Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit.

In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers of increasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in alpha-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule.     

Preclinical evaluation of a semi‐automated and rapid commercial electrophoresis assay for von Willebrand factor multimers

Background

The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time‐consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same‐day results.

Methods

Ten healthy volunteer plasma samples, in‐house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C).

Results

The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion.

Conclusions

The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second‐stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

     

Accelerated clearance alone explains ultra‐large multimers in von Willebrand disease Vicenza

See also Goodeve AC. Vicenza deciphered: modeling the von Willebrand disease enigma: commentary on accelerated clearance alone explains ultralarge multimers in VWD Vicenza. This issue, pp 1271–2. Summary. Background: von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra‐large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. Objectives: We addressed the question, whether the presence of ultra‐large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. Patients/methods: We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF‐R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. Results: We found that the half‐life of VWF after DDAVP was approximately one‐tenth of that after the administration of Haemate P, a source of exogenous wild‐type (WT) VWF (0.81 ± 0.2 vs. 7.25 ± 2.38 h). An analysis of recombinant mutant VWF‐R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half‐life to one‐tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra‐large multimers and a decrease of satellite band intensity. Conclusion: We conclude that accelerated clearance alone may explain all features of VWD Vicenza.     

Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Essentials

• Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered.
• Presence of mutant VWF may negatively affect phenotypes despite treatment.
• Inhibition of VWF by allele‐specific siRNAs targeting single‐nucleotide polymorphisms is effective.
• Allele‐specific inhibition of VWF p.Cys2773Ser improves multimerization.

Summary

Background

Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF‐containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant‐negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes.

Objectives

To investigate allele‐specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single‐nucleotide polymorphisms (SNPs) in VWF. This approach allows allele‐specific knockdown irrespective of the mutations causing VWD.

Methods

Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA‐mediated phenotypic improvements were determined in VWF‐expressing HEK293 cells.

Results

Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA‐mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser.

Conclusions

Allele‐specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele‐specific siRNAs in dominant‐negative VWD.

     

Multi‐step binding of ADAMTS‐13 to von Willebrand factor

Summary. Background: ADAMTS‐13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non‐catalytic domains in ADAMTS‐13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS‐13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS‐13. Methods: Immunoprecipiation of VWF–ADAMTS‐13 complexes using anti‐VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co‐immunoprecipitation demonstrated that ADAMTS‐13 binds to native multimeric VWF (K_d of 79 ± 11 nmol L^?1) with no measurable proteolysis. Upon shear‐induced unfolding of VWF, binding increased 3‐fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS‐13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS‐13 spacer, probably thrombospondin‐1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS‐13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS‐13 binds to circulating VWF and may therefore be incorporated into a platelet‐rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.     

Increased anticoagulant response to low‐molecular‐weight heparin in plasma from patients with advanced cirrhosis

Summary. Introduction: Cirrhotic patients may present thrombotic complications that warrant anticoagulant therapy. However, the efficacy of low‐molecular‐weight heparin (LMWH) in this clinical setting is still unclear. Aims/methods: To evaluate the in vitro effect of LMWH on thrombin generation (TG) in cirrhotic patients at different stages of liver disease. Thirty cirrhotics (10 Child Pugh A, 10 Child Pugh B and 10 Child Pugh C), 10 subjects with inherited type 1 antithrombin (AT) defect and 10 healthy controls were studied. TG was determined at baseline and with anti‐Xa levels after the addition of enoxaparin at 0.35 and 0.7 U anti‐Xa mL. The endogenous thrombin potential (ETP) ratio at 0.35 and 0.7 U anti‐Xa mL was obtained by dividing ETP with LMWH by ETP at baseline. Results: Mean AT levels in all cirrhotic subgroups and in patients with AT deficiency were significantly lower than in controls. The 0.35 ETP ratio was significantly lower in cirrhotic patients than in controls (0.26 ± 0.1 vs. 0.48 ± 0.1, P < 0.001) and the reduction paralleled the severity of liver disease, in spite of the concomitant decrease in AT and anti‐Xa activity. AT‐deficient subjects showed a significantly increased 0.35 ETP ratio compared with both cirrhotic patients and controls (0.69 ± 1 vs. 0.26 ± 0.1, P < 0.001, and vs. 0.48 ± 0.1, P = 0.04 respectively). LMWH at 0.7 U anti‐Xa mL completely inhibited TG in 9/30 cirrhosis patients with more advanced liver disease (Child Pugh B and C), whereas complete TG abolition was seen in only 1/10 controls. Conclusions: Cirrhotic patients show an increased response to LMWH, which correlates with the severity of liver disease, in spite of reduced AT and anti‐Xa activity levels. Thrombin generation may be a useful tool to monitor the response to LMWH in cirrhotic patients.     

