Affinity Of Von Willebrand Factor Research Articles

The VWF binding aptamer rondoraptivon pegol increases platelet counts and VWF/FVIII in type 2B von Willebrand disease

AbstractType 2B von Willebrand disease (VWD) is characterized by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high-molecular-weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/factor VIII (FVIII) levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in patients with type 2B VWD, we conducted a prospective phase 2 trial. Patients with type 2B VWD received 3 mg rondoraptivon pegol subcutaneously on study days 1, 4, and 7, followed by 6 to 9 mg every week until day 28. Five patients (male:female ratio = 3:2) were included. Rondoraptivon pegol rapidly tripled platelet counts from a median of 60 to 179 × 10E9/L (P < .001). Circulating VWF antigen increased from a median of 64% to 143%, which doubled FVIII activity levels from 67% to 134%. In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased, and HMW multimers appeared. These pronounced improvements reversed during washout of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase 2b/3 trial in such patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

The impact of aberrant von Willebrand factor-GPIbα interaction on megakaryopoiesis and platelets in humanized type 2B von Willebrand disease model mouse.

Type 2B von Willebrand disease (VWD) is caused by gain-of-function mutations in von Willebrand factor (VWF). Increased VWF affinity for GPIbα results in loss of high molecular weight multimers and enhanced platelet clearance, both contributing to the bleeding phenotype. Severity of the symptoms vary among type 2B VWD patients, with some developing thrombocytopenia only under stress conditions. Efforts have been made to study underlying pathophysiology for platelet abnormalities, but animal studies have been limited because of species specificity in the VWF-GPIbα interaction. Here, we generated a severe form of type 2B VWD (p.V1316M) knockin mice in the context of human VWF exon 28 (encoding A1 and A2 domains) and crossed them with human GPIbα transgenic strain. Heterozygous mutant mice recapitulated the phenotype of type 2B VWD in autosomal dominant manner and presented severe macrothrombocytopenia. Of note, platelets remaining in the circulation had extracytoplasmic GPIbα shed-off from the cell surface. Reciprocal bone marrow transplantation determined mutant VWF produced from endothelial cells as the major cause of the platelet phenotype in type 2B VWD mice. Moreover, altered megakaryocyte maturation in the bone marrow and enhanced extramedullary megakaryopoiesis in the spleen were observed. Interestingly, injection of anti-VWF A1 blocking antibody (NMC-4) not only ameliorated platelet count and GPIbα expression, but also reversed MK ploidy shift. In conclusion, we present a type 2B VWD mouse model with humanized VWF-GPIbα interaction which demonstrated direct influence of aberrant VWF-GPIbα binding on megakaryocytes.

A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD

AbstractGain-of-function (GOF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα to von Willebrand factor (VWF) interaction, and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GOF GP1BA variants generate a high-affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and GPIbα structure. Chinese hamster ovary cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen‐deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GOF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα, inducing a conformation with high affinity for VWF.

Type 2N VWD: Conclusions from the Spanish PCM-EVW-ES project.

Type 2N von Willebrand disease (VWD) is characterized by a decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). Abnormal binding of FVIII to VWF (VWF:FVIIIB), results in low FVIII plasma levels, which can lead to a misdiagnosis of mild haemophilia A. Accurate diagnosis of type 2N VWD is essential for appropriate genetic counselling and therapy. This disease can be distinguished from haemophilia A by in vitro assays (measurement VWF:FVIIIB activity) and/or genetic analysis. To identify the current challenges in the diagnosis and treatment of this type of VWD and provide an in-depth description of the phenotypes and mutations identified. Twenty-eight patients had at least one type 2N mutation, and 13 of these had a type 2N mutation combined with other variations. Three type 2N mutations were detected: p.Arg816Trp, p.Arg854Gln, and p.Arg763Ser. Two of these are the most frequently described mutations worldwide. This mutational spectrum differs from the broad spectrum seen in neighbouring France, where at least eight distinct 2N mutations have been found. In the PCM-EVW-ES cohort, 11 asymptomatic type 2N carriers with borderline FVIII plasma levels would probably have been excluded if the evaluation had been based on clinical and laboratory data only. Likewise, three patients with a severe phenotype would have been classified as homozygous for a 2N mutation if only the phenotype study had been performed. The high detection yield and affordability of next-generation sequencing support the use of this technology as a first-line diagnostic tool in this setting.

