Substitution of cysteine for phenylalanine 751 in mature von Willebrand factor is a novel candidate mutation in a family with type IIA von Willebrand disease

Summary Type IIA is a variant form of von Willebrand disease (vWD) characterized by the absence of von Willebrand factor (vWF) high molecular weight multimers in plasma. Most of the candidate missense mutations potentially responsible for type IIA vWD have been found clustered within a short segment of vWF, lying between Gly⁷⁴² and Glu⁸⁷⁵ of the mature subunit. The present work reports a single heterozygous T → G transversion in eight patients from a large type IIA vWD family, resulting in the substitution Phe⁷⁵¹→Cys. The absence of this mutation in 100 normal vWF genes as well as the lack, in these patients, of any other abnormality within the whole exon 28 encoding amino acids 463–921 of mature vWF, provide a strong support that this non-conservative mutation may be at the origin of the disease in this family. The presence of an additional cysteine at position 751 may induce a conformational change of the vWF subunit affecting either its ' in vivo ' sensitivity to proteolytic cleavage or, more likely, its intracellular transport as suggested by the abnormal multimeric pattern of platelet vWF observed in these patients.     

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.     

C1272S: a new candidate mutation in type 2A von Willebrand disease that disrupts the disulfide loop responsible for the interaction of VWF with platelet GP Ib-IX

Most of type 2A von Willebrand disease (VWD) mutations are clustered within the A2 domain of VWF, encoded by the 3' region of exon 28 of the von Willebrand factor (VWF) gene. A patient with lifelong and severe bleeding diathesis and laboratory data of type 2A VWD is described. The analysis of the complete exon 28 of the VWF gene showed a 3815 G-->C change within the A1 domain, resulting in the C1272S missense mutation in a heterozygous state. The substitution was not found in 100 normal alleles also examined and has not been described previously. This candidate mutation would interrupt the formation of the disulfide loop 1272-1458, which is important in maintaining the adequate conformation of the VWF functional domain that interacts with platelet glycoprotein Ib-IX. Gene expression of this candidate mutation is necessary to confirm its role.     

The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.     

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.     

A monomeric von Willebrand factor fragment, Leu-504--Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX [corrected]

von Willebrand factor interaction with glycoprotein Ib alpha (GPIb alpha) plays a critical role in the initial phase of platelet adhesion at high shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474--Pro-488 (peptide 153) and Ser-692--Pro-708 (peptide 154), inhibit von Willebrand factor--GPIb alpha interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504--Lys-728 [corrected], with a single intrachain disulfide bond linking residues Cys-509--Cys-695 and examined its ability to inhibit von Willebrand factor--GPIb alpha interactions and platelet adhesion at high shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetin-induced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an IC50 = 0.011-0.260 microM, significantly lower than the IC50 of peptide 153 or 154, IC50 = 86-700 microM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (IC50 greater than 500 microM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 microM and at 7.6 microM inhibited greater than 80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.     

Phenotype changes resulting in high-affinity binding of von Willebrand factor to recombinant glycoprotein Ib-IX: analysis of the platelet-type von Willebrand disease mutations

To maintain hemostasis under shear conditions, there must be an interaction between the platelet glycoprotein (GP) Ib-IX receptor and the plasma ligand von Willebrand factor (vWf). In platelet-type von Willebrand disease (Pt-vWD), hemostasis is compromised. Two mutations in the GPIbalpha polypeptide chain have been identified in these patients-a glycine-233 to valine change and a methionine-239 to valine change. For this investigation, these mutant proteins have been expressed in a Chinese hamster ovary cell model system. Ligand-binding studies were performed at various concentrations of ristocetin, and adhesion assays were performed under flow conditions. The Pt-vWD mutations resulted in a gain-of-function receptor. vWf binding was increased at all concentrations of ristocetin examined, and adhesion on a vWf matrix was enhanced in terms of cell tethering, slower rolling velocity, and decreased detachment with increasing shear rate. Two other mutations were also introduced into the GPIbalpha chain. One mutation, encompassing both the Pt-vWD mutations, created an increase in the hydrophobicity of this region. The second mutation, involving a valine-234 to glycine change, decreased the hydrophobicity of this region. Both mutations also resulted in a gain-of-function receptor, with the double mutation producing a hyperreactive receptor for vWf. These data further support the hypothesis that ligand binding is regulated by conformational changes in the amino-terminal region of GPIbalpha, thereby influencing the stability of the GPIbalpha-vWf interaction.     

