ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling.

Rapid Ca2+-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca2+ ionophores in megakaryoblastic HEL cells. Ca2+ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca2+, whereas exogenous Ca2+ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca2+ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms.

Generation of von Willebrand factor epitope libraries expressed in E. coli.

The von Willebrand factor (VWF) subunit is composed of several domains, often coinciding with structural regions, characterized through their specific interaction with a ligand. Since several monoclonal antibodies (MoAbs) have been shown to functionally interfere with one of the specific interactions, we have created libraries of bacterial clones expressing peptidic sequences of VWF to map antibodies directed against this protein. Randomly cleaved fragments of VWF cDNA have been cloned in a plasmid designed for the expression of small peptides as part of larger fusion proteins. The NovaTope system is a useful procedure for protein analysis, allowing screening of epitopes composed of contiguous amino acid residues. To map MoAbs with conformational discontinuous epitopes displayed on small as well as large peptidic domains, this technique had to be widely modified to obtain two VWF peptide libraries expressing two ranges of peptide length (15-70 and 100-300 amino acids). Screening with six MoAbs with an epitope in a known region was performed to control both libraries. Four MoAbs were mapped through the characterization of overlapping sequences for 5-10 different positively expressed clones respectively. Two of these mapped MoAbs had no known inhibitory effect and bind reduced VWF only. The fact that the two other MoAbs mapped VWF functional interactions with ligands, platelet GPIIb/IIIa and Factor VIII, respectively, demonstrate that our libraries are valuable tools to determine conformational epitopes.

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells.

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.

Oct-1 is involved in the transcriptional repression of the von willebrand factor gene promoter.

The negative regulation of transcription of the human von Willebrand factor (vWF) gene was investigated in human umbilical vein endothelial cells (HUVECs) and HeLa cells. A fragment spanning -89 to +244 nucleotides (nt), containing the first exon, is active in HUVECs only but not in HeLa cells. The activity of this promoter is sharply reduced by mutagenesis of the GATA binding site at +221. Extension of the upstream sequences from nt -89 to -142 and to -496 results in progressive reduction of the activity of the -89 to +244 promoter identifying a negative regulatory element between nt -142 and -89. A factor present in nuclear extracts from endothelial and nonendothelial cells binds to an AT-rich sequence located between nt -133 and -125. Mutagenesis of the AT-rich sequence interferes with nuclear protein binding and restores the activity of the -142 to +244 fragment to the level of the -89 to +244 promoter. Binding of the nuclear protein to the vWF AT-rich sequence in mobility shift assays is inhibited by competition with a consensus Oct-1 binding site and with a silencer octamer-like sequence from the vascular cell adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift experiments identified Oct-1 as the transcription factor that binds to vWF and VCAM-1 silencer elements. These results indicate that Oct-1 acts as a transcriptional repressor of promoters of genes expressed in endothelial cells.

Functional characterization of bovine von Willebrand factor gene promoter in bovine endothelial cells demonstrates species-specific properties.

Von Willebrand factor (vWF), a protein necessary for platelet adhesion and thrombus formation, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). We previously demonstrated that the sequences localized either in the 5'-flanking region or in the first exon of human (hu) vWF gene (vWF), which regulate the cell-specific transcription, are not conserved in the bovine counterpart. In order to look for cis-acting elements involved in the endothelial expression of bovine (bo) vWF, fragments including 113 base pairs (bp) of a sequence 5'-flanking the transcription start point (tsp, +1) and various deletions of the first 233 bp exon were linked in plasmids to the bacterial chloramphenicol (Cm) acetyltransferase gene (cat). These constructs were analyzed by transient transfection in calf pulmonary artery endothelial (CPAE), human epithelial (HeLa) from cervix and green monkey fibroblasts from kidney (COS) cells. The longest fragment, containing 229 bp of the first exon, was the most active, with identical cat expression in the three cell types. The CAT activity was equivalent to that measured by transfection of the same cells with the basal promoter (from bp -89 to +19) of hu vWF. Addition of upstream bo vWF sequences from bp -113 to -1362 resulted in progressive reduction of the activity of the -113/+229 fragment. The upstream negative regulatory domains between -1362 and -278 also repressed the heterologous thymidine kinase (tk) promoter in CPAE and HeLa cells. Comparison of results with those previously obtained by transfection of hu vWF promoter in bovine endothelial cells demonstrates that the cis-acting elements do not behave identically in bo vWF promoter. In particular, positive tissue-specific elements able to overcome the negative regulation in endothelial cells could not be found in bo vWF between bp -1362 and +229.