Platelet dysfunction in platelet-type von Willebrand disease due to the constitutive triggering of the Lyn-PECAM1 inhibitory pathway

Platelet-type von Willebrand disease (PT-VWD) is an inherited platelet disorder. It is characterized by macrothrombocytopenia and mucocutaneous bleeding, of variable severity, due to gain-of-function variants of GP1BA conferring to glycoprotein Ibα (GPIbα) enhanced affinity for von Willebrand factor (VWF). The bleeding tendency is conventionally attributed to thrombocytopenia and large VWF-multimer depletion. However, while some indications suggest that platelet dysfunction may contribute to the bleeding phenotype, no information on its characteristics and causes are available. The aim of the present study was to characterize platelet dysfunction in PT-VWD and shed light on its mechanism. Platelets from a PT-VWD patient carrying the p.M239V variant, and from PT-VWD mice carrying the p.G233V variant, showed a remarkable platelet function defect, with impaired aggregation, defective granule secretion and reduced adhesion under static and flow conditions. VWF-binding to GPIbα is known to trigger intracellular signaling involving Src-family kinases (SFK). We found that constitutive phosphorylation of the platelet SFK Lyn induces a negative-feedback loop downregulating platelet activation through phosphorylation of PECAM1 on Tyr686 and that this is triggered by the constitutive binding of VWF to GPIbα. These data show, for the first time, that the abnormal triggering of inhibitory signals mediated by Lyn and PECAM1 may lead to platelet dysfunction. In conclusion, our study unravels the mechanism of platelet dysfunction in PT-VWD caused by deranged inhibitory signaling. This is triggered by the constitutive binding of VWF to GPIbα which may significantly contribute to the bleeding phenotype of these patients.

Fibronectin binding to von Willebrand factor occurs via the A1 domain

BackgroundCollagen interactions with von Willebrand factor (VWF) perform an important role in initiation of hemostasis. ObjectivesWe hypothesized that in addition to collagen, other extracellular matrix (ECM) proteins such as fibronectin can bind VWF. MethodsFibronectin‐VWF interactions were measured by ELISA using both plasma‐derived and recombinant VWF–containing variants in specific domains. Inhibition was measured by antibody competition using antibodies directed against both VWF and fibronectin. Binding affinities were measured by the Octet Biosensor for fibronectin and collagen IV. ResultsFibronectin was able to bind both plasma‐derived and recombinant wild‐type VWF. This interaction was inhibited by both anti‐VWF antibodies and collagen types III and IV. Several VWF A1 domain variants in the region of the collagen IV binding site also demonstrated absent fibronectin binding, as did variants with defects in high‐molecular‐weight multimers. Binding affinity testing showed fibronectin has a strong affinity for VWF, in a range similar to that of collagen IV. Fibronectin binds VWF via a restricted region of the A1 domain. This interaction requires high‐molecular‐weight multimers and is similar to that seen with vascular collagens. ConclusionsTherefore, VWF would appear to be the common factor linking platelet adhesion to various ECM proteins and facilitating hemostasis under conditions of ECM exposure. [Display omitted]

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products

BackgroundTherapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product‐related impurity, whose properties and levels in rFVIII products should be investigated. ObjectiveTo isolate and characterize the FVIIIFT fraction in rFVIII products. MethodsProtein fractions unable (FVIIIFT) and able (FVIIIEL) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results and ConclusionsA robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%‐21.5%) in all products. Compared with FVIIIEL, FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low‐density lipoprotein receptor‐related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Characterization of Protein Unable to Bind Von Willebrand Factor in Recombinant Factor VIII Products: Can We Reduce Their Immunogenicity?

Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab.

Von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the USA. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggest that TAGX-0004 possesses better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

Status of Recombinant Factor VIII Concentrate Treatment for Hemophilia a in Italy: Characteristics and Clinical Benefits.

