Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.     

von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.     

Enhanced botrocetin-induced type IIB von Willebrand factor binding to platelet glycoprotein Ib initiates hyperagglutination of normal platelets

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPIb induced by these two substances have been reported. We have recently shown that the GPIb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.     

A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the Al disulphide loop (Cys509-Cys695) discriminates between type 2A and type 2B von Willebrand disease

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin-induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin-treated platelets nor on vWF-dependent platelet aggregation induced with ristocetin and asialo-vWF-mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512–673 sequence of the Al disulphide loop of vWF, MoAb B724 was used as second antibody in a two-site ELISA to test a series of patients with type 1, 2 A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the Al disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.     

Effects of different amino-acid substitutions in the leucine 694-proline 708 segment of recombinant von Willebrand factor

Summary. Type 2B von Willebrand disease (vWD) is characterized by an increased affinity of von Willebrand factor (vWF) for binding to platelet glycoprotein lb (Gplb). Most type 2B candidate mutations are clustered in the 509-695 disulphide loop but three of them (H505D, L697V and A698V) are outside this loop. We confirm here that the A698V mutation is a type 2B mutation by its expression in Cos-7 cells. As the L697V and A698V type 2B mutations both induce the presence of a valine residue in the 694-708 sequence, we created and expressed different mutated recombinant vWFs (rvWFs), in substituting the other leucine and alanine residues of this sequence (at positions 694, 701 and 706) into valine residues. V694rvWF and V706rvWF displayed decreased ristocetin-induced GpIb binding showing that it is not always the presence of a valine residue that may explain the increased affinity of type 2B vWF for GpIb. We also compared the interaction with platelets of V697rvWF and V698rvWF to those obtained with rvWFs reproducing two prevalent type 2B mutations located in the loop (R543W and V553M). We show that the two mutations located in the loop are more reactive than the two mutations identified outside the loop.     

The von willebrand factor domain-mediating botrocetin-induced binding to glycoprotein IB lies between Val449 and Lys728

Botrocetin, a component of Bothrops jararaca venom, induces von Willebrand factor (vWF)-dependent platelet agglutination and has been proposed as an alternative agent to ristocetin for evaluating vWF function. However, important differences between the vWF-platelet interactions induced by these two agents have suggested that different regions of vWF and the platelet may be involved in the interactions induced by the two agonists. We have recently demonstrated that binding of vWF to the platelet glycoprotein (GP) Ib receptor, either induced by ristocetin or as occurs spontaneously with asialo-vWF or vWF from IIb von Willebrand disease, is mediated by a domain residing on a 52/48- kilodalton (kD) tryptic fragment of vWF. This fragment extends from amino acid residue Val (449) to Lys (728). We have now found that this 52/48-kD fragment blocks botrocetin-induced binding of vWF to platelets and completely inhibits botrocetin-induced platelet agglutination. These results provide evidence that the vWF domain-mediating, botrocetin-induced platelet agglutination lies within the region delimited by this fragment and is therefore close to or identical with that which mediates ristocetin-induced binding and spontaneous binding of vWF to platelet GPIb. Anti-GPIb monoclonal antibodies also blocked agglutination, which showed that botrocetin, like ristocetin, induces binding of vWF to the GPIb receptor.     

A new mutation,S1285F,within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.     

Cleavage of human von Willebrand factor by porcine pancreatic elastase

von Willebrand factor (vWF) was purified from pooled normal plasma, radiolabeled with iodine then cleaved by porcine pancreatic elastase. Cleavage was monitored by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-agarose electrophoresis, crossed immuno- electrophoresis, ristocetin and botrocetin cofactor activities, and ristocetin and botrocetin induced binding to fixed washed platelets. Cleavage of vWF by porcine elastase was dependent on both the concentration of porcine elastase and period of incubation. Incubation of vWF with concentrations of porcine elastase greater than 20 U/mL for 30 minutes resulted in loss of the 240 Kd vWF subunit and a corresponding loss of the high molecular weight multimers and formation of fragments with molecular weights on reduced SDS-PAGE of 150, 125, 115 Kd and minor bands at 44, 28, and 24 Kd and a band at 20 Kd, which increased in intensity as either the concentration of porcine elastase or the period of incubation increased. More than 50% of the ristocetin cofactor activity was lost during a five-minute incubation of vWF with 0.56 U/mL porcine elastase when no detectable structural changes in the vWF molecule had occurred. Botrocetin cofactor activity was more resistant. Similarly botrocetin induced vWF binding to platelets was retained by a fraction produced by digestion of vWF with porcine elastase, which contained predominantly a 20 Kd fragment of vWF. This fragment retained insignificant amounts of ristocetin related functions and therefore represents a useful piece of the vWF molecule for further exploration of the site involved in botrocetin induced binding to platelets.     

