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Abstract: Treatment options for the bleeding disorder von Willebrand disease type 2B (VWD2B) are insufficient and fail to address the negative effects of circulating mutant von Willebrand factor (VWF). The dominant-negative nature of VWD2B makes functionally defective VWF an interesting therapeutic target. Previous in vitro studies have demonstrated the feasibility of allele-selective silencing of mutant VWF using small interfering RNAs (siRNAs) targeting common single nucleotide polymorphisms (SNPs) in the human VWF gene, an approach that can be applied irrespective of the disease-causing VWF mutation. This study aims to extend this concept to a heterozygous VWD2B mouse model (c.3946G>A; p.Val1316Met) here using mouse strain-specific genetic differences as proxy for human SNPs. Homozygous VWD2B C57BL/6J (2B-B6) mice were crossed with homozygous wild-type 129S1/SvImJ (129S) mice to create heterozygous 2B-B6.129S F1 offspring. These 2B-B6.129S mice were intravenously injected with endothelial-specific lipid nanoparticles loaded with the allele-selective siVwf.B6 or control and 96 hours later, lung Vwf messenger RNA, plasma VWF levels, and phenotypic characteristics were evaluated. Treatment with siVwf.B6 reduced total VWF levels by 50%, with an expected selective reduction in mutant VWF protein. This coincided with normalization of multimeric structure, improved VWF collagen binding/VWF antigen ratio, and normalized bleeding times in two-thirds of heterozygous 2B-B6.129S mice. Being a novel approach in the field of hemostasis, we proved, for VWD, in mice, the concept of selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single nucleotide variation rather than the disease-causing mutation. For dominant-negative VWD, this offers potential for a customized therapeutic strategy., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Endothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease., Objectives: Therefore, we aimed to determine phenotypic and gene expression differences between control ECFCs., Methods: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed., Results: Through hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel-Palade bodies. Migration speed in cluster 1 was also significantly higher., Conclusion: We observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel-Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact., Materials and Methods: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding., Results: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times., Conclusions: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: YKJ and JCJE – Dutch Thrombosis Foundation (TSN) grant #2018-01. NAL and JCJE – Netherlands Organization for Scientific Research (NWO) NWO-AES grant #18712. SNJL and JCJE – Netherlands Organization for Scientific Research (NWO) NWA-ORC grant #1160.18.038. JCJE – CSL Behring research funding. RJD, ESE, JED, BJMvV, CVD declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An imbalance in von Willebrand factor (VWF) may either lead to bleeding (von Willebrand disease, VWD) or thrombosis. Both disorders have shortcomings in the currently available treatments. VWF itself could be a potential therapeutic target because of its role in both bleeding and thrombosis. Inhibiting VWF gene expression through allele-selective silencing of VWF with small interfering RNAs (siRNAs) could be a personalized approach to specifically inhibit mutant VWF in VWD or to normalize increased VWF levels in thrombotic disorders without complete VWF knockdown. Therefore, we investigated a method to allele-selectively silence the VWF gene in mice as a therapeutic strategy. Fourteen candidate siRNAs targeting murine Vwf of either the C57BL/6J (B6) or the 129S1/SvImJ (129S) strain were tested in vitro in cells expressing B6- and 129S-Vwf for inhibitory effect and allele-selective potential. Together with a nonselective siVwf, 2 lead candidate siRNAs, siVwf.B6 and siVwf.129S, were further tested in vivo in B6 and 129S mice. Efficient endothelial siRNA delivery was achieved by siRNA encapsulation into 7C1 oligomeric lipid nanoparticles. Treatment with the nonselective siVwf resulted in dose-dependent inhibition of up to 80% of both lung messenger RNA and plasma VWF protein in both mouse strains. In contrast, the allele-selective siVwf.B6 and siVwf.129S were shown to be effective in and selective solely for their corresponding mouse strain. To conclude, we showed efficient endothelial delivery of siRNAs that are highly effective in allele-selective inhibition of Vwf in mice, which constitutes an in vivo proof of principle of allele-selective VWF silencing as a therapeutic approach., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell. One thus needs to analyze large numbers of organelles for valid quantification. Properly evaluating these parameters requires an automated, unbiased method to process and quantitatively analyze microscopy data. Here, we describe two pipelines, run by CellProfiler software, called OrganelleProfiler and OrganelleContentProfiler. These pipelines were used on confocal images of endothelial colony forming cells (ECFCs), which contain unique secretory organelles called Weibel-Palade bodies (WPBs), and on early endosomes in ECFCs and human embryonic kidney 293T (HEK293T) cells. Results show that the pipelines can quantify the cell count, size, organelle count, organelle size, shape, relation to cells and nuclei, and distance to these objects in both endothelial and HEK293T cells. Additionally, the pipelines were used to measure the reduction in WPB size after disruption of the Golgi and to quantify the perinuclear clustering of WPBs after triggering of cAMP-mediated signaling pathways in ECFCs. Furthermore, the pipeline is able to quantify secondary signals located in or on the organelle or in the cytoplasm, such as the small WPB GTPase Rab27A. Cell profiler measurements were checked for validity using Fiji. To conclude, these pipelines provide a powerful, high-processing quantitative tool for the characterization of multiple cell and organelle types. These pipelines are freely available and easily editable for use on different cell types or organelles., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Laan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF . The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes., Competing Interests: J.B. received research funding from CSL Behring and is an employee of Sobi. F.A. received research funding from CSL Behring and a travel grant from Sobi. F.W.G.L received research funding from CSL Behring, Takeda/Shire, and uniQure. He is member of a DSMB for Roche. He is consultant for Takeda, Biomarin, and uniQure of which fees go to the institution. J.E. received research funding from CSL Behring. J.E. reports grants from Landsteiner Foundation for Blood Transfusion Research, during the conduct of the study.other authors declare no conflicts of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion., (© 2020 by The American Society of Hematology.)

