Requirements for cellular co-trafficking of factor VIII and von Willebrand factor to Weibel–Palade bodies

Background:  von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. Objectives:  In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. Methods:  Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. Results:  Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding ( K _d = 0.065 ± 0.008 n m ) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel–Palade bodies (WPBs). Conclusions:  Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.     

Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype

Summary. Background: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII‐binding site of von Willebrand factor (VWF), located in the D′and D3 domains on the N‐terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. Objectives: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. Methods: The plasma VWF phenotype was studied, transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild‐type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel–Palade body‐like granules when transfected with WT VWF. Results: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel–Palade body‐like granules. Conclusions: Our results demonstrate that a homozygous R854W mutation in the D′ domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.     

Shear stress is required for the endocytic uptake of the factor VIII‐von Willebrand factor complex by macrophages

Summary. Background: Low‐density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. Objectives: It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte‐derived macrophages. Methods: A confocal microscopy study was employed to study the uptake by monocyte‐derived macrophages of a functional green fluorescent FVIII‐GFP derivative in the presence and absence of VWF. Results: The results revealed that FVIII‐GFP is internalized by macrophages. We found that FVIII‐GFP co‐localizes with LDL receptor‐related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII‐GFP. However, FVIII‐GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII‐VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII‐GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII‐GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII‐GFP and VWF was inhibited. Conclusion: The results show that shear stress is required for macrophages to internalize both constituents of the FVIII‐VWF complex.     

Formation of platelet‐binding von Willebrand factor strings on non‐endothelial cells

Summary. Background and Objective: Von Willebrand factor (VWF) forms strings on activated vascular endothelial cells that recruit platelets and initiate clot formation. Alterations in VWF strings may disturb hemostasis. This study was aimed at developing a flexible model system for structure–function studies of VWF strings. Methods: VWF strings were generated by inducing exocytosis of pseudo‐Weibel–Palade bodies from VWF‐transfected HEK293 cells, and the properties of these strings under static conditions and under flow were characterized. Results: Upon exocytosis, VWF unfurled into strings several hundred micrometers in length. These strings could form bundles and networks, and bound platelets under flow, resembling authentic endothelial VWF strings. Anchorage of the platelet‐decorated VWF strings was independent of P‐selectin and integrin α_Vβ₃. Translocation of platelets along the strings, elongation and fragmentation of the strings frequently occurred under flow. Furthermore, VWF variants with the p.Tyr87Ser and p.Cys2773Ser mutations, which are defective in multimer assembly, did not give rise to VWF strings. Also, insertion of the green fluorescent protein into VWF inhibited string formation. Conclusions: HEK293 cells provide a flexible and useful model system for the study of VWF string formation. Our results suggest that structural changes in VWF may modulate string formation and function, and contribute to hemostatic disorders.     

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII

Summary.  von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within α-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet α-granules. Isolex^® selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the α-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2–6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.     

Reduced von Willebrand factor secretion is associated with loss of Weibel–Palade body formation

Background:  von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. Objectives:  We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel–Palade body (WPB)‐like granules. Methods:  Transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild‐type VWF. Expression was also examined in HEK293 cells that form WPB‐like granules when transfected with wild‐type VWF. Results:  Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB‐like granules, while R2663C was capable of WPB‐like granules. Heterozygous expression of VWF variants had a negative impact on wild‐type VWF with a reduction in elongated WPB‐like granules in co‐transfected cells. Conclusions:  Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB‐like granules.     

Lack of desmopressin (DDAVP) response in men with hemophilia A following liver transplantation

Summary.  Although hemophilia A, a congenital disorder caused by defective or deficient factor VIII:C (FVIII), is cured by liver transplantation, the exact site of hepatic FVIII production is unknown. Further, while intracellular co-localization of FVIII and von Willebrand factor (VWF) is required for in vitro FVIII secretion, whether it is required for in vivo FVIII secretion is not known. An ideal setting to study this problem is in individuals with hemophilia A following liver transplantation, as their FVIII is synthesized primarily in hepatic, but not extrahepatic endothelial cells, while VWF is synthesized primarily in extrahepatic vascular endothelium. Following liver transplantation for end-stage liver disease, three hemophilic men showed VWF, but no FVIII response to (DDAVP) infusion. By contrast, both VWF and FVIII increased in a non-hemophilic transplant recipient after DDAVP. These findings support a model in which intracellular co-localization of FVIII and VWF is necessary for in vivo FVIII secretion after DDAVP.     