Relative importance of the glycoprotein Ib-binding domain and the RGD sequence of von Willebrand factor for its interaction with endothelial cells.

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the alpha vbeta3 integrin and the RGD sequence of von Willebrand factor (vWF). To define the potential involvement of glycoprotein Ib alpha (GPIb alpha) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet alphaIIb beta3, and deltaA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIb alpha. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to delta A1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 microg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-alpha (TNF alpha), reported to upregulate the expression of the putative endothelial GPIb alpha, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIb alpha, blocking vWF interaction with platelet GPIb alpha, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the alpha vbeta3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to deltaA1-rvWF (50% inhibition at a concentration of 11 and 15 microg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIb alpha-binding domain, we were unable to detect endothelial surface expression of GPIb alpha by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIb alpha mRNA was undetectable in endothelial cells, even after stimulation by TNF alpha. These studies indicate that GPIb alpha is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an alpha vbeta3-dependent, GPIb alpha-independent mechanism.

Primary structure of the propeptide and factor VIII-binding domain of bovine von Willebrand factor.

A 2811 base-pair cDNA, encoding the amino-terminal part of the bovine pre-pro-von Willebrand factor, was characterized and sequenced. The deduced amino acid sequence shares significant homology with the human von Willebrand antigen II and the amino-terminal part of the factor VIII-binding domain of von Willebrand factor. In contrast to human, there is no RGD motif in the bovine von Willebrand antigen II. High levels of Cys, characteristic of D domains, are also found in bovine and the Cys position is markedly conserved between the two species.

Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene.

von Willebrand factor (vWF) gene expression is restricted to endothelial cells and megakaryocytes. Previous results demonstrated that basal transcription of the human vWF gene is mediated through a promoter located between base pairs -89 and +19 (cap site: +1) which is functional in endothelial and non endothelial cells. Two DNA repeats TTTCCTTT correlating with inverted consensus binding sites for the Ets family of transcription factors are present in the -56/-36 sequence. In order to analyse whether these DNA elements are involved in transcription, human umbilical vein endothelial cells (HUVEC), bovine calf pulmonary endothelial cell line (CPAE), HeLa and COS cells were transfected with constructs containing deletions of the -89/+19 fragment, linked to the chloramphenicol acetyl transferase (CAT) reporter gene. The -60/+19 region exhibits significant promoter activity in HUVEC and CPAE cells only. The -42/+19 fragment is not active. Mutations of the -60/+19 promoter fragment in the 5' (-56/-49) Ets binding site abolish transcription in endothelial cells whereas mutations in the 3' (-43/-36) site does not. The -60/-33 fragment forms three complexes with proteins from HUVEC nuclear extracts in electrophoretic mobility shift assay which are dependent on the presence of the 5' Ets binding site. Binding of recombinant Ets-1 protein to the -60/-33 fragment gives a complex which also depends on the 5' site. The -60/+19 vWF gene core promoter is transactivated in HeLa cells by cotransfecting with Ets-1 or Erg (Ets-related gene) expression plasmids. In contrast to the wild type construct, transcription of the 5' site mutants is not increased by these expressed proteins. The results indicate that the promoter activity of the -60/+19 region of the vWF gene depends on transcription factors of the Ets family of which several members like Ets-1, Ets-2 and Erg are expressed in endothelium. Cotransfection of Ets-1 and Erg expression plasmids is sufficient to induce the -60/+19 vWF promoter activity in HeLa cells.

Comparison of the 5'-flanking sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains with species-specific Alu-type repeats.

