Enhancement of factor VIII-von Willebrand factor ristocetin cofactor activity by monoclonal antibodies

Five monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any cross-reaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.     

Structural characterization of urinary von Willebrand factor by using monoclonal antibodies

The presence of von Willebrand factor (vWF) related antigens has been previously described in urine. By gel filtration, three immunoreactive peaks have been demonstrated having Mr about 350 kDa, 60 kDa and 20 kDa. In this report, a structural characterization of the urinary fragments of vWF was studied by using monoclonal antibodies having known specificities towards the plasma sub-unit. The biggest urinary fragments contain the C-terminal portion and the central portion of the sub-unit. The other two urinary fragments come from the central part of the sub-unit. Antibodies to the N-terminus of the sub-unit do not react with any of the urinary fragments. These results led to a picture for degradation of vWF that is complex and may involve sequential degradation by several proteases. Patients with different types of vWD show qualitatively the same vWF degraded material in their urines, suggesting that whatever the mechanisms for degradation of vWF they are intact in these patients. Quantitatively, it seems that in patients receiving factor VIII-vWF concentrates there is an increased tendency to vWF excretion, which may be due to the presence of denatured material in the concentrates infused to these patients.     

The anti-von Willebrand factor aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease

Blockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59-130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40 x 10(9)/l (38-58 x 10(9)//l) to a maximum of 146 x 10(9)//l (107-248 x 10(9)//l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.     

Fc-receptor dependent platelet aggregation induced by monoclonal antibodies against platelet glycoprotein Ib or von Willebrand factor

In this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ibalpha to the platelet Fc-receptor (FcgammaRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbalpha, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcgammaRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbalpha to the FcgammaII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs IC1E7 and B724, both known to enhance vWF binding to GPIbalpha in the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIb MoAb, indicating that again the platelet activation results from signaling through FcgammaRII crosslinked to vWF bound to GPIbalpha. In addition, both the anti-GPIb MoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIb and anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIb and FcgammaRII, suggesting a role for the FcgammaII-receptor in GPIb-related signaling.     

Platelet-type von Willebrand disease platelet aggregating factor: a novel functional assay of von Willebrand factor

Blood platelets from patients with platelet-type von Willebrand disease (vWD) aggregate upon the addition of human von Willebrand factor (vWf) in the absence of ristocetin or other stimulating factors. We measured quantitatively the ability of vWf to induce directly aggregation of platelet-type vWD platelets (platelet-type vWD platelet aggregating factor [PT-PAF]). Cryoprecipitate and factor VIII concentrates were used as a source of vWf of various multimeric composition. The PT-PAF activity was dependent on the multimer size of vWf, like in the case of ristocetin cofactor (RCof) activity. However, PT-PAF activity was not equivalent to RCof activity and the relative PT-PAF/RCof ratio ranged from 1.00 to 0.18 in the materials studied. The preparations containing the higher-molecular-weight multimers had higher PT-PAF/RCof ratio. These findings suggest that PT-PAF activity is a functional expression of more highly polymerized multimers of vWf as compared with RCof activity. Measurement of PT-PAF would serve as a novel functional assay of vWf.     

Murine monoclonal antibodies to human factor XI

Murine monoclonal antibodies to human factor XI (F.XI) are described. The monoclonal antibodies (2-1, 4-1, 7-1 and 10-1) consisted of IgG1. 4-1 inhibited the activation of F.XI completely in the presence of high molecular weight kininogen and kaolin and the others did so partially, whereas these antibodies had no effect on the activation of F.XI with activated factor XII (beta-XIIa). Four antibodies had no effect directly on the amidolytic activity of activated F.XI (F.XIa). 10-1 inhibited the activation of factor IX in coagulant assay for F.XIa by Mannhalter. And 4-1 and 7-1 did so partially, whereas 2-1 did not. In immunoblotting analysis, all antibodies bound to F.XI, its reduced form and F.XIa. All were directed against the heavy chain of F.XI. All antibodies recognized F.XIa-alpha 1 antitrypsin complex.     

Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor

Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (K(D) approximately nM range) and recognised sequences V(357)-F(360) on the acidic a1 region and E(724)-L(731) on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC(50): 77 nM and 40 nM respectively), and (b) VWF (IC(50): 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab')(2) fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.     

Visualization of von Willebrand factor multimers by enzyme-conjugated secondary antibodies

A method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with primary vWF antiserum, peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.     