Characterization of the interaction between von Willebrand factor and osteoprotegerin

BACKGROUND AND OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. METHODS AND RESULTS: In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. CONCLUSIONS: Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.     

Heme binds to factor VIII and inhibits its interaction with activated factor IX

Summary. Background: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results: Heme bound to several sites on FVIII with high apparent affinity. Heme‐binding inhibited FVIII procoagulant activity in a dose‐dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme‐mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme‐binding protein known for its protective activity in hemolytic conditions. Conclusions: Our data identify FVIII as a novel heme‐binding protein. Occupation of high affinity heme‐binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme‐mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.     

Standardization of factor VIII and von Willebrand factor in plasma: calibration of the WHO 5th International Standard (02/150).

Calibration of the 5th International Standard factor (F)VIII/von Willebrand factor in plasma (02/150) (5th IS) for five parameters [factor VIII: coagulant activity (FVIII:C); FVIII: antigen (FVIII:Ag), von Willebrand factor: antigen (VWF:Ag), von Willebrand factor: ristocetin cofactor (VWF:RCo), von Willebrand factor: collagen binding (VWF:CB)] was achieved through an international collaborative study involving 37 laboratories. Estimates calculated relative to the previous 4th IS and locally prepared normal plasma pools were not significantly different for estimates of FVIII:Ag, VWF:Ag, VWF:RCo and VWF:CB and hence mean values calculated relative to the 4th IS of 0.94, 0.91, 0.78 and 0.94 IU ampoule(-1), respectively, were assigned. However, estimates for FVIII:C relative to the fresh normal pools (mean 0.61 IU ampoule(-1)) were significantly lower than estimates relative to the 4th IS (mean 0.68 IU ampoule(-1)). In consideration of the good stability of FVIII:C in the 4th IS and the variability of estimates relative to the local pools it was agreed to assign the mean value obtained relative to the 4th IS of 0.68 IU ampoule(-1). For all five parameters the interlaboratory variability (geometric coefficient of variation, GCV%) was larger for estimates calculated relative to the normal pools (range 12.6-16.5%) when compared with estimates calculated relative to the 4th IS (range 3.5-8.3%). An accelerated degradation study performed in six laboratories indicated that the five calibrated parameters are extremely stable when ampoules are stored at -20 degrees C. Mean estimates of predicted loss per year at -20 degrees C ranged from 0% for VWF:CB to 0.029% for VWF:RCo. The 5th IS (02/150) was established by the World Health Organization in November 2003.     

Type 2M von Willebrand disease variant characterized by abnormal von willebrand factor multimerization

We describe a von Willebrand disease (VWD) variant characterized by low plasma and platelet von Willebrand factor (VWF), impaired ristocetin-induced VWF binding to platelet glycoprotein Ib (GPIb), and abnormal VWF multimer pattern not associated with the absence of large forms. A C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene was found; this mutation introduces a cysteine at the codon for Arg 611 of mature VWF. In addition to the decreased factor VIII (FVIII) and VWF levels, ristocetin-induced platelet aggregation (RIPA) was almost absent, and VWF ristocetin cofactor activity (VWF:RCo) was significantly more decreased than VWF antigen. The patients (mother and son) also showed a defect in VWF collagen-binding activity. Plasma VWF multimers were decreased, with no limit in the size of large forms, and the normal discontinuous multimer organization was replaced by a diffuse smear, especially detectable in the large forms. This picture was emphasized by 1-deamino-8-D -arginine vasopressin (DDAVP) infusion, so that the abnormal VWF multimers appeared to have a molecular weight higher than those present in, or released by, human umbilical vein endothelial cells. DDAVP also increased FVIII and VWF levels but did not normalize the GPIb-dependent VWF functions expressed as RIPA and VWF:RCo. We include this variant in type 2M VWD, focusing on the abnormality in GPIb-dependent VWF function. We advance that this defect depends on the mutation in the GPIb binding domain of VWF rather than the abnormal VWF multimer pattern.     