A novel type 2A (Group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF) associated with an absence of high-molecular-weight multimers. In this study, sequence analysis of the VWF gene from a Type 2A VWD patient showed a novel, heterozygous T-->A transversion at nucleotide 4510, resulting in the non-conservative substitution of L1503Q in the mature VWF subunit. This substitution, which was not found in 55 unrelated normal individuals, was reproduced by in vitro site directed mutagenesis of a full-length VWF cDNA and was subsequently expressed in COS-7 cells. The corresponding recombinant mutant VWF protein was partially retained in COS-7 cells yet the full spectrum of multimers was observed, suggesting that the absence of the highest molecular weight multimers results from increased proteolysis. The recombinant mutant VWF protein was digested with the ADAMTS13 protease from VWF-depleted plasma and the aberrant VWF multimer pattern was observed. These results suggest that the L1503Q substitution induces a conformational change in the VWF protein, which increases the protein's susceptibility to proteolysis. A three-dimensional model of the A2 domain demonstrates that the L1503Q mutation and the physiological proteolytic cleavage site for ADAMTS13 (Y(1605)-M(1606)) are localized close together in two adjacent parallel beta-sheets. The mutation L1503Q does not significantly disrupt the conformation of the protein; thus the subtle loss of multimers in this patient may be due to altered interactions with the ADAMTS13 protease.     

Inhibition of von Willebrand factor-induced platelet agglutination by ADP does not result from reduced binding of total von Willebrand factor or its larger multimers

Earlier experiments showed that platelet agglutination induced by von Willebrand factor (vWf) plus ristocetin was greatly diminished if adenosine diphosphate (ADP) was added first in the presence of ethylenediaminetetraacetic acid (to prevent aggregation). Platelets treated with ADP and then fixed also agglutinated less than control fixed platelets. The studies reported here demonstrate that ADP did not decrease ristocetin-induced binding of vWf whether binding was measured on suspended platelets with iodine 125-labeled vWf or on suspended or agglutinated platelets with the use of any of three 125I-labeled monoclonal antibodies that bind to vWf but that do not interfere with ristocetin-induced agglutination. Equal amounts of vWf were eluted from ristocetin/vWf-treated platelets when they were resuspended without ristocetin, whether or not the platelets had been exposed to ADP, and the vWf recovered in either case was composed only of large multimers. No evidence for an agglutination site other than glycoprotein Ib could be demonstrated by measuring agglutination of a mixture of platelets fixed after inhibition with antibody against glycoprotein Ib and platelets fixed after inhibition with ADP. We conclude that inhibition of agglutination by ADP must involve the way in which vWf is bound, because it does not result from a decreased amount or from a difference in multimer size of bound vWf.     

ADAMTS‐13: double trouble for von Willebrand factor

See also Yeh HC, Zhou Z, Choi H, Tekeoglu S, May W III, Wang C, Turner N, Scheiflinger F, Moake JL, Dong JF. Disulfide bond reduction of von Willebrand factor by ADAMTS‐13. This issue, pp 2778–88.