ADAMTS13 activity and genetic mutations in Japan.

Thrombotic thrombocytopenic purpura (TTP), a life threatening disease, can be induced by congenital or acquired deficiency of plasma metalloprotease ADAMTS13. Since the publication of the first genetic analysis in patients with congenital ADAMTS13 deficiency in 2001, more than 100 genetic defects in the ADAMTS13 gene have been reported worldwide. Genetic analysis in patients with ADAMTS13 deficiency has greatly contributed to the understanding of the etiology of TTP. A rapid and quantitative assay method for the plasma ADAMTS13 activity was developed recently in 2005 and opened a new area of TTP research - namely genetic research using a general population to evaluate age and gender differences of ADAMTS13 activity as well as phenotype - genotype correlations of genetic polymorphisms and estimation of a homozygote or a compound heterozygote ADAMTS13 deficiencies. The Japanese general population study included 3616 individuals with an age between 30 - 80 years confirming other studies that while ADAMTS13 activity decreased with age, VWF antigen increased and VWF antigen levels are lowest in blood group O indviduals, whereas ADAMTS13 activity levels were not associated with the AB0 blood group. 25 polymorphisms with a minor allele frequency of more than 0.01 were found, among them 6 missense mutations and 19 synonymous mutations, except P475S missense polymorphisms that was only idenitified in an East Asian population, characterized by reduced ADAMTS13 activity. Prevalence of congenital ADAMTS13 deficiency in the Japanese population was estimated about one individual in 1.1 x 106 to be homozygote or compound heterozygote for ADAMTS13 deficiency. So far more than 40 mutations in Japanese congenital TTP patients were found, but R193W, Q449*, C754Afs*24 (c.2259delA) and C908Y were identified in more than four patients suggesting the precipitaion of these mutations in the Japanese population.

Crystal structure and enzymatic activity of an ADAMTS-13 mutant with the East Asian-specific P475S polymorphism.

BACKGROUND: An East Asian-specific P475S polymorphism in the gene encoding ADAMTS-13 causes an approximately 16% reduction in plasma ADAMTS-13 activity. OBJECTIVES: To demonstrate the impact of this dysfunctional polymorphism by characterizing the structure and activity of the P475S mutant protein. METHODS: We determined the crystal structure of the P475S mutant of ADAMTS-13-DTCS (DTCS-P475S, residues 287-685) and compared it with the wild-type structure. We determined the enzymatic parameters of ADAMTS-13-MDTCS (residues 75-685) and MDTCS-P475S, and further examined the effects of denaturants and reaction temperature on their activity. We also examined the cleavage of shear-treated von Willebrand factor (VWF) by MDTCS-P475S. RESULTS: MDTCS-P475S showed a reaction rate similar to that of wild-type MDTCS, but showed two-fold lower affinity for the peptidyl substrate, indicating that the Pro475-containing V-loop (residues 474-481) in the CA domain is a substrate-binding exosite. Structural analysis showed that the conformation of the V-loop was significantly different in DTCS-P475S and the wild type, where no obvious interactions of Ser475 with other residues were observed. This explains the higher susceptibility of the enzymatic activity of MDTCS-P475S to reaction environments such as denaturants and high temperature. MDTCS-P475S can moderately cleave shear-treated VWF. CONCLUSIONS: We have provided structural evidence that the P475S polymorphism in ADAMTS-13 leads to increased local structural instability, resulting in lowered affinity for the substrate without changing the reaction rate. The moderate activity of ADAMTS-13-P475S for shear-treated VWF is sufficient to prevent thrombotic thrombocytopenic purpura (TTP) onset.

Natural history of Upshaw-Schulman syndrome based on ADAMTS13 gene analysis in Japan.

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.

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13).

