Kenji Soejima

Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity

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.

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

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.

Pregnancy-induced thrombocytopenia and TTP, and the risk of fetal death, in Upshaw-Schulman syndrome: a series of 15 pregnancies in 9 genotyped patients

Upshaw–Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) due to mutations in the gene that encodes for ADAMTS13 (ADAMTS13), but its clinical signs may be mild or absent during childhood. We have identified 37 patients with USS (24 females, 13 males) belonging to 32 families. The nine women from six families who were diagnosed during their first pregnancy are the focus of this report. Six of the nine women had episodes of thrombocytopenia during childhood misdiagnosed as idiopathic thrombocytopenic purpura. Thrombocytopenia occurred during the second–third trimesters in each of their 15 pregnancies, with 16 babies (one twin pregnancy), often followed by TTP. Of 15 pregnancies, eight babies were stillborn or died soon after birth, and the remaining seven were all premature except one, who was born naturally following plasma infusions to the mother that had started at 8 weeks’ gestation. All nine USS women had severely deficient ADAMTS13 activity. ADAMTS13 analyses demonstrated that eight women were compound heterozygotes of Y304C/G525D (2 siblings), R125VfsX6/Q1302X (2 siblings), R193W/R349C (2 siblings), I178T/Q929X, and R193W/A606P; one woman was homozygous for R193W. Only the R193W mutation has been previously reported. These observations emphasize the importance of measuring ADAMTS13 activity in the evaluation of thrombocytopenia during childhood and pregnancy.

Effect of ADAM28 on Carcinoma Cell Metastasis by Cleavage of von Willebrand Factor

BACKGROUND A disintegrin and metalloproteinase 28 (ADAM28) is implicated in tumor growth and metastasis in human breast and non-small cell lung carcinomas. We explored the mechanism of ADAM28-mediated metastasis by searching for new substrates of ADAM28. METHODS We used a yeast two-hybrid system to screen the human lung cDNA library for ADAM28-binding proteins and identified von Willebrand factor (VWF) as a potential candidate. Binding was confirmed using yeast two-hybrid and protein binding assays, and ADAM28-mediated cleavage of VWF was analyzed by immunoblotting. Exogenous VWF-induced apoptosis in vitro was examined in human lung carcinoma (PC-9 and Calu-3), breast carcinoma (MDA-MB231 and MCF-7), renal cell carcinoma (Caki-2 and 769P), and hepatocellular carcinoma (HepG2) cells, and expression of ADAM28 was assessed by reverse transcription-polymerase chain reaction and immunoblotting. Effect on lung metastasis of PC-9 and MDA-MB231 cells was assessed by knockdown of ADAM28 expression using short hairpin RNAs (ADAM28-shRNA) and small interfering RNAs (ADAM28-siRNA), and inhibition of activity using neutralizing anti-ADAM28 antibody, in a mouse xenograft model by in vivo imaging (n = 9 mice per group). All statistical tests were two-sided. RESULTS ADAM28 could bind to and cleave native VWF. Cells with very low ADAM28 expression (MCF-7, 769P, and HepG2) were susceptible to VWF-induced apoptosis, whereas cells with high expression (PC-9, Calu-3, MDA-MB231, and Caki-2) were resistant. Knockdown of ADAM28 expression in PC-9 and MDA-MB231 cells by shRNA showed increased carcinoma cell apoptosis mainly in lung blood vessels and statistically significantly decreased lung metastasis at week 3 after injection (PC-9-control [n = 9 mice] vs PC-9-ADAM28-shRNA [n = 9 mice]: mean count = 198 × 10(6) vs 37 × 10(6) photons/s, difference = 161 × 10(6) photons/s, 95% confidence interval = 134 × 10(6) to 188 × 10(6) photons/s, P < .001). Similar inhibition of lung metastasis was observed with ADAM28-siRNA and anti-ADAM28 antibody. CONCLUSION ADAM28 cleaves and inactivates proapoptotic VWF in carcinoma cells and enhances lung metastasis probably by promoting carcinoma cell survival within the blood vessels.

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood

Background: Platelet collagen receptor GPVI likely functions as a dimer rather than a monomer. Results: Preformed GPVI dimers, but not monomers, in resting platelets bind specific collagen sequences and are essential for platelet adhesion and activation. Conclusion: Constitutive GPVI dimers on resting platelets support platelet adhesion to collagen and activation. Significance: Resting platelets bind collagen through GPVI dimers, allowing immediate initiation of thrombus formation. The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

ADAMTS-13 attenuates thrombus formation on type I collagen surface and disrupted plaques under flow conditions