Identification of two mutations (Arg611Cys and Arg611His) in the A1 loop of von Willebrand factor (vWF) responsible for type 2 von Willebrand disease with decreased platelet-dependent function of vWF [see comments]

We report the identification of von Willebrand factor (vWF) gene mutations within exon 28 occurring in three unrelated families with an infrequent form of type 2 von Willebrand disease (vWD). A C-->T transition and a G-->A transition, both at the codon for arginine 611 of the mature vWF subunit, were found. They result in either a cysteine or an histidine substitution, respectively. Patients were found to be heterozygous for these substitutions and the vWD was transmitted dominantly. These substitutions have been reproduced by in vitro mutagenesis of full-length cDNA of vWF and transiently expressed in Cos- 7 cells. The corresponding recombinant vWFs (rvWF) exhibited decreased expression and a significant decrease in the high molecular weight multimeric forms. In addition, ristocetin- and botrocetin-induced binding of mutated rvWFs to platelets were markedly decreased as compared with that for the wild-type rvWFs. Thus, the structural and functional characterization of both mutated rvWFs confirmed that the two nucleotide substitutions identified at position 611 of the mature subunit of vWF are real mutations. Although they are located in the A1 loop containing most of the type 2B mutations inducing increased affinity of vWF for platelet glycoprotein Ib, they are responsible for abnormal vWF with decreased platelet-dependent function.     

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.     

Two novel type 2N von Willebrand disease-causing mutations that result in defective factor VIII binding, multimerization, and secretion of von Willebrand factor

Two novel mutations, a T-to-C transition at nucleotide 2612 and a T-to-G transversion at nucleotide 3923 of the von Willebrand factor (vWF) complementary DNA, were detected by analysis of the vWF gene in DNA from members of 2 families with atypical von Willebrand disease. The T2612C transition predicts substitution of cysteine by arginine at amino acid position 788 (C788R), and the T3923G transversion predicts substitution of cysteine by glycine at position 1225 (C1225G) of pre-pro-vWF. The patients homozygous for the C788R and C1225G mutations both had a partial vWF deficiency (0. 18 IU/mL and 0.07 IU/mL vWF antigen, respectively); vWF in plasma from patients homozygous for either the C788R or the C1225G mutation failed to bind factor VIII and lacked high molecular weight multimers. Recombinant (r) vWF molecules having the C788R or C1225G mutation were expressed in COS-7 cells. Both rvWF C788R and rvWF C1225G exhibited significantly impaired secretion and failed to bind factor VIII. Recombinant vWF C788R in COS-7 culture medium showed a severe reduction in high molecular weight multimers, whereas rvWF C1225G showed a very mild reduction in high molecular weight multimers when compared with wild-type rvWF. (Blood. 2000;95:2000-2007)     

Inhibition of Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX Complex, Heparin and Sulfatides by Polyanionic Compounds. The Mechanism of Modulation of the Adhesive Function of von Willebrand Factor