von Willebrand factor (vWF), a multimeric glycoprotein important for hemostasis, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). Recent studies from two laboratories, including ours, were published regarding the cell-specific transcription of reporter genes controlled by the human (hu) vWF promoter in transfected bovine (bo) endothelial cells and cells of non-endothelial origins. In order to verify that the regulatory domains previously characterized in the 5' region of hu vWF are also present in bo vWF, we have sequenced 1.9 kb upstream from the cap site, plus five exons. The comparison of human and bovine exons two to five shows homology of 83% at the nucleotide (nt) level and 78% at the deduced amino-acid sequence level. The bovine and human exons one, which are non-coding and span 233 and 250 bp, respectively, are only 64% homologous. In the first exon, potentially involved in endothelial-cell-specific transcription, the binding site for factor Sp1 is present in bo vWF, whereas the GATA sequence is replaced by a GACA sequence. The sequence corresponding to the human basal promoter, located between nt -89 and +19, is well conserved with 82% homology. However, the human TAATTA sequence (at nt -32) considered to be a TATA box, is replaced by TCATTA, and the CCAAT element at nt -18 is replaced by CCTGT. Among domains involved in transcription, the negative regulatory domain located 5' from the core promoter is highly conserved. The bovine sequence upstream from the first intron can be aligned with the human sequence up to nt -656 which is located in a polymorphic poly(GT)18-26 sequence. At this site, the bovine DNA contains an insertion of 523 bp which corresponds to a bovine Alu-type art2 repeat of 331 bp flanked by bovine microsatellites. The art2 sequence is an Alu-type repeat in artiodactyls with at least 100,000 copies in the bovine genome. Upstream from this insertion, 368 bp of the bovine sequence can be aligned with the human counterpart up to a 9-bp element which flanks an human Alu repeat which is absent from the bovine DNA. Upstream of the human Alu insertion and a duplicate of the 9-bp element, the two sequences are again homologous.

Cyclic adenosine monophosphate-dependent mechanisms induce von Willebrand factor expression in the Dami megakaryoblastic cell line.

It has been proposed that cyclic adenosine monophosphate (cAMP) is involved in the differentiation of several cell types and this study analysed whether von Willebrand factor (vWf) synthesis, which is a marker of the megakaryocyte maturation of these cells, would be enhanced by agents acting on cAMP formation. Different compounds known to stimulate cAMP accumulation in cells were used: dibutyryl cAMP (db-cAMP), isobutyl-methylxanthine (IBMX) or pentoxifylline (PTX) and forskolin. Treatments with db-cAMP or IBMX (10-1,000 microM) induced a dose-dependent increase in vWf synthesis. Associations of IBMX with forskolin produced a synergistic enhancement in vWf synthesis. PTX alone did not enhance vWf synthesis but a latent effect was revealed in the presence of forskolin or db-cAMP. The increase in vWf mRNA shown by Northern blot analysis demonstrates that the protein synthesis correlates with the transcript expression after db-cAMP or IBMX treatments. vWf synthesis paralleled the accumulation of cAMP in the cells. Moreover vWf expression induced by combination of IBMX with forskolin was associated with a moderate increase in the percentage of GPIIb/IIIa positive cells and in the ploidy level related to an important inhibition of cell growth. These data provide evidence that agents acting on cAMP metabolism induce vWf synthesis in the Dami megakaryoblastic cells.

Optimization of the transfection of human endothelial cells by electroporation.

A method for efficient transfection of human umbilical vein endothelial cells (HUVEC) is presented here. The procedure involves cell synchronization followed by electroporation and allows the detection of the activity of low-strength promoters. Sodium butyrate, added to the culture medium after electroporation, strongly potentiates the transcriptional activity of viral promoters. The method can be successfully applied to analyze the transcriptional activity of different promoters linked to reporter genes or to express foreign genes controlled by viral promoters.

The role of the 5'-flanking region in the cell-specific transcription of the human von Willebrand factor gene.

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5' of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5' end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5'-flanking region interact to regulate vWF gene expression.

Primary structure of the factor VIII binding domain of human, porcine and rabbit von Willebrand factor.

von Willebrand factor (vWF) binds to Factor VIII (FVIII) and the FVIII binding domain has been localized to the amino-terminal of the vWF subunit. The DNA sequence coding for part of the vWF precursor (provWF) including the FVIII binding domain has been compared in man, pig and rabbit. The sequenced fragment corresponds to nucleotides 2416 to 2886 of the human vWF cDNA and encodes for the last 41 amino acids of the propolypeptide, the cleavage site and the first 116 amino acids of the mature vWF subunit. The homology of the three deduced amino acids sequences is remarkable: 88% between porcine and human and 87% between rabbit and human sequences. Four contiguous amino acids are lacking in the rabbit propolypeptide (-10 to -7) when compared to the human and porcine sequences. The cleavage site of the propolypeptide is conserved in the three species as well as amino acids where mutations in the human gene lead to a binding defect of vWF to FVIII. The Asn-94 N-glycosylation site is present in the human and rabbit sequences but absent in the pig sequence.

