Claudine Mazurier

Biological effect of desmopressin in eight patients with type 2n (‘Normandy’) von Willebrand disease

Summary It is generally thought that the plasma increase in factor VIII (FVIII) after desmopressin (dDAVP) infusion is related to the plasma increase in von Willebrand factor (vWF), which is the plasma carrier for FVIII. The aim of this study was to evaluate the FVIII and vWF responses in patients with type 2N vWD, characterized by the mild FVIII deficiency related to markedly decreased affinity of vWF for FVIII. At different times after one intravenous dose of dDAVP (0.3 or 0.4 μg/kg) we measured the FVIII coagulant activity, FVIII antigen, vWF antigen and ristocetin cofactor activity, in eight patients with either Arg91Gln or Arg53Trp amino acid substitution in mature vWF. In all the patients, whatever their mutation, the dDAVP infusion resulted in a 2.3. ± 0.7 ‐fold increase of vWF and a variable rise (9.5 ± 7.7 times) of FVIII, whereas the vWF capacity to bind FVIII was not improved. The FVIII response was more transient than vWF response, and FVIII half disappearance time was evaluated to the approximately 3h. The data indicate that the stabilizing effect of vWF on FVIII is not responsible for the FVIII increase induced by dDAVP. The clinical implication of this study is that, in the 2N vWD patients, dDAVP may be a useful prophylactic or curative treatment when the test dose has been shown to be effective to reach a haemostatic FVIII level.

Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: consequences for therapy and genetic counselling

A plasma von Willebrand factor (vWf) defect limited to its failure to bind factor VIII (FVIII) was previously characterized in a woman with FVIII deficiency and normal primary haemostasis. By using in vitro tests we found a similar pattern in three siblings of another family previously thought to be affected with mild haemophilia A. Furthermore, a decrease in vWf ability to bind FVIII was found in the parents and the brother of the three patients. This decrease was consistent with heterozygous expression of a recessive vWf gene abnormality. FVIII deficiency was corrected by infusion with a vWf concentrate almost devoid of FVIII coagulant activity. FVIII recovery and half‐life thus obtained showed that this treatment was more effective than a FVIII infusion performed by way of comparison. These results indicate that this vWf defect may account for FVIII deficiency in patients without the usual laboratory and clinical features of von Willebrands disease. Changes in therapy and genetic counselling following the new diagnosis in this family emphasize the need to search for such a vWf defect in patients in whom FVIII deficiency is not obviously X‐linked.

Identification of two point mutations in the von Willebrand factor gene of three families with the ‘Normandy’variant of von Willebrand disease

Summary Plasma von Willebrand factor (vWf) is a multidomain multimerized glycoprotein which has a dual role in haemostasis: it promotes platelet adhesion to subendothelium and is the carrier of blood coagulation factor VIII (FVIII). We previously characterized a functional defect of vWf, limited to its ability to bind FVIII, in two families whose affected members have the same phenotype that mimics mild haemophilia A and was tentatively named von Willebrands disease (vWD) ‘Normandy’. A homozygous point mutation C T converting Thr 28 to Met in mature vWf subunit was identified in one of these patients who was born of third‐cousin parents. In the present studies we report two unrelated new cases of vWD ‘Normandy’and characterize, using the analysis of the vWf gene intron 40 region containing a variable number of tandem repeats, the recessive inheritance of the disease in two affected families without known consanguinity. Exons 18–24 of the vWf gene encoding for the first 311 amino acids of mature vWf subunit were amplified by the polymerase chain reaction method and sequenced. Two new missense mutations, both corresponding to a C T transition and predicting respectively an Arg 53 Trp and an Arg 91 Gln substitution, were characterized. The three patients from family 1 were homozygous for the first‐mentioned mutation while the patient from family 3 was homozygous for the second. The patient from family 2 was found a compound heterozygote for the two mutations. None of the two point mutations reported, both destroying a MspI restriction site, could be detected in DNA from 50 normal controls screened by restriction endonuclease analysis. Our data show that different mutations may be found in patients with the ‘Normandy’phenotype. The mutations characterized so far are all localized on the N‐terminal region of mature vWf subunit, within or near the major FVIII binding domain, and some of them occur within the epitope of monoclonal antibodies inhibiting the vWf/FVIII interaction. These observations suggest a causal relationship between these mutations and the vWD ‘Normandy’phenotype.

Primary structure of the major O-glycosidically linked carbohydrate unit of human Von Willebrand Factor

A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz1H-NMR spectroscopy to be: NeuAc(α2-3)Gal(β1-3)[NeuAc(α2-6)]GalNAc-ol.