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., (© 2018 International Society on Thrombosis and Haemostasis.)

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., (© 2015 International Society on Thrombosis and Haemostasis.)

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., (© 2015 International Society on Thrombosis and Haemostasis.)

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion., (© 2014 John Wiley & Sons Ltd.)

During posttranslational modifications of von Willebrand factor (VWF), the VWF propeptide (VWFpp) is cleaved. The ratio between VWFpp and VWF antigen (VWF:Ag) and the ratio between factor VIII (FVIII:C) and VWF:Ag may be used to assess synthesis and clearance of VWF. We analyzed the contribution of VWFpp and ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag in the pathophysiological characterization of type 1 von Willebrand disease (VWD) in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD) study. The VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were increased among patients compared with unaffected family members and healthy controls. The VWFpp/VWF:Ag ratio was higher in individuals heterozygous for missense mutations than in those heterozygous for null alleles. In contrast, the FVIII:C/VWF:Ag ratio was highest among heterozygotes for VWF null alleles. The ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag indicate that the pathophysiological mechanisms of type 1 VWD include reduced production and accelerated clearance of VWF, but that often a combination of both mechanisms is implicated.

Background: Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies., Design and Methods: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate., Results: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser., Conclusions: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.

The procoagulant function of activated factor V (FVa) is inhibited by activated Protein C (APC) through proteolytic cleavages at R306, R506 and R679. Recombinant FVa mutated at all three APC-cleavage sites, FVa-GQA, was still inactivated by APC through at least two cleavages in the heavy chain of FVa; relatively rapid cleavage at R(x1) close to residue 506 and slower cleavage at R(x2) nearby residue 306. We investigated the exact location of these two cleavages, by substitution of arginines by glutamine within the R(x1)-region (R501, R505 or R510) and the R(x2)-region (R313, R316, R317 or R321). Immunoblot and kinetic analyses of the inactivation of activated R(x1)-mutants by APC revealed that using mutant FVa-GQA-505Q no R(x2)-R(x1) fragment was formed and that the inactivation reaction was first order with a rate constant of 1.0 x 10(4) M(-1) s(-1), similar to the rate constant of R(x2) cleavage (k(2)=1.3 x 10(4) M(-1) s(-1)). No single arginine could be pinpointed identified as R(x2). Individual replacement of arginine by glutamine at positions 313, 316, 317 or 321 in FV-GQA-505Q did not result in the disappearance of R(x2) as judged from kinetic and immunoblot analyses. However, replacement of all four arginines by glutamine completely prevented formation of the R(x2)-R(709) fragment. We conclude that substitution of arginine 506 by glutamine as in FV-Leiden, leads to the detection of a novel cleavage site at arginine 505 (R(x1)). Substitution of arginine 306 by glycine, like in FV-Cambridge, reveals several alternative cleavage sites near arginine 306, which together constitute a secondary cleavage site., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

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.

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.

The R2 haplotype of the FV gene spans from exon 8 through 25 and comprises several strongly linked polymorphisms in the FV gene, including some missense mutations. Carriership of the R2-FV allele has been associated with reduced plasma FV levels, increased FV1/FV2 ratios and mild APC resistance. Some studies have reported that carriership of the R2-FV allele is associated with an increased risk of venous thombosis. At this moment, the individual contribution to the R2-associated phenotypes of the different mutations linked to the R2 haplotype of FV is unclear. The main objective of our study was to obtain insight in the influence of the R2-related Asp2194Gly mutation on FV expression, FV structure and FV function using Bdomainless rFV mutants. Replacing Asp at position 2194 by Gly resulted in a more than threefold reduction of rFV expression compared to rFV wild-type. Therefore, we propose that the R2-linked Asp2194Gly mutation is an important determinant of the association of the R2-FV allele with lower FV levels. Furthermore, the light chains from Asp2194Gly containing rFV mutants showed similar molecular weights as the light chains of the non-glycosylated rFVwt or the plasma FV2 isoform, indicating that glycosylation at Asn2181 is not stimulated by the presence of a glycine in position 2194. Finally, the apparent K(d) for dissociation of the FXaVa complex (K(1/2Xa)) was not higher in rFV mutants with the Asp2194Gly mutation than for rFVwt, suggesting that also the affinity for negatively charged phospholipids is not affected by substitution of Asp into Gly at position at 2194.

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.

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.

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.