Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen.

We report the identification of a new mutation of von Willebrand Factor (VWF) gene within exon 30 occurring in two related patients (mother and daughter) with a hemorrhagic syndrome. A T-->A transvertion at nucleotide 5441 was found changing the serine 968 to threonine of the mature VWF subunit (S1731T of the preproVWF). The Ser968Thr mutation is located within the VWF A3 domain which interacts with type I and III collagens. Both patients were found to be heterozygous for the mutation. The propositus (daughter) exhibited a slightly prolonged bleeding time, levels of VWF:Ag and VWF:RCo at the lower limit of normal, contrasting with normal levels of VIII:C. Her mother exhibited borderline bleeding time and moderately decreased levels of VWF and VIII:C. In both patients multimeric structure of VWF and ristocetin- as well as botrocetin-induced binding of VWF to GPIb were normal; however both patients repeatedly showed decreased binding of VWF to collagen. The Ser968Thr substitution was reproduced by site-directed mutagenesis on the full-length cDNA of VWF. The mutated recombinant VWF (rVWF), T968rVWF, and the hybrid S/T968rVWF were transiently expressed by COS-7 cells. Both rVWF exhibited normal multimeric pattern and normal ristocetin- as well as botrocetin-induced binding to GPIb. T968rVWF showed significantly decreased binding to collagen while the hybrid S/T968rVWF bound to collagen in a similar way to that of the patients' plasma VWF. Thus, our data demonstrate that the Ser968Thr mutation of the VWF A3 domain is clearly responsible for the abnormal binding of VWF to collagen observed in both patients. The Ser968Thr substitution of the VWF is the first mutation identified in two patients leading to a decreased affinity of VWF for collagen and a normal multimeric structure.

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

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.

Characterization of recombinant von Willebrand factors mutated on cysteine 509 or 695.

The interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be "locked" into active or inactive conformations in regard to the binding to platelet GPIb receptor.

Epitope mapping of inhibitory monoclonal antibodies to human von Willebrand factor by using recombinant cDNA libraries.

Two recombinant expression libraries containing small (300-600 base pairs) cDNA fragments of von Willebrand Factor (vWF) were screened in order to map the epitope of monoclonal antibodies (MAbs) to vWF. Among eleven MAbs tested, seven were effectively mapped. The epitopes of MAbs 418 and 522, which inhibit the binding of vWF to Factor VIII (FVIII), were localized between Leu 2 and Arg 53 and between Glu 35 and Ile 81 of the vWF subunit respectively, within the N-terminal trypsin fragment called SpIII-T4 [amino acids (aa) 1-272] which contains a binding domain for FVIII. The epitope of MAb 710, which inhibits the binding of vWF to glycoprotein Ib (GPIb), was identified between Ser 593 and Ser 678 on the tryptic 52/48 kDa fragment (aa 449-728) which contains binding domains for GPIb, collagen, heparin, sulfatides and subendothelium extracellular matrices. The epitope of MAb 723, which does not interfere with any known function of vWF, was localized between Ser 523 and Gly 588. The epitopes of MAb 505 and MAb 400, which inhibit the binding of vWF to collagen, were identified between Leu 927 and Arg 1114 within the SPI fragment (aa 911-1365) corresponding to the central part of the vWF subunit. The epitope of MAb 9, which inhibits the binding of vWF to GPIIb/IIIa, was identified in the C-terminal part of the vWF subunit between Gln 1704 and Asp 1746, the latter being the third aa of the RGD sequence common to adhesive proteins and serving as a recognition site for integrin receptors.

Fine epitope mapping of monoclonal antibodies to the NH2-terminal part of von Willebrand factor (vWF) by using recombinant and synthetic peptides: interest for the localization of the factor VIII binding domain.

Two different approaches were used in order to define the epitope of three monoclonal antibodies (MoAbs) against the NH2-terminal part of the mature subunit of von Willebrand factor (vWF) which contains its factor VIII (FVIII) binding site. First, a vWF cDNA fragment library using the bacteriophage lambda gt11 expression vector was screened with radiolabelled MoAbs. The epitope of each MoAb was defined, following sequence analysis, by the overlapping DNA sequence of immunoreactive clones. MoAb 32B12, a potent inhibitor of FVIII/vWF interaction, binds within the Glu35-Ile81 sequence of vWF subunit. MoAb 14A12, a non-inhibitory antibody, recognizes a sequence within Thr141-Val220. MoAb 31H3, a partial inhibitory antibody, gives no positive clone. In the second method, a panel of 24 synthetic pentadecapeptides corresponding to the first NH2-terminal 105 amino acid residues was used to block the binding of inhibitor MoAbs to immobilized vWF in an ELISA system. The localization of MoAb 32B12 epitope was confirmed and restricted to the Met51-Ala60 sequence. The MoAb 31H3 binding to vWF is inhibited by two synthetic peptides with the overlapping sequence Cys66-Gly76. All these data confirm that the FVIII binding site of vWF is not limited to the binding area (Thr78-Thr96) of the previously described MoAbs inhibiting FVIII/vWF interaction but is composed of several key sequences.

Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from qualitative abnormalities of von Willebrand factor (vWF) characterized by an absence in plasma of high molecular weight vWF multimers and an abnormal reactivity of vWF towards platelet glycoprotein (GP) Ib, which is decreased in type IIA and increased in type IIB. In this report, we describe the case of a patient having a IIA vWD phenotype associated with an intermittent thrombocytopenia atypical in this subtype but observed in type IIB vWD. The patient plasma vWF showed an absence of high molecular weight and intermediate multimers and had a decreased binding capacity to GPIb. The affinity of botrocetin was normal for plasma vWF from the propositus. Analysis of the propositus vWF gene showed the presence of a substitution Val 551 to Phe of the mature vWF subunit. This mutation is localized within a 509-695 disulphide loop of the vWF that plays an important role in the binding to GPIb and is where most of the molecular defects described so far were associated with type-IIB vWD. We have reproduced the Val 551 Phe substitution onto the vWF cDNA, expressed it in COS-7 cells, and performed structural and functional analysis of the mutant recombinant protein (rvWFPhe 551). The rvWFPhe 551 had a normal multimeric structure and showed the capacity to spontaneously interact with GPIb. Botrocetin had a decreased affinity for rvWFPhe 551. In conclusion, the Val 551 Phe mutation modifies the affinity of vWF for platelet GPIb, as does a type IIB mutation, and may be responsible for the thrombocytopenia of the patient and the clearance of the high molecular weight and intermediate-sized multimers of vWF from the plasma. The study of the rvWFPhe 551 has confirmed the discrepancy between the IIA phenotype and the IIB genotype of the patient.

A novel case of compound heterozygosity with "Normandy"/type I von Willebrand disease (vWD). Direct demonstration of the segregation of one allele with a defective expression at the mRNA level causing type I vWD.

We report the case of a family with type I von Willebrand disease (vWD), characterized by a quantitative defect in von Willebrand factor (vWF), associated with a defective binding of vWF to factor VIII (FVIII) also called the "Normandy" variant of vWD. PCR products from genomic DNA of the family members were analysed in the region coding for the binding domain of vWF to FVIII. It showed that the proposita and one of her sons were heterozygous for the Arg91Gln missense mutation, abolishing an MspI restriction enzyme site located in exon 20. The transcription of the normal and mutated alleles was tested by the amplification of cDNA after reverse transcription of platelet mRNA in this region. A total lack of expression of the normal allele was observed in the proposita, who appeared as a compound heterozygous with one allele mutated at Arg91 and a "silent" expression of the other one. The segregation of the "silent" allele was studied in the family with the exonic BstEII RFLP both at the DNA and mRNA levels. The proposita has transmitted her "silent" allele to her daughter and to another son. As this son was informative for this RFLP, the absence of expression of the allele could be demonstrated at the mRNA level, providing evidence that this defect was responsible for his type I vWD.

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.

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.

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.

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.

Specific binding of human fibrinogen to cultured human fibroblasts. Evidence for the involvement of the E domain.

Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37 degrees C with 125I-fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I-fibrinogen and MRC5 was observed. The binding was time-dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non-labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non-immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast-bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B beta and gamma chains of the fibrinogen molecule. An apparent affinity constant, Ka = 6.7 +/- 0.2 X 10(6) M-1, was estimated from binding isotherms. After a 30-min incubation time the interaction between 125I-fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non-labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I-fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin-degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I-fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.

The binding of fibrinogen to its platelet receptor.

Specific receptors for fibrinogen can be induced on platelets by a variety of stimuli. In this study, two independent approaches have implicated the D domain of fibrinogen in its interaction with the platelet. Immunochemically purified Fab fragments of anti-fibrinogen and anti-D inhibited 125I-fibrinogen binding to platelets in a dose-dependent fashion and platelet aggregation. In contrast, Fab fragments of anti-E produced only a slight inhibition of fibrinogen binding and nonimmune Fab fragments had no effect. Fibrinogen was digested with plasmin in the presence of 5 mM calcium and fragment D and E of Mr 100,000 and 50,000, respectively, were isolated. This D fragment inhibited 125I-fibrinogen binding to platelets in a concentration-dependent fashion, whereas the E fragment was ineffective. With 125I-fibrinogen at 0.17 microM, nonlabeled fibrinogen inhibited binding by 50% at 0.7 microM, whereas 170 microM fragment D was required to produce 50% inhibition. D fragment of Mr 80,000, generated in the absence of calcium, was noninhibitory. These observations provide strong evidence for the participation of the D domain in the binding of fibrinogen by its platelet receptor and suggest that the recognition site is lost in the conversion of the Mr 100,000 to 80,000 D species.