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Summary. Background: Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological regulator of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) activity. A number of studies have shown that elevated levels of PAI-1 are related to pathological states such as an increased risk of arterial thrombotic events and a poor prognosis for cancer patients; however, there are few reports about PAI-1 deficiency in humans because the disorder is very rare. Objective: To understand the in vivo impact of a complete PAI-1 deficiency, Serpine1 )/) mice were generated; a number of in vivo studies have been conducted to elucidate the function of PAI-1 using Serpine1 )/) mice. The phenotypes demonstrated in Serpine1 )/) mice, however, were quite different from those in humans. Therefore, it is necessary to find out and analyze SERPINE1 deficiency in humans. Patient and methods: The patient is a 47-year-old woman who has had multiple episodes of major bleeding. Although most of the patientÕs blood coagulation factors were functionally normal, her PAI-1 antigen levels were undetectable. Therefore, DNA sequencing of the SERPINE1 gene were analyzed. Results: The proband had a homozygous 1-bp duplication (C) at exon 3 (c.356dupC; p.Ile120AspfsX42). Both wild-type PAI-1 (42.7 kDa) and mutated (Mut) PAI-1 (14.7 kDa) were expressed in COS-1 cells, although the level of Mut PAI-1 expressed in the cell lysates was much lower. Wild-type PAI-1 was observed in the culture supernatant, whereas no Mut PAI-1 was detected in the supernatant. Conclusions: Considering the results of the present study, the translation of mouse studies to humans must be performed with great care.

Pregnant women show a low level of protein S (PS) in plasma,which is known to be a risk for deep venous thrombosis. 17␤-Estradiol (E 2 ), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E 2 in HepG2-ER␣ cells, which stably express ER␣, and also the genomic ER signaling pathway, which modulates the liganddependent repression of the PS␣ gene (PROS1). We observed that E 2 repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E 2 down-regulated PROS1 promoter activity and that this E 2 -dependent repression disappeared upon the deletion or mutation of two adjacent GCrich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ER␣. In a chromatin immunoprecipitation assay, we found ER␣-Sp protein-promoter interaction involved in the E 2 -dependent repression of PROS1 transcription. Furthermore, we demonstrated that E 2 treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E 2 might repress PROS1 transcription depending upon ER␣-Sp1 recruiting transcriptional repressors in HepG2-ER␣ cells and, consequently, that high levels of E 2 leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Summary. Background: Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/). However, only 10% of these large deletions have been fully characterized at the nucleotide level. Patients and methods: We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated. Results: Inverse shifting-PCR (IS-PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild-type (WT) pattern in the IS-PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h-2 or -3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy. Conclusions: These gene rearrangements might result from double-strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non-homologous end joining or break-induced replication; thus leading to large F8 deletions in severe hemophilia A patients.