Acquired type II von Willebrand's disease: demonstration of a complexed inhibitor of the von Willebrand factor-platelet interaction and response to treatment

An acquired von Willebrands disease developed in two patients in association with a monoclonal gammopathy plus a Sjögrens syndrome and a chronic lymphocytic leukaemia (CLL). In both cases a plasma inhibitor to von Willebrand factor (vWf) was suspected and characterized after plasma gel filtration. The inhibitor was shown to be entirely complexed with vWf and was only demonstrated after complex dissociation by heating. The inhibitor was able to inhibit the binding of125I‐vWf to platelets in the presence of ristocetin in both cases and to thrombin‐stimulated platelets in one case. In the two patients, the highest molecular weight multimers (HMWM) of vWf were absent when assessed by sodium dodecyl‐sulphate agarose plasma electrophoresis. Intravenous infusion of 1‐deamino‐(8‐D‐arginine) vasopressin (DDAVP) resulted in the appearance of the HMWM in both cases and of the satellite bands of each multimer subunit which were lacking prior to the infusion in one patient. After transfusion of a VIII/vWf concentrate containing a significant amount of HMWM, there was a rapid plasma clearance of the vWf‐related activities and of the HMWM when compared to that seen in a patient with type III constitutional vWD. We conclude that in the two patients studied the coagulation defect was related to the presence of a circulating inhibitor to vWf which could be responsible for the disappearance of the HMWM from plasma.

Two novel mutations, Q1053H and C1060R, located in the D3 domain of von Willebrand factor, are responsible for decreased FVIII-binding capacity

Summary. In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D′ domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site‐directed mutagenesis and transient expression in COS‐7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

Structure determination of the major asparagine-linked sugar chain of human factor VIII - von Willebrand factor

N‐glycosidically‐linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were seperated by high‐voltage electrophoresis. Five fraction were obtained, one of them representing 60% of the total amount of the N‐glycosidically‐linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H‐NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N‐acetyllactosaminic type.

Molecular Genetics of Type 2 von Willebrand Disease

Type 2 von Willebrand disease (VWD) is characterized by a wide heterogeneity of functional and structural defects. These abnormalities cause either defective von Willebrand factor (VWF)-dependent platelet function in subtypes 2A, 2B, and 2M or defective VWF-factor VIII (FVIII) binding in subtype 2N. The diagnoses of types 2A, 2B, and 2M VWD may be guided by the observation of disproportionately low levels of ristocetin cofactor activity or collagen-binding capacity relative to VWF antigen. The abnormal platelet-dependent function is often associated with the absence of high molecular weight (HMW) multimers (type 2A, type 2B), but the HMW multimers may also be present (type 2M, some type 2B), and supranormal multimers may exist (“Vicenza” variant). The observation of a low FVIII-to-VWF:Ag ratio is a hallmark of type 2N VWD, in which the FVIII levels depend on the severity of the FVIII-binding defect. Today, the identification of mutations in particular domains of the pre-pro-VWF is helpful in classifying these variants and providing further insight into the structure-function relationship and the biosynthesis of VWF. Thus, mutations in the D2 domain, involved in the multi-merization process, are found in patients with type 2A, formerly named IIC VWD. Mutations located in the D’ domain or in the N terminus of the D3 domain define type 2N VWD. Mutations in the D3 domain characterize Vicenza and IIE patients. Mutations in the A1 domain may modify the binding of VWF multimers to platelets, either increasing (type 2B) or decreasing (type 2M, 2A/2M) the affinity of VWF for platelets. In type 2A VWD, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding, impairing VWF secretion or increasing its susceptibility to proteolysis. Finally, a mutation localized in the carboxy-terminus CK domain, which is crucial for the dimerization of the VWF subunit, has been identified in a rare subtype 2A, formerly named IID.

Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents

Summary. The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0·6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.

Preliminary results on the carbohydrate moiety of factor VIII/Von Willebrand factor (FVIII/vWf)

Human FVIII/vWf, purified 9 000 fold, was prepared from therapeutic concentrates by gel filtration and by immuno-affinity chromatography on insolubilized immunoglobulins isolated from a rabbit immunized with the plasma of a patient devoid of FVIII R:Ag. These preparations which contain coagulant activity and agglutinate normal washed human platelets in the presence of ristocetin are immunologically pure. The carbohydrate moiety of this highly purified FVIII/vWf was submitted to analysis by gas liquid chromatography and thin layer chromatography before and after hydrazinolysis and alkaline-borohydride treatment. The total carbohydrate content is 14.4 p. cent (w/w). Man and GalNAc residues were identified, this result indicating the coexistence of N- and O-glycosidically linked glycans (70 and 30 p. cent respectively). After hydrazinolysis it was demonstrated that the N-glycosidically linked glycans do not contain GalNAc residues. One major glycan belonging to the N-acetyllactosaminic type with a bi-antennary structure has been characterized by thin layer chromatography. The alkaline-borohydride treatment procedure reduced all the FVIII/vWf GalNAc to GalNAc-ol residues, demonstrating that they are all involved in the linkage of the O-glycans with the peptide chain and consequently they cannot be in oligosaccharidic sequences inducing A-blood group activity. Furthermore, at least 10 O-glycosidically linked glycans were identified by thin layer chromatography. Thus, the high degree of heterogeneity of the FVIII/vWf carbohydrate moiety requires further structural studies in order to precise which class of glycans is involved in the biological activity of FVIII/vWf.