L-精氨酸对人凝血因子Ⅷ基因表达的影响

目的研究L-精氨酸对人凝血因子Ⅷ（FⅧ）基因转录及表达的影响。方法将B结构域（氨基酸760～1639）缺失的人FⅧcDNA（BDDhFⅧcDNA）插入至质粒载体pcDNA6／V5-HisA，构建重组载体pcDNA6／V5-HisA-BDDhFⅧ，转染人脐静脉内皮细胞（HUVEC），加入L-精氨酸（终浓度10mmol／L）培养72h后，用ELISA方法检测细胞上清液中的人FⅧ抗原（FⅧ：Ag），一期凝血酶法检测FⅧ的促凝活性（FⅧ：C）。用Northern blot检测HUVEC中人FⅧ基因的转录。用PCR扩增BDDhFⅧcDNA的A1、A2、A3、C1和C2结构域基因，分别插入至pcDNA6／V5-HisA，构建五种载体pcDNA6／V5-HisA-BDDhFⅧ-A1、pcDNA6／V5-BDDhF Ⅷ-A2、pcDNA6／V5-HisA-BDDhF Ⅷ-A3、pcDNA6／V5-HisA-BDDhF Ⅷ-C1和pcDNA6／V5-HisA-BDDhF Ⅷ-C2，分别转染HUVEC后，加入L-精氨酸（终浓度10mmol／L）培养72h。膨胀裂解法分离细胞核，进行细胞核连缀（Run-on）反应，狭线印迹杂交检测A1、A2、A3、C1和C2结构域基因的转录。结果经L-精氨酸诱导后，HUVEC中人FⅧ基因表达明显增强[FⅧ：Ag为（146．08±4．78）ng／ml，10^6细胞诱导24h后FⅧ：C为（0．752±0．009）U／ml]，约是未经L-精氨酸诱导[FⅧ：Ag为（34．66±3．98）ng／ml，10^6细胞诱导24h后FⅧ：C为（0．171±0．006）U／ml]的4倍（P〈0．01）。Northern blot检测显示，加L-精氨酸培养之后，HUVEC中人BDDFⅧ mRNA的转录也较未加L-精氨酸显著增强，而只转染pcDNA6／V5-HisA的HUVEC中无人BDDFⅧ mRNA的转录存在。Run-on及狭线印迹杂交显示，加L-精氨酸培养后，A1和A2结构域基因的转录均较未经L-精氨酸诱导者明显增强，也显著强于A3、C1和C2结构域基因的转录，而A3、C1和C2结构域基因的转录在L-精氨酸诱导前后均无明显变化。结论L-精氨酸通过促进人FⅧ的A1和A2结构域基因的转录进而增强FⅧ在体外的转录和表达，因而在血友病A的基因治疗研究中，L-精氨酸是一个很有前途的诱导FⅧ转录和表达的物质。     

Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.     

von Willebrand factor variant p.Arg924Gln marks an allele associated with reduced von Willebrand factor and factor VIII levels

Summary. Background: von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies. Objectives: To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD. Methods: One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM‐1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells. Results: c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843‐8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co‐inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF‐FVIII binding and normal clearance of VWF and FVIII. Conclusions: The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels.     