Comparison of the primary structure of the functional domains of human and porcine von Willebrand factor that mediate platelet adhesion.

Porcine von Willebrand factor (vWF) directly aggregates human platelets in vitro indicating a conformational difference between the human and porcine molecules. We amplified and directly sequenced 1242 nucleotides of porcine vWF cDNA that encodes functional domains which mediate the binding of vWF to platelets and subendothelium. The deduced amino acid sequence corresponds to residues 473-891 of the human mature vWF subunit and is 79% homologous with the human protein. Significant differences are found in two discontinuous segments thought to be involved in the binding of vWF to platelet glycoprotein Ib. Porcine vWF lacks four contiguous residues in the first segment and has two positively charged arginine residues in the second. Three point mutations associated with human type IIB von Willebrand disease in the first segment of a botrocetin binding site are at the same position as mismatches between the pig and human. The second segment of the botrocetin site is highly conserved while the third segment shows only a 60% homology.

Expression of von Willebrand factor in porcine vessels: heterogeneity at the level of von Willebrand factor mRNA.

The expression of the von Willebrand factor (vWF) gene by cultured endothelial cells from the porcine pulmonary artery, aorta, and lung was compared at the levels of messenger (m)RNA and antigen. Steady-state levels of vWF mRNA were determined by dot-blot analysis using a partial human vWF cDNA as the hybridization probe; vWF mRNA from cultured aortic endothelial cells, and vWF antigen secreted into the culture supernatants were barely detectable. In contrast, vWF mRNA and antigen from the pulmonary artery endothelial cells were approximately eight to nine times that demonstrated by aortic cells. Levels of vWF mRNA and antigen in cultured lung cells were intermediate of those found in the pulmonary artery and aorta and correlated with the estimated number of cells demonstrated to be of endothelial origin in the mixed cell populations grown from the lung. Differences between the levels of vWF mRNA found in cultured cells from the pulmonary artery and those found in the aorta were maintained in cells processed directly from these vessels. Correlation between the levels of vWF mRNA and antigen in endothelial cells from different vessels of the pig suggests that the differential control of vWF synthesis is at the level of transcription. Furthermore, maintenance in cultured cells of the difference in transcription rates that were observed in vivo suggests that vWF gene expression is not exclusively regulated through environmental factors.

Identification of a CpG mutation in the coagulation factor-IX gene by analysis of amplified DNA sequences.

In a family with no previous bleeding history, the sister of a single, severely affected haemophilia B patient requested carrier detection and prenatal diagnosis. In Southern blots, using Taq I digested DNA and a factor-IX cDNA probe, a normal invariant band at 1.6 kb was missing in the haemophiliac suggesting the loss of the Taq I site at the 5' end of exon h. A 162 bp sequence which includes the suspected mutant region was amplified by the polymerase chain reaction in each DNA. Two oligonucleotide probes were synthesized and differed by only one base pair which substituted a T for C in the normal Taq I recognition sequence. The amplified DNA was dot-blotted and hybridized with the labelled probes. The altered sequence hybridized to DNA from the affected individual, his sister and her fetus and not to DNA from the normals. The mutation, involving the haemophiliac, his mother, his sister and her fetus, transforms a CGA codon that encodes for arginine in the catalytic domain of the protein into a UGA stop codon.

The human gene for von Willebrand factor. Identification of repetitive Alu sequences 5' to the transcription initiation site.

The region at the 5' end of von Willebrand factor gene was cloned by screening genomic libraries with a partial von Willebrand factor cDNA probe and oligonucleotides complementary to areas of von Willebrand factor mRNA at the extreme 5' end of the untranslated region. The sequence 2158 bp upstream of the transcription initiation site, the first exon and first exon-intron junction is reported. The first exon includes the entire 5' untranslated sequence (250 bp) and the translation initiation codon starts the second exon, suggesting an unusual control mechanism for the cell specific expression of von Willebrand factor. An AT-rich region resembling a TATA box is found 32 bp upstream of the transcription initiation site. At -1030 and -1806 nucleotides 5' of the TATA box are two repetitive Alu sequences of approximately 300 bp. Recombinant events at these Alu sequences could result in some clinical forms of von Willebrand disease involving transcriptional defects.

Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe.

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.