gasser's triad 2 ; MAHA, thrombocytopenia, and renal insufficiency. Thus, the comprehensive term "TTP/HUS" or "thrombotic microangiopathy" 3 has frequently been used in clinical practice.Recent advances in elucidating the proteolytic processing of plasma von Willebrand factor (VWF) multimers have established assays for the activity of VWF-cleaving protease and its inhibitor (autoantibody). [4][5][6][7] These assays have largely made it possible to distinguish TTP from HUS, because the former has defective VWF-cleaving activity, whereas the latter has VWF-cleaving activity. 6,7 Studies by several groups of investigators have led to the identification of this enzyme as a new metalloprotease belonging to the ADAMTS (a disintegrinlike and metalloprotease with thrombospondin type 1 motif) family, which has been designated ADAMTS13. [8][9][10][11][12] This enzyme is produced in the liver. [10][11][12] The deduced amino acid residue number is 1427, and the gene contains 29 exons and is located on chromosome 9q34. [10][11][12] Upshaw-Schulman syndrome (USS) was originally reported as a disease complex with repeated episodes of thrombocytopenia and hemolytic anemia that quickly respond to infusions of fresh frozen plasma (FFP). [13][14][15][16] Clinical signs often develop in the patients during the newborn period or early infancy. In fact, the ea...

Senescence accelerated mice P8 (SAMP8) show significant age-related deteriorations in memory and learning ability in accordance with early onset and rapid advancement of senescence. Brains of SAMP8 mice reveal an ageassociated increase of PAS-positive granular structures in the hippocampal formation and astrogliosis in the brain stem and hippocampus. A spongy degeneration in the brain stem appears at 1 month of age and reaches a maximum at 4-8 months. In addition, clusters of activated microglia also appear around the vacuoles in the brain stem. β/A4(Aβ) protein-like immunoreactive granular structures are observed in various regions and increase in number markedly with age. Other age-associated histological changes include cortical atrophy, neuronal cell loss in locus coeruleus and lateral tegmental nuclei, intraneuronal accumulation of lipopigments in Purkinje cells and eosinophilic inclusion bodies in thalamic neurons. A blood-brain barrier dysfunction and astrogliosis are also prominent with advancing age in the hippocampus. These changes are generally similar to the pathomorphology of aging human brains and characterized by their association with some specific glioneuronal reactions. As for the hallmarks of Alzheimer brains, tau morphology has not yet been confirmed regardless of the age-related increase in phosphorylated tau in SAMP8 mice brains, but early age-related Aβ deposition in the hippocampus has recently been published. SAMP8 mice are, therefore, not only a senescence-accelerated model but also a promising model for Alzheimer's disease and other cognitive disorders.

Human quiet standing is often modeled as a single inverted pendulum rotating around the ankle joint, under the assumption that movement around the hip joint is quite small. However, several recent studies have shown that movement around the hip joint can play a significant role in the efficient maintenance of the center of body mass (COM) above the support area. The aim of this study was to investigate how coordination between the hip and ankle joints is controlled during human quiet standing. Subjects stood quietly for 30 s with their eyes either opened (EO) or closed (EC), and we measured subtle angular displacements around the ankle (thetaa) and hip (thetah) joints using three highly sensitive CCD laser displacement sensors. Reliable data were obtained for both angular displacement and angular velocity (the first derivative of the angular displacement). Further, measurement error was not predominant, even among the angular acceleration data, which were obtained by taking the second derivative of the angular displacement. The angular displacement, velocity, and acceleration of the hip were found to be significantly greater (P<0.001) than those of the ankle, confirming that hip-joint motion cannot be ignored, even during quiet standing. We also found that a consistent reciprocal relationship exists between the angular accelerations of the hip and ankle joints, namely positive or negative angular acceleration of ankle joint is compensated for by oppositely directed angular acceleration of the hip joint. Principal component analysis revealed that this relationship can be expressed as: thetah=gammathetaa with gamma=-3.15+/-1.24 and gamma=-3.12+/-1.46 (mean +/-SD) for EO and EC, respectively, where theta is the angular acceleration. There was no significant difference in the values of y for EO and EC, and these values were in agreement with the theoretical value calculated assuming the acceleration of COM was zero. On the other hand, such a consistent relationship was never observed for angular displacement itself. These results suggest that the angular motions around the hip and ankle joints are not to keep the COM at a constant position, but rather to minimize acceleration of the COM.