Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene.

The endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed -5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of -5.5HS coupled to either PP or the SV40 promoter were able to direct beta-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The -5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell-specific transcriptional regulation that reflects the common origin of these cell types.

Proteolytic inactivation of ADAMTS13 by thrombin and plasmin.

The multimeric size and the function of circulating von Willebrand factor are modulated via its proteolytic cleavage by the plasma metalloproteinase, ADAMTS13. It is unclear how ADAMTS13 activity is regulated within the vascular system. In the absence of a regulatory mechanism, ADAMTS13 activity might compromise platelet adhesion at sites of vascular injury. We hypothesized that at sites of vascular injury, ADAMTS13 activity could be regulated locally by coagulation proteinases. Initiation of coagulation in human plasma resulted in the disappearance of added full-length recombinant ADAMTS13. This loss was inhibited by hirudin. Using purified proteins, we showed that ADAMTS13 is proteolyzed at several cleavage sites by thrombin in a time- and concentration-dependent manner. Furthermore, this proteolysis ablated ADAMTS13 activity against purified von Willebrand factor. Preincubation of thrombin with soluble thrombomodulin, but not heparin, inhibited the proteolysis of ADAMTS13, suggesting the involvement of thrombin exosite I (and not exosite II) in ADAMTS13 recognition. Plasmin also cleaved ADAMTS13 into similar fragments, resulting in the loss of ADAMTS13 activity. This study demonstrates the susceptibility of ADAMTS13 to proteolytic inactivation and suggests possible roles for thrombin and plasmin at sites of vascular injury.

Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites.

The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demonstrated the presence of a hypersensitive site in the 5' flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937) known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5' flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at -368, -232, -226, -201, -146, and -102 bp relative to the translation start site. With the exception of the site at -102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5' flanking region is critical for normal hEPCR gene expression in endothelial cells.

Partial epilepsy with pericentral spikes: a new familial epilepsy syndrome with evidence for linkage to chromosome 4p15.

The genetic analysis of simple Mendelian epilepsies remains a key strategy in advancing our understanding of epilepsy. In this article, we describe a new family epilepsy syndrome, partial epilepsy with pericentral spikes, which we map to chromosome 4p15. We distinguish it clinically, electrophysiologically, and genetically from previously described Mendelian epilepsies. The family described is a large Brazilian kindred of Portuguese extraction in which affected family members manifest a variety of seizure types, including hemiclonic, hemitonic, generalized tonic-clonic, simple partial (stereotyped episodes of epigastric pain), and complex partial seizures consistent with temporal lobe epilepsy. The syndrome is benign, either requiring no treatment or responding to a single antiepileptic medication. Seizure onset is in the first or second decades of life, with seizures in individuals up to the age of 71 years and documented encephalogram changes up to the age of 30 years. A key feature of partial epilepsy with pericentral spikes is a characteristic encephalogram abnormality of spikes or sharp waves in the pericentral region (centroparietal, centrofrontal, or centrotemporal). This distinctive encephalogram abnormality of pericentral spikes unites these several seizure types into a discrete family epilepsy syndrome. As with other familial epilepsies, the inherited nature of this new syndrome may be overlooked because of the variability in penetrance and seizure types among affected family members.