A qualitative and quantitative analysis of von Willebrand factor contained in a very high-purity plasma-derived FVIII concentrate.

BACKGROUND AND OBJECTIVES: We studied the structural and functional properties of von Willebrand factor (VWF) molecules present in a very high-purity plasma-derived factor VIII concentrate (VHP pdFVIII - Factane® ) because several observations suggest that the presence of VWF in factor VIII (FVIII) preparations may decrease their immunogenicity. MATERIALS AND METHODS: Ten marketed batches of VHP pdFVIII (Factane® ) with levels of VWF ranging from 15 to 39 IU/100 IU FVIII were analysed. The VWF multimeric pattern was studied by agarose gel electrophoresis. The binding of VWF to FVIII was studied by gel filtration and ELISA. The binding of VWF to GPIb was analysed by ELISA. RESULTS: The results showed that high-molecular-weight multimers of VWF were present in VHP pdFVIII (Factane® ). VWF subunits maintain a triplet structure similar to that of normal plasma. Regardless of the VWF content, all FVIII molecules of each batch were co-eluted with VWF, and no free FVIII was detectable. By immunoassays, VWF was found to be able to bind to FVIII and platelet GPIb in a similar manner to that of VWF in normal plasma. CONCLUSIONS: In all the VHP pdFVIII (Factane® ) batches studied, regardless of the level of VWF, the structure and capacity of VWF binding to FVIII and to platelet GPIb were fully preserved.

A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates.

BACKGROUND: Since the early 1990 s the Committee for Proprietary Medicinal Products has set the mandatory requirement that all manufacturing processes for blood products include two virus removal/inactivation steps that are complementary in their action. OBJECTIVES: The objective was to develop a manufacturing process for factor VIII (FVIII) including two complementary steps of viral inactivation/elimination. METHODS: A 35-15 nm nanofiltration step was added to a former FVIII manufacturing process that included solvent/detergent (S/D) treatment to generate a new FVIII concentrate called Factane. The impact of nanofiltration on the structural and functional characteristics of FVIII, as well as virus/transmissible spongiform encephalopathy reduction factors were assessed. RESULTS: Using an innovative approach, FVIII was successfully nanofiltered at 35-15 nm, while the biological properties of the active substance were unmodified. FVIII coagulant and antigen content for Factane and previous S/D-treated FVIII (FVIII-LFB, commercialized as Facteur VIII-LFB) were comparable. The FVIII one-stage chromogenic and coagulant/antigen ratios confirmed that nanofiltered FVIII was not activated. After nanofiltration, the copurified von Willebrand factor (vWF) was reduced but vWF/FVIII binding properties were unaffected. Phospholipid binding and thrombin proteolysis studies displayed no differences between Factane and FVIII-LFB. The rate of factor Xa generation was slightly lower for Factane when compared to FVIII-LFB. Viral validation studies with different viruses showed no detectable virus in the filtrate. CONCLUSIONS: Nanofiltration of FVIII at 15 nm is feasible despite the large molecular weight of FVIII and vWF. Nanofiltration has been proven to be highly effective at removing infectious agents while preserving the structural and functional integrity of FVIII.

In vitro study of a triple-secured von Willebrand factor concentrate.

BACKGROUND AND OBJECTIVES: Patients suffering from von Willebrand disease who are not responsive to desmopressin require substitutive treatment. This study was part of the development of a second-generation plasma-derived von Willebrand factor (VWF) concentrate, the manufacturing process of which includes two complementary viral-inactivation/elimination steps that are effective against non-enveloped viruses. MATERIALS AND METHODS: VWF was purified from solvent/detergent-treated cryoprecipitate through a combination of anion-exchange and affinity chromatography. The VWF preparation was diluted and filtered through filters with pore size of 35 nm. After concentration and formulation, the product was freeze-dried and further heated at 80 degrees C for 72 h. Tests were performed to evaluate the effects of nanofiltration and dry heating on VWF multimeric structure and function. RESULTS: Nanofiltration and dry heating of the formulated product increased viral safety but did not modify VWF multimeric structure. Furthermore, these steps did not alter the ability of VWF to bind to platelet glycoprotein Ib, collagen and Factor VIII. CONCLUSIONS: We have perfected a large-scale manufacturing process to produce a human plasma-derived VWF concentrate that boasts high specific activity and is very safe for the treatment of patients with von Willebrand disease.