The interaction of the multimeric glycoprotein von Willebrand Factor (vWF) with its platelet membrane receptor, the glycoprotein (GP) Ib-IX complex plays a key role in the initial adhesion of platelets to the vascular subendothelium at high shear blood flow. The GP Ib-IX-binding site is only expressed following activation of vWF, a process that regulates vWF-mediated platelet adhesion. Binding of vWF to the GP Ib-IX complex involves the vWF A1 internal repeat domain, which also contains distinct binding sites for sulfatides, heparin, and the non-physiological modulators of the vWF-GP Ib-IX interaction, ristocetin and botrocetin. With the ultimate aim of further defining the mechanism of vWF modulation, we have analyzed the ability of various polyanionic compounds, including aurintricarboxylic acid, Evans blue, fucoidan, and a range of sulfated and phosphorylated sugars, to inhibit specific binding of purified vWF to immobilized sulfatides and heparin, and the ristocetin- and botrocetindependent binding of vWF to the platelet GP Ib-IX complex. Firstly, it was confirmed using a solid-phase binding assay that, like sulfatides, heparin specifically bound to a purified 39/WkiloDalton fragment of vWF (Leu-480 to Gly-718) that encompasses the A1 domain. Secondly, the ability of a number of polyanionic compounds to inhibit binding of vWF to heparin, but not to immobilized sulfatides, supported previous data suggesting that heparin and sulfatides bind to distinct sites on vWF. In addition, aurintricarboxylic acid, Evans blue and fucoidan all inhibited binding of vWF to both heparin and sulfatides with similar ICso values. Thirdly, many of the compounds tested that inhibited binding of vWF to heparin also effectively inhibited both ristocetin- and botrocetin-dependent binding of vWF to the GP Ib-IX complex on platelets, whereas none of the compounds tested blocked vWF binding to sulfatides and GP Ib-IX but not heparin. The majority of compounds tested inhibited the vWF-platelet interaction to a comparable degree in the presence of ristocetin or botrocetin, suggesting a similar mechanism for inhibition irrespective of the modulator used. These combined experiments provide evidence for an electrostatic model of vWF modulation, and suggest that the heparin-binding domain of vWF may be an important regulatory site involved in the adhesion of vWF to the platelet GP Ib-IX complex.     

Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody

An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil- T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF- binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF- binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those of the Ab occurring in inhibitor von Willebrand's disease in which vWF inhibitor and binding values were similar, with a strong anamnestic response. The findings indicate that the vWS Ab binds to an epitope on the molecular vWF in such a way that causes only limited inhibition of vWF/ristocetin function and no inhibition of vWF/botrocetin function, suggesting that these two functional domains are at separate sites.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

Leu 697-->Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease

Type IIB von Willebrand disease is characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers from plasma and enhanced platelet agglutination of platelet-rich-plasma in the presence of low concentrations of ristocetin. We identified, in two related patients, a C-->G transversion resulting in the substitution of Valine for Leucine at position 697 of the mature subunit of vWF. We reproduced this mutation in vWF cDNA and expressed the recombinant protein in Cos-7 cells. The subunit composition and multimeric structure of mutated protein (rvWFLeu697Val) were similar to the wild- type recombinant (WTrvWF). Ristocetin-induced binding of rvWFLeu697Val to platelets was markedly increased in the presence of low doses of ristocetin and slightly increased with botrocetin as compared with that for WTrvWF, whereas collagen binding was not affected by the mutation. These data show that the Leu 697-->Val substitution is not a rare polymorphism but is responsible for the subtype IIB characteristic abnormalities identified in the two affected patients; however, it is not located in the area of vWF (amino acid 540 to amino acid 578) where most of the other type IIB mutations have already been reported.     

Functional analysis of a type IIB von Willebrand disease missense mutation: increased binding of large von Willebrand factor multimers to platelets.

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.     

Effect of recombinant von Willebrand factor reproducing type 2B or type 2M mutations on shear-induced platelet aggregation

The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds(-1)) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds(-1). Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds(-1) confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD. (Blood. 2000;95:3796-3803)     

Characterization of murine anti-glycoprotein Ib monoclonal antibodies that differentiate between shear-induced and ristocetin/botrocetin-induced glycoprotein Ib-von Willebrand factor interaction

Platelet adhesion to vascular subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein (GP) Ib-von Willebrand Factor (vWF) interaction. The aim of this study was to characterize the murine monoclonal antibodies (MoAbs) 27A10 and 28E6, both raised against purified GPIb. The MoAb 27A10 is a potent inhibitor of shear-induced platelet adhesion to collagen type I in a flow chamber at shear rates of 1,300 and 2,700 s(-1). 20 microg/ml of MoAb 27A10, furthermore, could completely block shear-induced aggregation in a modified Couette viscometer at shear rates of 1,000 and 4,000 s(-1). On the other hand, MoAb 27A10 had a negligible effect on botrocetin-induced GPIb-vWF binding and is only a poor inhibitor of the ristocetin-dependent interaction. In contrast, MoAb 28E6 did abolish both the ristocetin- and botrocetin-induced GPIb-vWF binding, whereas it did not block the shear-induced interaction. Thus, we identify here two anti-GPIb MoAbs 27A10 and 28E6 that either preferentially inhibit the shear-induced or the ristocetin/botrocetin-induced platelet-vWF interaction. With these tools it should be possible to more clearly define the mechanisms by which platelets bind to vWF in vivo.     

Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size

We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.