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.

Angiogenic characteristics of blood outgrowth endothelial cells from patients with von Willebrand disease.

BACKGROUND: Endothelial von Willebrand factor (VWF) inhibits angiogenesis. Accordingly, blood outgrowth endothelial cells (BOECs) isolated from von Willebrand disease (VWD) patients showed enhanced in vitro angiogenesis when compared with healthy control BOECs. Characterization of the angiogenic response of VWD BOECs is limited and differences between the different types of VWD have not been investigated in detail. OBJECTIVES: The aim of this study was to further explore the potential pathogenic effect of VWF mutations on angiogenesis. METHODS: BOECs were isolated from four healthy individuals, 10 patients with VWD and one heterozygous carrier of a type 2N mutation. Cell migration and tube formation were measured. RESULTS: Migration velocity and total tube formation were similar between VWD patients and controls in general. BOECs from the type 3 VWD patient and one type 2B patient showed increased migratory velocity and tube formation compared with BOECs from other patients and healthy controls. Directional migration was impaired in eight out of 10 VWD BOECs and the ability to form tubes was limited to early passage numbers, but not for BOECs from healthy controls. CONCLUSION: BOECs can be a useful tool for ex vivo assessment of endothelial cell function in patients with different types of VWD, but possible limitations, such as early loss of angiogenic capacity, should be recognized. BOECs from most VWD patients consistently showed impairment in the directionality of migration. This is the first report on angiogenic properties of a type 3 VWD BOEC, which showed increased in vitro angiogenesis.

No evidence for a direct effect of von Willebrand factor's ABH blood group antigens on von Willebrand factor clearance.

BACKGROUND: One of the major determinants of von Willebrand factor (VWF) plasma levels is ABO blood group status, and individuals with blood group O have ~ 25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated. OBJECTIVES: To determine whether clearance of VWF is directly dependent on the presence of ABH antigens on VWF. METHODS: Three type 3 von Willebrand disease (VWD) patients were infused with Haemate-P, and the relative loading of VWF with ABH antigens at different time points was measured. VWF-deficient mice were injected with purified plasma-derived human VWF obtained from donors with either blood group A, blood group B, or blood group O. RESULTS: In mice, we found no difference in clearance rate between plasma-derived blood group A, blood group B and blood group O VWF. Faster clearance of the blood group O VWF present in Haemate-P infused in type 3 VWD patients would have resulted in a relative increase in the loading of VWF with A and B antigens over time. However, we observed a two-fold decrease in the loading with A and B antigens in two out of three patients, and stable loading in the third patient. CONCLUSION: There is no direct effect of ABH antigens on VWF in VWF clearance. We demonstrate that, in a direct comparison within one individual, blood group O VWF is not cleared faster than blood group A or blood group B VWF. Clearance differences between blood group O and non-blood group O individuals may therefore be related to the blood group status of the individual rather than the ABH antigen loading on VWF itself.

Characterization of an immunologic polymorphism (D79H) in the heavy chain of factor V.

BACKGROUND: During the study of a family with hereditary factor (F)V deficiency (FV Amersfoort, 1102 A > T in exon 7) we identified an individual with 5% FV heavy chain antigen (FV(HC)) and 50% FV light chain antigen (FV(LC)). Further testing revealed that apart from the FV Amersfoort allele a second variant FV allele was segregating in this family, which encodes for a FV molecule with a reduced affinity for mAb V-23 used in the FV heavy chain ELISA (ELISA(HC)). OBJECTIVE: Identification and characterization of the molecular basis responsible for the reduced affinity of the variant FV for mAb V-23. METHODS: Family members of the proband were screened for mutations in the exons coding for the heavy chain of FV, after which the recombinant variant FV could be generated and characterized. Next, the cases and controls of the Leiden Thrombophilia Study (LETS) were genotyped for carriership of the variant FV. RESULTS: In the variant FV allele a polymorphism in exon 3 (409G > C) was identified, which predicts the replacement of aspartic acid 79 by histidin (D79H). Introduction of this mutation in recombinant FV confirmed that it reduces the affinity for binding to mAb V-23. The substitution has no effect on FV(a) stability and Xa-cofactor activity. In Caucasians the frequency of the FV-79H allele is approximately 5%. Analysis of the LETS revealed that the FV-79H allele is not associated with FV levels (FV(LC)), activated protein C sensitivity (using an activated partial thromboplastin time-based test) or risk of venous thrombosis (OR 1.07, CI 95: 0.7-1.7). CONCLUSION: The D79H substitution in FV should be considered as a neutral polymorphism. The monoclonal antibody V-23, which has a strongly reduced affinity for FV-79H, is not suitable for application in diagnostic tests.