The current interest in recombinant factor VIII (rFVIII) products stems from the fact that they offer a technological solution to prolonging the half-life of and reducing the risk of formation of alloantibodies (inhibitors) against FVIII in treated patients with hemophilia A (HA). The Italian health care system has authorized the use of a wide range of rFVIII concentrates of the first, second, and third generation, as well as new innovative rFVIII preparates with an extended half-life (EHL) (Kogenate FS®-Bayer, belonging to the second generation and replaced since 2017 by a product consisting of the same modified molecule; because it is only available until the end of the current year, it will not be considered in this review). Some of these products have unique pharmacodynamic and pharmacokinetic (PK) profiles, including an EHL. The first-generation full-length rFVIII (FL-rFVIII), octocog alfa (Recombinate® Baxter/BIOVIIIx), although the oldest rFVIII product, has several desirable features. Third-generation products include two modified octocog alfa molecules (Advate®, Shire; Kovaltry®, Bayer) as well as the B domain-deleted rFVIII (BDD-rFVIII) moroctocog alfa (ReFacto®-Pfizer). The B domain-truncated (BDT-rFVIII) turoctocog alfa (NovoEight®, Novo Nordisk), the BDD-rFVIII simoctocog alfa (Nuwiq®, Kedrion), the single-chain BDT-rVIII lonoctocog alfa (Afstyla®, CSL Behring), and the BDD-rFVIIIFc efmoroctocog alfa (Elocta®, Sobi-Biogen) are new, innovative products. Simoctocog alfa, because its peculiarities, is considered a fourth-generation rFVIII concentrate. Turoctocog alfa, simoctocog alfa, and lonoctocog alfa have a high affinity for von Willebrand factor (vWF) that reduces renal clearance and prolongs the half-life of rFVIII. Efmoroctocog alfa, a first-in-class rFVIII-Fc fusion protein (rFVIIIFc), has a half-life 1.5–1.8 times longer than that of conventional plasma-derived FVIII (pd-rFVIII) and other rFVIII products. Clinical studies have evaluated the efficacy, safety, and inhibitor development of all these innovative concentrates in both previously treated (PTPs) and untreated patients (PUPs). This review considers the rFVIII products that are indicated for the treatment of patients with severe HA, focusing on those that are commercially available in Italy. Their PK characteristics, immunogenicity, and clinical benefits are discussed and compared.

Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

AbstractRecombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

Mechanisms and Therapeutic Modulation of the Bleeding Tendency in Genetically-Engineered Von Willebrand Disease Type 2B Mice

Von Willebrand disease type 2B (VWD2B) is characterized by an abnormal increased affinity of von Willebrand factor (VWF) for platelets and related to mutations in the A1 domain of VWF. In patients, the VWD2B-mutant p.V1316M is specifically associated with a severe phenotype including hemorrhagic tendency, loss of high molecular weight VWF multimers, thrombocytopenia and platelet dysfunction related to defective αIIbβ3 integrin activation (Casari et al. JCI 2013, 123:5071). Genetically-engineered mice carrying the p.V1316M mutation (V1316M+/+ mice) perfectly mimic the human phenotype. We used this murine model to investigate the relative contribution of platelet count and platelet dysfunction to the occurrence of bleeding tendency and explore new therapeutic options.Bleeding tendency was evaluated using a novel tail-vein transection bleeding model involving the specific transection of the lateral vein at standardized depth and diameter (0.7 and 2.3mm respectively). Time (TVT) and blood loss were measured in wild-type (WT) and V1316M+/+ mice at baseline and after platelet count alteration.Compared to WT mice (platelet count: 1019±154G/L, n=58), V1316M+/+ mice (platelet count: 455±139G/L, n=52) have a significant increase (p<0.01) in TVT (25±9min, n=12 vs 2±1min, n=14) and blood loss (448±273µl vs 34±19µl). Induced thrombocytopenia (after infusion of anti-GPIb antibody) in WT mice (platelet count: 347±93G/L, n=6), to a similar level of V1316M+/+ mice, did not induce a hemorrhagic tendency (TVT: 2±1min, blood loss: 14±13µl). An increased bleeding phenotype in WT mice was exclusively observed in case of severe thrombocytopenia (platelet count: 70±25G/L, TVT>30min, blood loss: 532±168ml, n=6). Conversely, romiplostim-treated V1316M+/+ mice with normalized platelet count (platelet count: 866±100G/L, n=8) did not improve their bleeding tendency (TVT: 21±8min, blood loss: 448±282ml). These results suggest the bleeding phenotype in V1316M+/+ mice is independent of their thrombocytopenia, and could be related to the p.V1316M-associated defect in platelet function in general, and αIIbβ3 integrin activation in particular.Based on the hemostatic efficacy of recombinant activated factor VII (rFVIIa) in Glanzmann thrombasthenia, we tested rFVIIa in thrombocytopenic V1316M+/+ mice.A single infusion of rFVIIa (3 mg/kg) induced a complete and immediate correction of the bleeding phenotype (TVT: 4±2min, blood loss: 69±25ml, n=6). Thrombin generation measured in platelet-rich plasma before and after treatment showed a significant reduction of the lag time (1.13min, n=12 vs 1.8min, n=8 respectively, p<0.01).In conclusion, genetically-engineered mice carrying the VWD2B p.V1316M mutation display a bleeding phenotype that is not explained by their reduced platelet count, but is possibly related to the impaired platelet function induced by this VWF mutant. The bleeding tendency was rapidly and completely controlled by using rFVIIa. Based on these promising data, it would be of interest to consider rFVIIa as a therapeutic option in VWD-2B patients characterized by a severe hemorrhagic phenotype. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Type 2N Von Willebrand Disease Mutations Using Ιn Vitro and Ιn Vivo Mouse Models