Plaque disruption with subsequent thrombus formation is a major cause of atherothrombotic diseases and von Willebrand factor (VWF), which is cleaved by ADAMTS-13, plays a critical role in thrombus formation. However, the role of ADAMTS-13 during thrombogenesis on atherosclerotic vessel remains unknown. We examined the localization of ADAMTS-13 in coronary thrombi obtained from patients with acute myocardial infarction. We also investigated the roles of ADAMTS-13 in thrombus formation using type I collagen-coated flow chambers (100S(-1) and 1500S(-1)) and on injured neointima of rabbit femoral arteries. ADAMTS-13 was present in thrombi of human coronary arteries, where it co-localized with VWF. In a flow chamber, both the average of the surface covered by platelet adhesion and the long axes of platelet thrombi were significantly augmented by an antibody to the ADAMTS-13 disintegrin-like domain (WH2-22-1A) at a shear rate of 1500s(-1), but not by an antibody to the ADAMTS-13 thrombospondin 1-3 domain (WH10). WH2-22-1A also reduced the activity of plasma ADAMTS-13 to cleave large VWF multimers during perfusion. Thrombi on injured neointima were induced by repeated balloon injury of rabbit femoral arteries, and were composed of platelet and fibrin, like human coronary thrombi. WH2-22-1A significantly augmented thrombus formation on injured neointima. These results suggest that the disintegrin-like domain of ADAMTS-13 functions in attenuating thrombus growth on diseased arteries exposed to a high shear rate.

Changes in plasma von Willebrand factor and ADAMTS13 levels associated with left atrial remodeling in atrial fibrillation.

INTRODUCTION Previous studies have shown raised plasma von Willebrand factor (VWF) levels in patients with atrial fibrillation (AF). However, little is known about changes of VWF associated with VWF-cleaving protease (ADAMTS13) in AF. The aim of this study was to examine the relationship between changes in plasma VWF and ADAMTS13 levels, and left atrial remodeling in AF patients. MATERIALS AND METHODS We measured plasma VWF and ADAMTS13 antigen levels in 70 paroxysmal AF (PAF) patients, 56 chronic AF (CAF) patients, and 55 control subjects. RESULTS Plasma VWF levels (mU/ml) were significantly higher in CAF and PAF patients compared with the controls (2103 +/- 743, 1930 +/- 676, 1532 +/- 555, respectively, P < 0.0001 in CAF vs. controls, P = 0.001 in PAF vs. control), while ADAMTS13 levels (mU/ml) were significantly lower in CAF and PAF patients compared with the controls (795 +/- 169, 860 +/- 221, 932 +/- 173, respectively, P = 0.0002 in CAF vs. controls, P = 0.04 in PAF vs. control). The VWF/ADAMTS13 ratio was significantly higher in patients with CAF than PAF or controls (2.81 +/- 1.30, 2.34 +/- 0.92, 1.73 +/- 0.83, respectively; P = 0.01 in CAF vs. PAF, P < 0.0001 in CAF vs. controls). There was a significant correlation between the VWF/ADAMTS13 ratio and left atrial diameter (positive correlation; r = 0.275, P = 0.0002) and left atrial appendage flow velocity (negative correlation; r = -0.345, P = 0.0018). CONCLUSIONS These findings suggest that the imbalance between plasma VWF and ADAMTS13 levels caused by left atrial remodeling might be closely associated with intra-atrial thrombus formation in AF patients.

The 99 and 170 Loop-modified Factor VIIa Mutants Show Enhanced Catalytic Activity without Tissue Factor

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. Thek cat/K m value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm − 1s− 1) was 3-fold higher than that of wild-type VIIa (30.3 mm − 1 s− 1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K m value but not to an increase in the k cat value. On the other hand, the k cat/K m value for S-2288 hydrolysis by VIIa-39 (17.9 mm − 1 s− 1) was 18-fold higher than that of wild-type (1.0 mm − 1 s− 1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K m value but also to an increase in the k cat value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229–17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k cat value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K m andk cat values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity towardS-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.

Novel Antibody for the Treatment of Transthyretin Amyloidosis

Familial amyloidotic polyneuropathy (FAP) is a systemic amyloidosis mainly caused by amyloidogenic transthyretin (ATTR). This incurable disease causes death ∼10 years after onset. Although it has been widely accepted that conformational change of the monomeric form of transthyretin (TTR) is very important for amyloid formation and deposition in the organs, no effective therapy targeting this step is available. In this study, we generated a mouse monoclonal antibody, T24, that recognized the cryptic epitope of conformationally changed TTR. T24 inhibited TTR accumulation in FAP model rats, which expressed human ATTR V30M in various tissues and exhibited non-fibrillar deposits of ATTR in the gastrointestinal tracts. Additionally, humanized T24 (RT24) inhibited TTR fibrillation and promoted macrophage phagocytosis of aggregated TTR. This antibody did not recognize normal serum TTR functioning properly in the blood. These results demonstrate that RT24 would be an effective novel therapeutic antibody for FAP.

The homozygous p.C1024R- ADAMTS13 gene mutation links to a late-onset phenotype of Upshaw-Schulman syndrome in Japan

The homozygous p.C1024R- ADAMTS13 gene mutation links to a late-onset phenotype of Upshaw-Schulman syndrome in Japan -