The activated protein C (APC)-resistant phenotype of APC cleavage site mutants of recombinant factor V in a reconstituted plasma model.

Recently, new missense mutations in the activated protein C (APC) cleavage sites of human factor V (FV) distinct from the R506Q (FV Leiden) mutation have been reported. These mutations affect the APC cleavage site at arginine (Arg) 306 in the heavy chain of activated FV. Whether these mutations result in APC resistance and are associated with a risk of thrombosis is not clear. The main objective of the present study was to identify the APC-resistant phenotype of FV molecules with different mutations in APC cleavage sites. To study this, recombinant FV mutants were reconstituted in FV-deficient plasma, after which normalized APC-sensitivity ratios (n-APC-SRs) were measured in activated partial thromboplastin time-based and Russell's Viper Venom time-based APC-resistance tests. The mutations introduced in FV were R306G, R306T, R506Q, R679A and combinations of these mutations. Based on the APC-sensitivity ratios, we conclude that the naturally occurring mutations at Arg306 (i.e. FV HongKong and FV Cambridge) result in a mildly reduced sensitivity for APC (n-APC-SR, 0.74-0.87), whereas much lower values (n-APC-SR, 0.41-0.51) are obtained for the mutation at Arg506 (FV Leiden). No effect on the n-APC-SR was observed for the recombinant FV mutant containing the single Ala679 mutation. Because reduced sensitivity for APC, not due to FV Leiden, is a risk factor for venous thrombosis, these data suggest that mutations at Arg306 might be associated with a mild risk of venous thrombosis.

Mutation in blood coagulation factor V associated with resistance to activated protein C.

Activated protein C (APC) is a serine protease with potent anticoagulant properties, which is formed in blood on the endothelium from an inactive precursor. During normal haemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa (ref. 2). To do this efficiently the enzyme needs a nonenzymatic cofactor, protein S (ref. 3). Recently it was found that the anticoagulant response to APC (APC resistance) was very weak in the plasma of 21% of unselected consecutive patients with thrombosis and about 50% of selected patients with a personal or family history of thrombosis; moreover, 5% of healthy individuals show APC resistance, which is associated with a sevenfold increase in the risk for deep vein thrombosis. Here we demonstrate that the phenotype of APC resistance is associated with heterozygosity or homozygosity for a single point mutation in the factor V gene (at nucleotide position 1,691, G-->A substitution) which predicts the synthesis of a factor V molecule (FV Q506, or FV Leiden) that is not properly inactivated by APC. The allelic frequency of the mutation in the Dutch population is approximately 2% and is at least tenfold higher than that of all other known genetic risk factors for thrombosis (protein C (ref. 8), protein S (ref. 9), antithrombin10 deficiency) together.

Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacterial lipopolysaccharide and phorbolester.

The monocyte is the only circulating cell type capable of initiating blood coagulation by the expression of tissue factor (TF). The mechanism and kinetics of TF mRNA and TF activity induction in human peripheral blood monocytes (HPBM) in response to bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) were investigated. Northern blot analysis showed that both LPS and PMA induce a transient accumulation of TF mRNA in HPBM, that reaches maximum levels after 3-6 h and rapidly declines thereafter. Nuclear run-on experiments demonstrated that the accumulation of TF mRNA requires de novo transcription of the TF gene. Since cycloheximide alone also caused an increase of TF mRNA levels and gene transcription it is concluded that the transcriptional activation of the TF gene does not require protein synthesis. Using specific protein kinase inhibitors, it was further demonstrated that activation of the protein kinase C pathway is involved in the induction of TF mRNA in HPBM. The accumulation of TF mRNA in LPS-stimulated HPBM is followed by an increase of TF activity on the cell surface. The kinetics of TF mRNA induction were found to be very similar in HPBM stimulated with LPS or PMA. However, in the latter case TF activity appeared considerably later on the cell membrane than in the LPS-stimulated cells. Non-stimulated HPBM contain very low levels of mRNA of the tissue factor pathway inhibitor (TFPI). No induction of TFPI (mRNA, activity or antigen) in HPBM after LPS or PMA treatment was demonstrated. This seems to be in contrast with the earlier observation that the human monocyte cell line U937 produces significant amounts of TFPI in response to treatment with LPS and PMA.