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G Piétu

Publications and source records attributed to G Piétu.

17 recordsLinked to original sources
PubMed1992

Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa.

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Antibodies, Monoclonal↗
PubMed1992

Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.

The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgII is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Molecular study of von Willebrand disease: identification of potential mutations in patients with type IIA and type IIB.

The defective von Willebrand Factor (vWF) in type IIA von Willebrand disease (vWD) has decreased binding affinity for platelet membrane glycoprotein Ib (GPIb) while in type IIB vWD, the abnormal vWF has increased affinity for this receptor. Segments of exon 28 of the vWF gene were amplified by the polymerase chain reaction and sequenced in two patients with type IIA and two patients with type IIB vWD. One type IIB patient showed an arginine to tryptophan substitution at amino acid residue 543 in the mature vWF and the other patient had a valine to methionine change at residue 553. Including these two new cases, substitutions at residues 543 and 553 now account for more than half of the documented mutations in patients with type IIB vWD. One patient with type IIA vWD showed an isoleucine to threonine change at amino acid 865. This substitution has been reported in another patient with type IIA vWD. The other patient showed a novel proline to serine change at residue 885. The C to T nucleotide transition which causes the amino acid change was not found in over 100 normal chromosomes tested by allele specific oligonucleotide hybridization and was linked to type IIA vWD in the family. This potential mutation is more carboxyterminal in the vWF subunit than other reported mutations in type IIA vWD. It is apparent that mutations associated with type IIA vWD are not as tightly grouped as defects in type IIB vWD, supporting the evidence that the type IIA vWD phenotype is generated by diverse mechanisms.

Base Sequence↗
PubMed1991

Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease.

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5' end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

Base Sequence↗
PubMed1991

von Willebrand factor: structure and function.

Von Willebrand factor (vWF) is an adhesive, multimeric glycoprotein present in plasma, platelets, and subendothelium, which has two main functions: (1) it serves as a carrier for factor VIII and (2) it plays a crucial role in platelet adhesion to subendothelium, acting as a "bridge" between platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa and subendothelial components such as collagen and heparin. vWF is involved at high shear rates in the initial contact of platelets with the subendothelium, in their subsequent spreading, and in thrombus formation. The three pools of vWF (plasma, platelets, and subendothelium) are necessary for optimal adhesion. Specific fragments of vWF involved in binding to platelets, collagen, heparin, and factor VIII have been mapped by using a series of proteases and well-characterized monoclonal antibodies to distinct epitopes of vWF. Several groups, including ours, have identified at least eight functional domains on the 270-kd subunit that consists of 2,050 amino acids. The importance of the binding domains to GP Ib and to collagen is illustrated by the role of vWF fragment SpIII (amino acids 1 through 1,365) in promoting platelet adhesion to collagen. The role of the vWF-GP Ib axis and of the vWF-GP IIb/IIIa axis in platelet-vessel wall interactions has been demonstrated through the study of patients, monoclonal antibodies, recombinant fragments, and synthetic peptides. We have recently expressed fragments of vWF complementary DNA in Escherichia coli. One of these recombinant fragments, which spans amino acids 449 through 730, binds to platelets in the presence of ristocetin, to collagen, and to heparin and has the property of inhibiting ristocetin-induced platelet agglutination. The second fragment, which spans amino acids 914 through 1,364, binds to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1991

[Triplane fractures of the upper head of tibia. Apropos of 2 cases].

The authors reported 2 cases of Salter-Harris type IV fracture of the proximal tibial epiphysis in which the fracture line mimicked that of triplane fractures of the distal tibia. X rays and CT aspects were detailed. A surgical management is proposed according to the risk of evolving varus deformity.

Adolescent↗
PubMed1990

Expression in Escherichia coli of a recombinant fragment (Ile 914-Leu 1364) of human von Willebrand factor containing a collagen binding domain.

Previous studies have shown that a collagen binding domain of human von Willebrand factor (vWF) resides on a fragment named SpI obtained by digestion with Staphylococcus aureus V8 protease which corresponds to residues Gly 911-Glu 1365 of the mature plasma vWF subunit. We have subcloned a fragment of a full-length cDNA encoding vWF into an expression vector which uses an inducible lambda PL promoter. The predicted product expressed by this plasmid is a fusion protein consisting of 16 amino acids (aa) of the lambda cII protein and aa Ile 914-Leu 1364 of human vWF. This fusion protein was shown to be expressed as insoluble inclusion bodies by induced E. coli harbouring the recombinant vector and was partially purified from bacterial debris and renatured. Partially purified bacterial extract run on SDS-polyacrylamide gels contained a major band representing 50-70% of the visualized proteins and corresponded to the predicted fusion protein. This band reacted with both polyclonal antibodies against human vWF and monoclonal antibodies (MAbs) which recognize the SpI fragment of plasma vWF. The radiolabelled partially purified bacterial extract was shown to bind specifically to human fibrillar collagen types I and III. This binding, which was a function of radiolabelled ligand and collagen concentrations, did not occur on monomeric denatured collagen. It was inhibited by both unlabelled bacterial extract and plasma vWF and also by a MAb against vWF SpI, which has the property of inhibiting vWF/collagen interaction. Our data demonstrate that this recombinant vWF SpI fragment, which can be obtained in large amounts, is a useful model for localizing the epitopes of monoclonal antibodies and then for studying the mechanism of vWF/collagen interaction.