Two monoclonal antibodies to D-dimer-specific inhibitors of fibrin polymerization

D-dimer of human fibrin was used as antigen to obtain monoclonal antibodies (mAbs). We have obtained 16 hybridomas producing mAbs of different specificity. Only two of these mAbs inhibited fibrin polymerization. They are of the IgG-class. One mAb (II-4d) inhibited fibrin polymerization to 100% and another (II-3b) to 60% at a molar ratio mAb/fibrin=1.0. Fab-fragments of these mAbs inhibited fibrin polymerization completely at the same molar ratio. The epitopes for the mAbs studied are situated in the NH2-terminal part of the gamma-chain in fibrin D-domain. Electron microscopy showed that fibrin was in monomeric form in the presence of these mAbs or their Fab-fragments. Thus, these mAbs stop the initial step of fibrin polymerization, i.e. protofibril formation. Only one site of protofibril formation is known now in COOH-terminal half of the D-domain gamma chain named "a" site, which is complementary to the "A" site in the central E-domain of fibrin molecule. Our experiment with immobilized GPRP showed that the "a" site in fibrin D-fragment preserved its binding activity to GPRP when the D-fragment was complexed with mAbs-inhibitors of fibrin polymerization. Thus, these two mAbs inhibit fibrin polymerization not by blocking the sites "a" but either by blocking another (not "a") specific site in D-domain or by steric hindrance of highly organized fibrin polymerization process.     

Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to vWF

A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.     

Specificity of monoclonal antibodies to factor VIII:C

Three monoclonal antibodies (42 IgG, 47 IgG, 56 IgG) towards factor-VIII:C (VIII:C) have been produced. In ELISA for VIII:C-antigen (VIII:CAg), 47 IgG showed higher affinity for VIII:CAg than 42 IgG and 56 IgG. In solid phase immunoisolation of iodinated VIII:C diluted in EDTA buffer, the three monoclonals, like human VIII:C inhibitors, bound the 77/80 kD-light chain of VIII:C. In the absence of EDTA, 56 IgG bound the heavy chain-light chain complex of VIII:C, while 47 IgG was only able to bind the light chain. When coupled on Sepharose, 56 IgG adsorbed coagulation active VIII:C, while 47 IgG was only able to adsorb coagulation inactive VIII:CAg. In coagulation assay 56 IgG inhibited with 20 BU/mg while 42 IgG and 47 IgG inhibited with 4 BU/mg. A mixture of 42 IgG and 56 IgG showed a synergistic effect and inhibited with 50 BU/mg total IgG. In radioimmunoassay a human VIII:C inhibitor was able to inhibit the VIII:C binding of 42 IgG and 56 IgG but not of 47 IgG. The monoclonals did not inhibit each other. On the contrary, 56 IgG increased the binding of 42 IgG to VIII:C.     

Two novel monoclonal antibodies (1.9.E and 4.11.C) against olfactory bulb ensheathing glia

We produced and characterized two monoclonal antibodies, termed 1.9.E and 4.11.C, that specifically recognize olfactory bulb ensheathing glia. Both antibodies were generated using the olfactory nerve layer (ONL) of newborn rat olfactory bulbs (P0, P1) as immunogens. The specificity of these antibodies was tested by immunofluorescence techniques on tissue sections and cultures of adult and neonatal rat olfactory bulbs, and by Western blot analysis. 1.9.E labeled the ONL and glomerular layer of the olfactory bulb (OB) of adult rats. In newborn rats, 1.9.E immunostained ensheathing cells from the ONL and peripheral olfactory fascicles. Furthermore, 1.9.E reacted with some processes of the radial glia in the periventricular germinal layer of the newborn rat. Although 4.11.C also specifically labeled ensheathing cells in the adult OB, it did not stain any cell type in the ONL of newborn rats. The lack of double labeling with either 1.9.E or 4.11.C and anti-olfactory marker protein (OMP) antibody, a specific marker for olfactory axons, indicated that none of the monoclonals recognized olfactory axons. Double immunostaining of adult OB cultures with 1.9.E or 4.11.C and anti-p75-nerve growth factor receptor revealed that both antibodies specifically recognized ensheathing glia in those cultures. Filaments were strongly labeled throughout the entire cytoplasm of ensheathing cells, suggesting that 1.9.E and 4.11.C immunoreacted with ensheathing glia cytoskeleton. 4.11.C stained a few Schwann cells in adult sciatic nerve sections. Moreover, 4.11.C immunostained cortical astrocyte cultures from newborn rats (P1). In Western blot analysis both antibodies recognized a major component, migrating with an apparent molecular weight of 60 kDa, from olfactory nerve and glomerular layer (ONGL) extracts of adult and neonatal rats. The pattern of immunoreactivity of 1.9.E and 4.11.C antibodies suggest that both antibodies are specific markers for olfactory ensheathing glia in the adult rat central nervous system (CNS). GLIA 24:352–364, 1998. © 1998 Wiley-Liss, Inc.     