Estimation of the carbohydrate moiety of von Willebrand factor in the plasma of patients with subtypes 2a and 2b of von Willebrand disease.

Von Willebrand disease (vWD) results from quantitative (types 1 and 3) or qualitative (type 2) deficiency of von Willebrand factor (vWF). This glycoprotein present in plasma is involved in platelet adhesion at the site of vascular injury and serves as the carrier of antihaemophilic A factor (FVIII). Whereas recent studies have identified mutations in patients suffering from type 2 vWD, the integrity of the carbohydrate moiety of vWF in these patients is still matter of debate. In order to analyse in the plasma milieu the carbohydrate content of plasma vWF from various well-characterized type 2 vWD patients, we developed a colorimetric assay in microtiter plate based on the use of peroxidase-conjugated lectins specific for either alpha 2-6 sialic acid or beta 1-4 galactose. Removal of sialic acid from purified plasma vWF induced significant changes in the reactivity of both lectins. The analysis of various normal plasmas showed no influence of the blood groups and allowed us to compare various vWD patients. The reactivity of lectins for plasma vWFs from two type 2A and six type 2B vWD patients harbouring different mutations was not statistically different from that of a pool of normal plasmas. We conclude that the alpha 2-6 sialic acid and beta 1-4 galactose content of plasma vWF is not altered in these patients affected with types 2A and 2B vWD.

In vitro evaluation of a very-high-purity, solvent/detergent-treated, von Willebrand factor concentrate.

A very highly purified von Willebrand factor (vWF) concentrate was analyzed in order to evaluate its suitability for the treatment of von Willebrand's disease (vWD). The functional activity of vWF assessed by its ristocetin cofactor activity (vWF:RCo) correlated with the level of vWF antigen (vWF:Ag), with the vWF:RCo/vWF:Ag ratios ranging from 0.56 to 1.02, and the specific activity being always greater than 50 IU vWF:RCo/mg protein. Electrophoretic analysis showed a normal pattern of high, intermediate and low-molecular-weight multimers of vWF. The biological activity of vWF was also evaluated by studying its ability to bind to collagen and to platelet receptors in the presence of either ristocetin or thrombin. Furthermore, these functional activities of vWF were confirmed by the capacity of this concentrate to induce platelet adhesion to collagen in a perfusion system. Moreover, the vWF present in this preparation was able to bind factor VIII. All these in vitro data suggest that this preparation is likely to be effective in the treatment of vWD patients.

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 this O-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz 1H-NMR spectroscopy to be: NeuAc(alpha 2-3)Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc-ol.

Adhesive properties of the carbohydrate-modified von Willebrand factor (CHO-vWF).

In this cooperative study, we explored the role of the carbohydrate moiety (CHO) of von Willebrand factor (vWF) in supporting platelet adhesion. Because of previous discrepant results, all purification steps and CHO modifications by various enzymes were critically evaluated. Under our conditions, CHO-modified vWF preparations contained less than 5% of the initial sialic acid ([Neu]-ase-vWF) and less than 45% ([Neu-Gal]-ase-vWF) or 21% ([Neu-Gal-eF]-ase-vWF) of the D-galactose. These preparations usually showed increased electrophoretic mobility but no significant loss of high-mol-wt multimers when proteolysis had been prevented. Some degree of proteolysis was noted in some carbohydrate-modified vWFs, but the degree of degradation observed did not correlate with the removal of D-galactose. Platelet adhesion to various matrices increased after removal of the terminal sialic acid ([Neu]-ase-vWF) and approximately 45% of the D-galactose ([Neu-Gal]-ase-vWF), but returned to normal values when greater than 70% of the total carbohydrate had been removed by endoglycosidase F [Neu-Gal-ef]-ase-vWF). These changes in reactivity were also reflected in the spontaneous aggregation in normal platelet-rich plasma (PRP) after CHO removal.