Weibel–Palade bodies: a window to von Willebrand disease

Weibel–Palade bodies (WPBs) are the storage organelles for von Willebrand factor (VWF) in endothelial cells. VWF forms multimers that assemble into tubular structures in WPBs. Upon demand, VWF is secreted into the blood circulation, where it unfolds into strings that capture platelets during the onset of primary hemostasis. Numerous mutations affecting VWF lead to the bleeding disorder von Willebrand disease. This review reports the recent findings on the effects of VWF mutations on the biosynthetic pathway of VWF and its storage in WPBs. These new findings have deepened our understanding of VWF synthesis, storage, secretion, and function.     

Factor VIII inhibitors: von Willebrand factor makes a difference in vitro and in vivo

Summary. Background: The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on the reactivity of FVIII inhibitory antibodies, referred to as inhibitors, is still controversial. Objective: To investigate the interaction among VWF, FVIII and FVIII inhibitory antibodies. Methods: Three sources of inhibitors were used for in vitro studies, including the plasma from immunized VWF^nullFVIII^null mice, purified plasma IgG from human inhibitor patients, or human monoclonal antibody from inhibitor patients’ B‐cell clones. Inhibitors were incubated with recombinant human FVIII (rhFVIII) either with or without VWF. The remaining FVIII activity was determined by chromogenic assay and inhibitor titers were determined. For in vivo studies, inhibitors and rhFVIII were infused into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Results: VWF has a dose‐dependent protective effect on FVIII, limiting inhibitor inactivation of FVIII in both mouse and human samples. A preformed complex of VWF with FVIII provides more effective protection from inhibitors than competitive binding of antibodies and VWF to FVIII. The protective effect of VWF against FVIII inactivation by inhibitors was further confirmed in vivo by infusing inhibitors and FVIII into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Conclusion: Our results demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo.     

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels

Summary. Objectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain‐of‐function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. Methods and Results: We genotyped the control populations of two population‐based studies for four AVPR2 variations: a‐245c, G12E, L309L, and S331S. Rare alleles of a‐245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G‐V2R or 12E‐V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three‐fold in 12E‐V2R–green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (K_D) was 4.5 nm [95% confidence interval (CI) 3.6–5.4] for 12E‐V2R–GFP and 16.5 nm (95% CI 10.1–22.9) for 12G‐V2R–GFP. AVP‐induced cAMP generation was enhanced in 12E‐V2R–GFP cells. Conclusions: The 12E‐V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVIII.     

A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF‐binding site distant from its epitope

Summary. Background: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab_BO2C11) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. Objectives: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. Methods: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab_BO2C11‐complexed. In silico predictions were confirmed by site‐directed mutagenesis and VWF‐binding assays of the mutated FVIII. Results: Binding of Fab_BO2C11 to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF‐binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. Conclusions: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti‐FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.     

Linkage analysis of factor VIII and von Willebrand factor loci as quantitative trait loci

Summary.   Elevated factor (F)VIII levels contribute to venous thrombotic risk. FVIII levels are determined to a large extent by levels of von Willebrand factor (VWF), its carrier protein which protects FVIII against proteolysis. VWF levels are largely dependent on ABO blood group. Subjects with blood group non-O have higher VWF and FVIII levels than individuals with blood group O. Apart from ABO blood group no genetic determinants of high FVIII levels have been identified, whereas clustering of FVIII levels has been reported within families even after adjustment for ABO blood group and VWF levels. We investigated the FVIII and VWF loci as possible quantitative trait loci (QTL) influencing FVIII and VWF levels. Two sequence repeats in the FVIII gene and three repeats in the VWF gene were typed in 52 FV Leiden families. Multipoint sib-pair linkage analysis was performed with the MAPMAKER/SIBS program. FVIII levels adjusted for VWF levels and age, and VWF levels adjusted for ABO blood group and age, were used for this linkage analysis. No linkage of FVIII levels to the FVIII locus was found, whereas we found evidence that the VWF locus contains a QTL for VWF levels [maximum likelihood no dominance variance lod score = 0.70 ( P  = 0.04) and non-parametric Z-score = 1.92 ( P  = 0.03)]. About 20% of the total variation in VWF levels may be attributed to this VWF locus.