BACKGROUND: Over the last 4 years ADAMTS-13 measurement underwent dramatic progress with newer and simpler methods. AIMS: Blind evaluation of newer methods for their performance characteristics. DESIGN: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS-13-deficient plasma (arbitrarily set at 0%) into one normal-pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested 'blind' 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. RESULTS: There were eight functional and three antigen assays. Linearity of observed-vs.-expected ADAMTS-13 levels assessed as r2 ranged from 0.931 to 0.998. Between-run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10-15% for five methods and up to 20% for the remaining three. F-values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS-13 levels (the higher the F-value, the better the capacity) ranged from 3965 to 137. Between-method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS-13 offer the best performance characteristics. CONCLUSIONS: New assays for ADAMTS-13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

Site-specific effect of estradiol on gene expression in the adipose tissue of ob/ob mice.

Sex hormones are likely to be involved in sex differences in adipose tissue distribution. To test whether estrogen regulates genes expressed in the adipose tissue in a site-specific manner, we studied the effect of exogenous estradiol on the gene expression in visceral and subcutaneous adipose tissues of male ob/ob mice. We screened genes expressed in a site- and sex-specific manner, and genes that were affected by exogenous estradiol by DNA chip analysis. They were verified by real-time PCR. Myosin heavy chain 2B (Myh4) and phosphoglycerate mutase muscle-specific subunit (Pgam) were expressed specifically in the subcutaneous adipose tissue, and uroplakin IIIb (UP3) was expressed specifically in the visceral adipose tissue. DEAD-box Y RNA helicase (DBY) and eukaryotic initiation factor 2 gamma Y (eIF2gamma Y) were expressed only in male adipose tissue. X-chromosome inactive specific transcript (Xist) was expressed only in female adipose tissue. When estradiol was subcutaneously administrated to male mice, the expression of monocyte-chemoattractant protein-1 (MCP-1) and androgen receptor (AR) genes was regulated in a site-specific manner. The difference in the amount of estrogen receptor did not account for the site-specific effect of estrogen. Our findings show that estrogen affects the expression of some adipocyte genes in a site-specific manner.

Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart.

RTP/Drg1/Cap43/rit42/TDD5/Ndr1/NDRG1 (referred to as NDRG1 hereafter) is a cytoplasmic protein involved in stress responses, hormone responses, cell growth, and differentiation. Recently, the mutation of this gene was reported to be causative for hereditary motor and sensory neuropathy-Lom. Here, we cloned two human cDNAs encoding NDRG3 and NDRG4, which are homologous to NDRG1. These two genes, together with NDRG1 and a previously deposited cDNA (designated NDRG2), constitute the NDRG gene family. The four members share 57-65% amino acid identity. NDRG4 was further characterized because its mRNA expression was quite specific in brain and heart, in contrast to the relatively ubiquitous expression of the other three members. NDRG4 mRNA consists of three isoforms, NDRG4-B, NDRG4-B(var), and NDRG4-H. Northern and Western blot analyses showed that NDRG4-B was expressed only in the brain, whereas NDRG4-H was expressed in both brain and heart. NDRG4-B(var) was a minor product. NDRG4 expression was more abundant in adult than fetal brain and heart and was markedly decreased in the Alzheimer's diseased brain. In situ hybridization showed that NDRG4 was localized in neurons of the brain and spinal cord. The NDRG4 gene contains 17 exons. mRNA expression of the three NDRG4 isoforms is regulated by alternative splicing and possibly by alternative promoter usage. The finely tuned expression of the NDRG gene family members suggests that they have different specific functions.

Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response.

Herp is a 54-kDa membrane protein in the endoplasmic reticulum (ER). The mRNA expression level of Herp is increased by the accumulation of unfolded proteins in the ER. Transcriptional changes designed to deal with this type of ER stress is called the unfolded protein response (UPR). Most mammalian UPR-target genes encode ER-resident molecular chaperones: GRP78, GRP94, and calreticulin. The promoter regions of these genes contain a cis-acting ER stress response element, ERSE, with the consensus sequence of CCAAT-N(9)-CCACG. Under conditions of ER stress, p50ATF6 (the active form of the transcription factor, ATF6) binds to CCACG when CCAAT is bound by the general transcription factor, NF-Y/CBF. Here, we report the genomic structure of human Herp and the presence of a new ER stress response element, ERSE-II, in its promoter region. The gene for Herp consists of eight exons, localized to chromosome 16q12.2-13. The promoter region contains a single ERSE-like sequence. In reporter gene assays, disruption of this cis-element resulted in a partial reduction of the transcriptional response to ER stress, suggesting that the element is functional for the UPR. These results also suggest the involvement of additional elements in the UPR. Further analysis, using an optimized plasmid containing an mRNA-destabilizing sequence, revealed ERSE-II (ATTGG-N-CCACG) as the second ER stress response element. Interestingly, ERSE-II was also dependent on p50ATF6, in a manner similar to that of ERSE, despite the disparate structure. The strong induction of Herp mRNA by ER stress would be achieved by the cooperation of ERSE and ERSE-II.

Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress.

Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a novel protein, Herp, containing a ubiquitin-like domain at the N terminus. mRNA expression of Herp was strongly up-regulated by inducers of ER stress, including mercaptoethanol, tunicamycin, A23187, and thapsigargin. The ER stress-dependent induction of Herp was also observed at the protein level. Immunochemical analyses using Herp-specific antibodies indicated that Herp is a 54-kDa, membrane-associated ER protein. Herp is the first integral membrane protein regulated by the ER stress response pathway. Both the N and C termini face the cytoplasmic side of the ER; this membrane topology makes it unlikely that Herp acts as a molecular chaperone for proteins in the ER, in contrast to GRP78 and other ER stress-responsive proteins. Herp may, therefore, play an unknown role in the cellular survival response to stress.

Phosphorylation of RTP, an ER stress-responsive cytoplasmic protein.

RTP, also called Drg1/Cap43/rit42/TDD5/Ndr1, was originally identified as a homocysteine-responsive gene product, and is now considered to be involved in stress responses, atherosclerosis, carcinogenesis, differentiation, androgen responses, hypoxia, and N-myc pathways. We raised an antiserum against a recombinant human RTP. Western blot analysis showed that RTP expression was induced in human umbilical vein endothelial cells under conditions causing endoplasmic reticulum stress. RTP was partially phosphorylated at seven or more sites. The phosphorylation was reversible, and was enhanced by an increased level of intracellular cAMP and inhibited by both a protein kinase A inhibitor and a calmodulin kinase inhibitor. Protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in cells at the early log phase, and then decreased with increasing cell density. These data demonstrated that RTP is a phosphorylated stress-responsive protein, and its phosphorylation may be related to cell growth.

Monkey hepatocytes efficiently express tissue factor pathway inhibitor (TFPI), in contrast with human and rat hepatocytes.

It has been reported that tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor that regulates the extrinsic blood coagulation pathway, is not expressed in human, bovine, rabbit, or rat liver. Here, we found that TFPI is efficiently expressed in Macaque monkey liver. Monkey hepatocytes were identified as the expression cells by Northern blot analysis. The hepatocytes were stained with anti-human TFPI antibody, as were endothelial cells of the small vessels. We isolated and sequenced the 5'-flanking 1.4 kb regions of monkey and human TFPI genes, and found them to show 92.6% identity in their nucleotide sequences. We measured their transcriptional activities using a luciferase reporter gene and showed that the activity of the monkey TFPI gene is higher than that of the human gene in monkey primary hepatocytes. Although the binding motif of hepatocyte nuclear factor-1 is present only in the monkey gene, the site does not seem to be involved in the transcriptional activity. Mutagenetic analyses revealed that the region from -138 to +28 in the monkey gene is important for the expression of TFPI in hepatocytes. The present study indicates that the expression of the monkey TFPI gene is regulated by different mechanisms from the human TFPI gene.

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers.

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.

Analysis of gene expression in homocysteine-injured vascular endothelial cells: demonstration of GRP78/BiP expression, cloning and characterization of a novel reducing agent-tunicamycin regulated gene.

An elevated plasma level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not yet been elucidated. In the present study, we have applied a modified nonradioactive differential display analysis to evaluate changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVEC). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, a stress protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in a homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. The remaining four were uncharacterized genes. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed reducing-agents and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP gene expression was induced in HUVEC after 4 h of incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercaptoethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissues. These observations indicate that homocysteine can alter the expressivity of multiple genes, including a stress protein and several novel genes. These responses may contribute to atherogenesis.

Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C.

Thrombomodulin is a cofactor protein on vascular endothelial cells that inhibits the procoagulant functions of thrombin and enhances thrombin-catalyzed activation of anticoagulant protein C. Thrombomodulin also accelerates the proteolytic activation of a plasma procarboxypeptidase referred to as thrombin-activable fibrinolysis inhibitor (TAFI). In this study, we describe structures on recombinant membrane-bound thrombomodulin that are required for human TAFI activation. Deletion of the N-terminal lectin-like domain and epidermal growth factor (EGF)-like domains 1 and 2 had no effect on TAFI or protein C activation, whereas deletions including EGF-like domain 3 selectively abolished thrombomodulin cofactor activity for TAFI activation. Provided that thrombomodulin EGF-like domain 3 was present, TAFI competitively inhibited protein C activation catalyzed by the thrombin-thrombomodulin complex. A thrombomodulin construct lacking EGF-like domain 3 functioned normally as a cofactor for protein C activation but was insensitive to inhibition by TAFI. Thus, the anticoagulant and antifibrinolytic cofactor activities of thrombomodulin have distinct structural requirements: protein C binding to the thrombin-thrombomodulin complex requires EGF-like domain 4, whereas TAFI binding also requires EGF-like domain 3.

Changes of gene expression by lysophosphatidylcholine in vascular endothelial cells: 12 up-regulated distinct genes including 5 cell growth-related, 3 thrombosis-related, and 4 others.

Lysophosphatidylcholine (lysoPC), a component of oxidatively modified lipoproteins, is present in atherosclerotic lesions, and its proatherogenic properties have been demonstrated. To gain an insight into lysoPC-mediated endothelial gene expression, we applied nonradioactive differential display analysis of mRNA from lysoPC-treated and untreated human umbilical vein endothelial cells. We identified 12 up-regulated distinct genes including 5 cell growth-related genes (two phosphatases CL100 and B23/hVH-3, gravin, activating transcription factor-4, and heparin-binding epidermal growth factor-like growth factor), 3 thrombosis-related genes (plasminogen activator inhibitor-1, tissue plasminogen activator, and thrombomodulin), and 4 others (stanniocalcin, NAD-dependent methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, BENE, and reducing agents and tunicamycin-responsive protein). We isolated a full-length cDNA of human gravin. The cDNA sequence of gravin was homologous with rat mitogenic regulatory gene or rat protein kinase C binding protein and substrate, suggesting that gravin would regulate cell growth. Thus, lysoPC apparently accelerates atherosclerosis by regulating the expression of a wide variety of genes. Our data suggest the involvement in atherogenesis of the genes hitherto regarded as atherosclerosis-unrelated.

Lysophosphatidylcholine decreases the synthesis of tissue factor pathway inhibitor in human umbilical vein endothelial cells.

Thrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 microM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

Nonradioactive differential display cloning of genes induced by homocysteine in vascular endothelial cells.

Hyperhomocysteinemia is known to be a risk factor for arteriosclerosis and thrombosis. To elucidate the mechanisms by which homocysteine may promote vascular diseases, we have applied a modified nonradioactive differential display analysis that evaluates changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVECs). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, suggesting that unfolded proteins would accumulate in the ER because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. Homology searches of the remaining four clones failed to retrieve any similar sequences with a known function. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed RTP (reducing agent and tunicamycin-responsive protein). Northern blot analysis revealed that RTP mRNA expression was induced in HUVECs treated with not only homocysteine but also 2-mercaptoethanol and tunicamycin, both of which are known to induce ER stress. RTP mRNA was ubiquitously expressed in human adult organs, and seemed to be regulated in mouse embryogenesis. Consequently, our differential display analysis revealed that homocysteine alters the expressivity of multiple proteins, especially ER stress-responsive ones. This potential ability of homocysteine may be involved in atherogenesis.