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 Willebrand's disease developed in two patients in association with a monoclonal gammopathy plus a Sjögren's 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 of 125I-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.

Characterisation of a monoclonal antibody to von Willebrand factor as a potent inhibitor of ristocetin-mediated platelet interaction and platelet adhesion.

We studied a murine monoclonal antibody (211 A6) to von Willebrand factor (vWF) with a view to investigating structure-relationship of plasma vWF. The specificity of this antibody has been substantiated by ELISA tests and indirect immunofluorescence. It reacts with purified vWF, normal plasma but not with plasma or platelets from a severe von Willebrand's disease patient. Monoclonal antibody 211 A6 is a potent inhibitor of ristocetin-induced platelet aggregation. The 125I-FVIII/vWF binding to platelets in presence of ristocetin is totally inhibited by low 211 A6 concentrations. Thrombin-induced binding of vWF to platelets is not affected by 211 A6. The ability of this antibody to inhibit platelet adhesion to subendothelium and to collagen was investigated with a perfusion model. The complete inhibition of platelet adhesion by 211 A6 questions the similarity or the interrelationship in vWF domains involved in ristocetin-induced platelet functions and platelet adhesion.

Primary structure of a new tetraantennary glycan of the N-acetyllactosaminic type isolated from human factor VIII/von Willebrand factor.

N-Glycosidically linked glycopeptides released by mild alkaline treatment of human factor VIII/von Willebrand factor (FVIII/vWF) were fractionated by serial affinity chromatography on columns of Sepharose linked to concanavalin A (ConA) and Lens culinaris agglutinin (LCA). The fraction which is not retained on ConA-Sepharose, but eluted from LCA-Sepharose contains a pure minor glycopeptide which was structurally analysed. Based on the results of methylation analysis and 500-MHz 1H-NMR spectroscopy, the following structure is proposed: (Formula: see text).

Improved characterization of plasma von Willebrand factor heterogeneity when using 2.5% agarose gel electrophoresis.

Discontinuous sodium dodecyl sulfate electrophoresis in large pore gels, followed by overlay with radiolabelled anti-von Willebrand factor (vWF) antibodies and by autoradiography, permits to analyze the multimeric structure of vWF. The aim of this study was to improve experimental conditions of this technique to satisfactorily resolve the minor forms of plasma vWF while still separating its high, intermediate, and low molecular weight predominant multimers. By using a 2.5% mixture of two selected agaroses, a single electrophoretic analysis of plasma clearly reveals the extreme complexity of the molecular forms of circulating vWF: each multimeric unit of plasma vWF may be separated into five bands, the central one being predominant. The multimeric distribution and "quintuplet" pattern obtained in the electrophoretic system described here permit a convenient classification of the different subtypes of von Willebrand's disease.

Effect of carbohydrate modifications of factor VIII/von Willebrand factor on binding to platelets.

This study compares the ability of unmodified and carbohydrate-modified forms of factor VIII/von Willebrand factor (FVIII/vWF) protein to bind to platelets in the presence of ristocetin or thrombin. Treatment of intact FVIII/vWF with alpha-D-neuraminidase results in more than 95% desialylation. Asialo FVIII/vWF retains total activity in ristocetin- and thrombin-mediated binding to platelets as demonstrated by direct and competitive binding assays. Examination of its multimeric pattern by sodium dodecyl sulfate-agarose electrophoresis reveals a normal multimeric structure. Treatment of intact FVIII/vWF with beta-D-galactosidase results in the removal of 20% of galactose (agalacto FVIII/vWF) whereas 55% of galactose is released from asialo FVIII/vWF (asialo agalacto FVIII/vWF). Agalacto and asialo-agalacto FVIII/vWF are both unable to bind to platelets in the presence of ristocetin. In contrast, they still bind to thrombin-stimulated human (except thrombasthenic) platelets. Removal of either ultimate (agalacto FVIII/vWF) or ultimate and penultimate (asialo-agalacto FVIII/vWF) galactose results in the same loss of the larger molecular weight multimers and in an increase of smaller multimers. These results suggest (1) that sialic acid does not play a significant role in ristocetin- or thrombin-mediated FVIII/vWF-platelets interactions and multimeric structure of FVIII/vWF (2) that ultimate beta-linked galactose residues are essential for the maintenance of a normal multimer organization (3) that ristocetin- and thrombin-mediated binding of FVIII/vWF to platelets differ in FVIII/vWF galactose requirement.