Structural determinants within platelet glycoprotein Ibalpha involved in its binding to von Willebrand factor

Platelet adhesion to subendothelial structures upon injury to a vessel wall is one of the first steps in a sequence of reactions critical for the formation of a haemostatic plug, or in diseased vessels for the development of an arterial thrombus. This adhesion process is mediated by an interaction between the glycoprotein (GP) Ib-V-IX complex on the platelet surface with von Willebrand Factor (vWF), associated with collagen on the subendothelial surface. After this initial adhesion, platelets will activate, resulting in recruitment of additional platelets and adherence to each other to form the platelet plug or developing thrombus. Several studies to date have attempted to identify the regions of the GPIb-V-IX complex that are critical for binding to vWF. The vWF binding site is contained in the 45 kDa N-terminal domain of the GPIbalpha chain. This N-terminal domain is characterized by a structural motif consisting of 7 leucine-rich repeats (LRRs), followed by a double disulphide-bonded loop and an anionic sulphated region. This review summarizes recent research efforts elucidating the characteristics of the GPIb-vWF interaction. Potential mechanisms that regulate the GPIb-vWF function are discussed, and advances in identifying functional sequences within GPIba involved in the binding to vWF are reviewed.     

Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type IIA von Willebrand disease. The patient's EC, compared with those from normal individuals, produced vWf that had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features characteristic of plasma vWf from patients with type IIA von Willebrand disease. The type IIA EC did produce a full spectrum of vWf multimers in both cell lysates and postculture medium, although the relative amounts of the largest species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites that contained approximately equal to 170-kDa proteolytic fragments, suggesting that this patient's functional defect is due to abnormal proteolysis and not to a primary failure of vWf subunit oligomerization. Moreover, the observed degradation appears to result from an abnormal vWf molecule and not elevated protease levels. These results suggest that this patient's von Willebrand disease phenotype is caused by increased proteolytic sensitivity of his vWf protein.     

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.     

Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers

Ultralarge von Willebrand factor (ULVWF) multimers have been implicated in the pathogenesis of the catastrophic microangiopathic disorder, thrombotic thrombocytopenic purpura. Spontaneous ULVWF binding to platelets has been ascribed to increased avidity due to the greatly increased number of binding sites for platelets (the A1 domain) per molecule. To address the mechanism of enhanced ULVWF binding to platelets, we used optical tweezers to study the unbinding forces from the glycoprotein Ib-IX (GP Ib-IX) complex of plasma VWF, ULVWF, and isolated A1 domain. The unbinding force was defined as the minimum force required to pull ligand-coated beads away from their attachment with GP Ib-IX-expressing cells. Beads coated with plasma VWF did not bind to the cells spontaneously, requiring the modulators ristocetin or botrocetin. The force required to break the ristocetin- and botrocetin-induced plasma VWF-GP Ib-IX bonds occurred in integer multiples of 6.5 pN and 8.8 pN, respectively, depending on the number of bonds formed. In contrast, beads coated with either ULVWF or A1 domain bound the cells in the absence of modulators, with bond strengths in integer multiples of approximately 11.4 pN for both. Thus, in the absence of shear stress, ULVWF multimers form spontaneous high-strength bonds with GP Ib-IX, while plasma VWF requires exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1 domain and greater than those of plasma VWF induced by either modulator. Therefore, we suggest that the conformational state of ULVWF multimers is more critical than their size for interaction with platelets.     

Abnormal platelet von Willebrand factor interaction in patients with TTP

Thrombotic thrombocytopenic purpura (TTP) is associated with abnormal platelet function and disturbances in coagulation; however, a specific causative factor is not defined. Plasma infusion or plasma exchange (PE) are thought to be of benefit in replacing a deficient plasma component or removing some toxic compound. In three patients with TTP, samples taken prior to initiation of PE showed high levels of vWF:Ag in the plasma (208, 264, and 321 U/dl), whereas the VIII:C levels were normal. The vWF:Ag multimer patterns of the plasma demonstrated a decrease in the amount of high molecular weight (HMW) forms. Analysis of the platelets from one patient also showed an increase in the HMW multimers. Platelets from all three patients showed a decreased ability to absorb vWF:Ag, with little or no absorption of the HMW forms. Following extensive PE and resolution of disease, the platelets regained their ability to absorb vWF:Ag in the one patient examined.     

Multimeric pattern discrepancy between platelet and plasma von Willebrand factor in type IIC von Willebrand disease

von Willebrand factor (vWF) from platelet lysate and plasma, collected in the presence of protease inhibitors, was studied in two patients with type IIC von Willebrand disease (vWD). Platelet and plasma vWF showed the smallest multimer increased, but the latter had a repeating single band whereas the former had a repeating "doublet." This platelet-plasma discrepancy observed for the first time in these patients suggests that the repeating "doublet" or single band described in other type IIC patients represent minor subgroups of type IIC vWD.     