Anti-human factor IX monoclonal antibodies specific for calcium ion-induced conformations

Four monoclonal antibodies have been produced, which are specific for the Ca²⁺ or Sr²⁺-induced conformation of human factor IX. Certain, but not all, γ-carboxyglutamic acid residues in factor IX are involved in the epitope expression together with the conformation stabilized by the adjacent region of Gla-domain and a disulfide bridge. All the antibodies interfered with the binding of factor IX to phospholipids and inhibited the procoagulant activity of factors IX and IXaβ.     

Platelet von Willebrand factor: Comparison with plasma von Willebrand factor

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that 1) the multimeric organization was different, 2) larger multimers were present, and 3) the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.     

Epitope specificity of anti-FVIII antibodies during immune tolerance therapy with factor VIII preparation containing von Willebrand factor

The study aimed at characterizing the putative changes in the epitope specificity of anti-FVIII antibodies during a successful immune tolerance treatment of the haemophilia A patient with the factor VIII (FVIII) preparation containing the von Willebrand factor (VWF). At the beginning of treatment, anti-FVIII inhibitory antibodies recognizing predominantly the light chain of FVIII were prevalent and persisted throughout the treatment. More detailed characterization of the FVIII antibody epitope specificity by using GST-fusion proteins corresponding to different FVIII domains revealed the prevalence of C1-domain-specific antibodies, while a remarkably lower amount of antibodies were targeted at the C2 and the a3 domains of the FVIII light chain and towards the A2 and the A1 domain of the FVIII heavy chain. The epitope specificity of antibodies remained rather unchanged throughout treatment except the elevated level of C2-domain-specific FVIII antibodies after a temporary interruption of treatment. The patient's antibodies were unable to interfere with the FVIII binding to VWF or to phospholipids, but inhibited FXa generation and the binding of FX to FVIII on the phospholipid monolayer. Thus, a unique pattern of the epitope specificity of FVIII antibodies and the mechanism to inhibit FVIII:C activity by FVIII-light-chain-specific antibodies were characterized.     

A novel method for quantitation of human von Willebrand factor messenger RNA

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700 quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism BigDye terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r(2)=0.912, p<0.001).     

Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies

Tissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F.VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F.VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F.VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F.VIIa contact region. It included residues Lys 165, Lys 166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent anticoagulants when infused to flowing human blood in an ex-vivo thrombosis model. Plasma fibrinopeptide A levels and fibrin deposition were completely inhibited. In contrast, 6B4 was a weak inhibitor in this ex-vivo thrombosis model, and HTF1 displayed no inhibition at all. These disparate activities were also reflected in TF-dependent F.X activation assays performed with human plasma. The potency differences could neither be explained by the determined binding affinities nor by the on-rates of antibodies. Therefore, the results suggest that antibody binding epitope and hence the particular mechanism of inhibition, is the main determinative factor of anticoagulant potency of anti-TF antibodies.     

Characterization of a novel mutation in the von Willebrand factor propeptide in a distinct subtype of recessive von Willebrand disease

von Willebrand factor (VWF) is a plasma protein that consists of a series of multimers of which the high-molecular-weight VWF multimers are the most potent in platelet adhesion and aggregation. The propeptide of the VWF (VWFpp) is known to be essential in the process of multimer assembly. Genetic studies were performed in a patient with a phenotype of von Willebrand disease (VWD) characterized by very low plasma factor VIII and VWF levels and a VWF consisting of only a dimeric band and total absence of all multimers in plasma. The patient was found to be homozygous for the novel C570S mutation, caused by a 1709G>C transition in exon 14 of the VWF gene coding for the propeptide. Three asymptomatic relatives were found to be heterozygous. In-vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on VWF multimerization. Our findings show that the C570S mutation in the VWFpp abolishes multimerization of VWF. The mutation probably disrupts the normal configuration of the VWFpp, which is essential for correct orientation of the protomers and ultimately multimerization. The mutant amino acid is located in a region that is highly conserved across several species which underlines its critical role. This variant constitutes a distinct subtype of recessive 2A VWD with the exclusive presence of the dimeric form of VWF in plasma.