The role of fibronectin in factor VIII/von Willebrand factor cryoprecipitation.

To evaluate the role of fibronectin (Fn) in factor VIII (FVIII) and von Willebrand factor (vWf) cryoprecipitation, factor VIII procoagulant activity, factor VIII coagulant antigen, factor VIII-related antigen and von Willebrand ristocetin cofactor activity were measured in cryoprecipitate and cryosupernatant from normal and Fn-depleted plasmas. Following cryoprecipitation of normal plasmas, most of the FVIII and almost all the FvWf recovered were found with a part of Fn and of fibrinogen in cryoprecipitate. Fn-depleted plasmas prepared either by affinity chromatography on gelatin or by immunoadsorption on monoclonal anti-Fn antibodies behaved differently: although their cryoprecipitate contained normal fibrinogen levels, neither FVIII nor FvWf was precipitated. Experiments performed with Fn-depleted plasma to which purified fibronectin had been added, and samples of plasma with decreased Fn levels (0.01 to 0.2 g/l) suggest that there is a relation between initial Fn level and the extent of FVIII/vWf cryoprecipitation. We conclude that Fn, like fibrinogen, is necessary to induce cryoprecipitation of FVIII/vWf and that an initial plasma level of 0.2 g/l is sufficient to obtain good recovery of FVIII/vWf in cryoprecipitate.

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 separated by high-voltage electrophoresis. Five fractions 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.

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.

Blood group A and B activity associated with factor VIII - von Willebrand factor.

This report presents further progress on the characterization of blood group activities associated with F VIII/vWf. Evidence is provided that a relationship exists between the nature of the soluble blood group substance of plasma and of F VIII/vWf isolated from this plasma. A mixture of F VIII/vWf from O blood group plasma with A and B substances leads to a purified F VIII/vWf with AB blood group activity. Furthermore, chemical analysis of glycans released from F VIII/vWf by alkaline-borohydride treatment or hydrazinolysis shows the absence of N-acetylgalactosamine residues in non-reducing terminal positions. These results suggest that the blood group activity of F VIII/vWf preparations may be related to minor contamination of this molecule by glycolipidic or glycoproteinic plasma components with A or B oligosaccharide structures.

[Factor VIII-related antigen (F VIII R : Ag). Purification by immuno-affinity chromatography]. / Etude de l'antigène lié au facteur VIII (F VIII R : Ag). Purification par chromatographie d'immuno-affinité.

Immuno-affinity chromatography on immunoglobulins from a rabbit immunised with the plasma of a patient devoid of factor VII R : Ag allows the elimination of certain contaminants from preparations already enriched in factor VIII. The biological activity and criteria of purity of the factor VIII R : Ag preparation obtained are presented.

[Physicochemical properties of an extracellular polysaccharide isolated from culture media of Bacillus amyloliquefaciens]. / Propriétés physicochimiques d'un polysaccharide extra-cellulaire isolé des moûts de fermentation de Bacillus amyloliquefaciens.

A new extracellular polysaccharide has been isolated by chromatography on anion exchanger of a fraction obtained from highly viscous culture media of Bacillus amyloliquefaciens. This polysaccharide is characterised by high molecular weight (1,000,000 dalton) and intrinsic viscosity (323 ml/g). It contains 24% neutral sugar (galactose and mannose 5:1), 35% glucuronic acid and 51.5% N-acetylhexosamines (N-actylglucosamine, N-acetylgalactosamine and N-acetylbacillosamine 6:9:1).