Base Sequence↗
PubMed1989

Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.

A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.

Binding Sites↗
PubMed1989

Localization within the 106 N-terminal amino acids of von Willebrand factor (vWF) of the epitope corresponding to a monoclonal antibody which inhibits vWF binding to factor VIII.

We have used an in vitro transcription-translation system to localize the epitope corresponding to a monoclonal antibody (MAb 418) to vWF which specifically inhibits its binding to F. VIII. We have subcloned 1.5 Kb of vWF cDNA encoding for the N-terminal part of the vWF subunit which contains a binding site for F.VIII into the Bluescribe expression vector. After in vitro transcription and translation using the resulting construction (pBS-TG3522), a polypeptide of mol wt 60,000 (AA 1-495) was immunoprecipitated with a polyclonal antibody to vWF and with MAb 418. The MAb 418 epitope was further localized by reducing the size of the cDNA insert. This resulted in the production of two polypeptides of 18,000 (AA 1 to 142) and 13,000 (AA 1 to 106) which both retained reactivity with MAb 418. Thus the epitope corresponding to MAb 418 is localized to the first 106 AA of the mature vWF subunit.

Antibodies, Monoclonal↗
PubMed1989

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.

Animals↗
PubMed1988

Haemophilia A in a female: study of a family using intragenic and extragenic restriction site polymorphisms.

Restriction fragment length polymorphisms (RFLPs) were studied in a large Algerian family which includes 6 haemophiliacs and a previously described case of female haemophilia A. The female propositus is 66 years old with a normal karyotype. Her parents are first cousins. Her 3 sons are haemophiliacs and her 3 daughters with affected children are obligate carriers. The proband has an excessive bleeding tendency and markedly reduced levels of F.VIII (VIII C 0.03 U/ml, VIII Ag 0.01 U/ml) with elevated vWF Ag (2.30 U/ml), similar to the levels observed in affected males from the family. Four RFLPs can be identified by Southern blotting after digesting genomic DNA with the restriction enzymes Bcl I, Bgl I, Kpn I/Xba I and Taq I and hybridization with a 647 bp Stu I/Sca I F.VIII genomic probe, a 1.8 Kb EcoRI F.VIII cDNA probe, a 1.0 Kb EcoRI/Sst I fragment of intron 22 and the extragenic probe ST 14, respectively. With these four RFLPs, the propositus was found to be homozygous for the alleles segregating in this family with the abnormal X-chromosome. The carrier status was proven in a granddaughter and excluded in another. In conclusion, this RFLP linkage analysis is another argument to suggest that the propositus, a rare case of female haemophilia, is homozygous for the abnormal gene.

Aged↗
PubMed1988

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.

Base Sequence↗
PubMed1988

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.

Adult↗
PubMed1988

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.

Animals↗
PubMed1986

Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies.

We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin-induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino-terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.

Antibodies, Monoclonal↗
PubMedDate not supplied

Inhibition of von Willebrand factor-platelet interaction by fibrinogen.

The prolonged bleeding time and frequent haemorrhagic episodes in patients with severe von Willebrand's disease (vWD) attest to the importance of von Willebrand factor (vWF) in platelet adhesive functions. In in vitro systems, vWF is directly involved in platelet adhesion at high shear rates, and this participation may be mediated by platelet receptors for vWF. Two apparently distinct mechanisms have now been identified for enhancing 125I-vWF interaction with stimulated platelets: one is induced by ristocetin and apparently mediated by platelet membrane glycoprotein Ib (GPIb); a second mechanism has been identified more recently and is induced by the physiological stimuli ADP and thrombin. The failure of thrombin to support 125I-vWF binding to thrombasthenic platelets, which contain GPIb but lack GPIIb/IIIa, suggests that the mechanism of this interaction may be distinct from ristocetin-induced binding. As ADP and thrombin are released at sites of platelet accumulation, it is possible that these agonists regulate the vWF-platelet interactions in vivo. To test this hypothesis, we have examined ADP- and thrombin-supported 125I-vWF binding to platelets in the plasma of severe vWD patients, and report here that it is inhibited by fibrinogen. Thus, in addition to its role in coagulation and platelet aggregation, fibrinogen influences the binding of vWF to thrombin- and ADP-stimulated platelets.

Adenosine Diphosphate↗