Distinct sequences of the glycoprotein Ib-binding domain of von Willebrand factor involved in shear induced platelet aggregation

Platelet aggregometry is an important technique which is frequently used by hemostaseologists and researchers. Gustav Born introduced the principle more than 40 years ago. Many different ways to perform aggregometry have been published. The results of aggregometry may become more comparable if some rules would be generally accepted. (1) The pre-analytical procedures are probably the most important factors which influence aggregometry results. Besides correct blood sampling important factors are the preparation of platelet-rich plasma (PRP), incubation of the PRP at room temperature and awareness of time-dependent changes of aggregometry results. (2) A major point concerns the agonists. Agonists of different sources have to be compared to verify that they lead to the expected results. Even different salts of ADP lead to different results and different collagen preparations lead to a large variation of aggregation response (3). The frequently used procedure of adjusting the platelet number in the PRP is cumbersome, affects platelet activation and is not necessary. (4) Aggregometers should comply with some simple rules. The changes in optical density should be linearized so that – if this is required – percentages can be given. The recorder speed should be standardized and all recorders should provide 1?cm/min. Calibration of the aggregometer sensitivity should be possible. (5) If aggregometry is used to define the response to antiaggregating agents agreement on the inducer concentrations is essential. If some rules are applied aggregometry is a relatively simple and reliable method, and well suited for clinical studies and for experimental research.     

Distinct sequences of the glycoprotein Ib-binding domain of von Willebrand factor involved in shear induced platelet aggregation

Shear-induced platelet aggregation (SIPA) requires von Willebrand factor (vWF) binding to the platelet receptors GPIb and alphaIIbbeta3. In order to determine the vW F sequences involved in SIPA at 4000/s, we studied the llb 3 effect of three monoclonal antibodies (mabs) 724, 713 and 328 to the A1 domain of vWF. We found that mab 724 induced an enhanced SIPA via a Fc gamma-receptor independent mechanism. In contrast, mab 713 and mab 328 could inhibit SIPA by 52 and 91% , respectively. Based on distinct effects on SIPA, we can propose the following working model for the interaction between vWF and GPIb: mabs 713 and 328, which block SIPA, may recognize an epitope that is involved in binding to GPIb, whereas mab 724, which increases SIPA in the presence of vWF, may mimic the effect of botrocetin when binding to vWF, by inducing an active conformation of vWF, which may be more sensitive to high shear rate.     

Dominant type 1 von Willebrand disease caused by mutated cysteine residues in the D3 domain of von Willebrand factor

No defects have been reported in moderately severe type 1 von Willebrand disease (vWD) with a clear autosomal dominant inheritance pattern, and the mechanism underlying this form of vWD remains obscure. We have studied a type 1 vWD family with such a dominant phenotype. The entire coding sequence of the von Willebrand factor (vWF) gene was analyzed by direct sequencing of DNA fragments amplified by polymerase chain reaction. Only one candidate mutation T(3445)-->C in exon 26 was detected that predicts a replacement of cysteine (C) at position 386 of the mature vWF subunit by arginine (R). Both mutant and normal vWF alleles were expressed as shown by analysis of platelet mRNA. This substitution segregates with vWD in the family and was not found in 100 unrelated individuals. The recombinant mutant vWF(C386R) was characterized by expression in 293T cells. The secretion of vWF(C386R) was greatly impaired due to retention in the endoplasmic reticulum. In cotransfections of normal and mutant vWF constructs, the vWF(C386R) subunits caused a dose-dependent decrease in the secretion of vWF. The multimer pattern remained nearly normal and consistent with a dominant vWD type 1 phenotype. The importance of the cysteine residues in the D3 domain of vWF in the pathogenesis of dominant type 1 vWD was further shown by the detection of another cysteine mutation, Cys367-->Phe, in two additional unrelated patients with a similar dominant type 1 vWD phenotype. We conclude that the loss of cysteine pairing in the D3 domain, leaving one free cysteine, can induce a purely quantitative deficiency of vWF by dominantly suppressing the secretion of normal vWF.     

Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency

In normal platelet α-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal α-granular multimerin and factor V, and normal α-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, α-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal α-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWF. The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.     

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.     

Role and initiation mechanism of the interaction of glycoprotein Ib with surface-immobilized von Willebrand factor in a solid-phase platelet cohesion process

To know the role and initiation mechanism of the interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF), we examined the effect of shear stress levels on platelet binding to vWF-coated plates using a cone-and-plate type viscometer capable of loading various levels of shear stress. The extent of platelet binding to immobilized vWF reached a plateau at the shortest period tested (20 seconds) under high shear stress (90 dyne/cm2), whereas 9 to 12 minutes was necessary for saturable platelet binding under static conditions. This shear effect, which was found to be dependent on the vWF-GP Ib interaction, was observed even under the lowest shear stress (1.5 dyne/cm2) examined. In contrast with the high shear effect previously reported to initiate the interaction of GP Ib with soluble vWF, these results indicate that relatively low levels of shear stress can promote the interaction of GP Ib with surface- immobilized vWF. This effect of shear stress was observed regardless of the manner in which vWF was immobilized, suggesting that immobilization itself and not, as previously hypothesized, a conformational change in vWF induced by direct adsorption to the surface is responsible for the enhanced GPIb binding. Thus, the present findings suggest that the vWF- GP Ib interaction contributes optimally to rapid platelet cohesion on a thrombogenic surface when vWF is in a static state and when platelets are moved by an appropriate rheological force such as low shear stress.     

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.