Introduction: Von Willebrand factor (VWF) is a multimeric glycoprotein that serves as the carrier for the essential coagulation cofactor, factor VIII (FVIII). Both plasma levels of VWF and its FVIII-binding ability can influence plasma levels of FVIII. Type 2N von Willebrand disease (VWD) is associated with a reduced binding affinity of VWF for FVIII, resulting in accelerated proteolysis and clearance of FVIII (plasma levels 5 – 30% of normal). Type 2N VWD is a recessive trait and patients are either homozygous or compound heterozygous for 2N alleles. We hypothesize that type 2N VWD mutations can alter the expression and FVIII-binding ability of VWF. In these studies, we characterize three type 2N VWD mutations in vitro and in a murine model. R854Q (20-30% FVIII) is the most common 2N allele and is associated with a mild phenotype, while R816W (<10% FVIII) is associated with a severe phenotype. The R763A mutation inhibits propeptide cleavage that likely sterically interferes with the FVIII-binding ability of VWF.Methods: Type 2N VWD mutations were generated in the murine VWF cDNA. Heterologous VWF synthesis/secretion was characterized in vitro using HEK 293T cells and in vivo using hydrodynamic gene transfer of the murine VWF cDNA into VWF deficient mice. Binding of FVIII to type 2N variants was assessed in vitro using a solid phase binding assay and in vivo in VWF deficient mice by a FVIII chromogenic activity assay.Results: In HEK 293 T cells, biosynthesis of type 2N VWD variants was not significantly different from wild type VWF while secretion of all type 2N VWD variants was decreased relative to wild type: R763A (66%, p=0.0043), R816W (53%, p=0.0004), R854Q (4%, p<0.0001). Immunofluorescent staining of transfected HEK 293 cells demonstrated impaired pseudo-Weibel Palade body formation for the R854Q variant. Western blot analysis under denaturing conditions demonstrated that approximately 50% of the secreted R763A protein remained attached to the propeptide. Multimeric profiles of plasma-derived type 2N VWD mutants were normal. In vitro binding of plasma-derived murine type 2N VWD mutants to recombinant human FVIII was reduced relative to wild type VWF: R763A (56%, p=0.0009), R816W (10%, p<0.0001), R854Q (46%, p=0.0002).Type 2N VWD mutants were expressed alone or in a compound heterozygous state (R816W/R854Q) in VWF deficient mice. A trend of lower VWF:Ag levels were observed for type 2N VWD mutants relative to wild type (average 4.8 U/mL) after 14 days: R763A (35.7%), R816W (53.1%), R854Q (21.3%), except for compound heterozygous condition R816W/R854Q (103%).Plasma levels of FVIII:C are significantly reduced in VWF deficient mice (15-20% of normal). We measured the ability of hydrodynamically expressed type 2N VWD mutants to stabilize endogenous FVIII:C in VWF deficient mice. Hepatic expression of wild type VWF stabilized endogenous plasma FVIII:C, resulting in a significant increase in FVIII:C after 14 days (7.7-fold increase above baseline, p=0.0002). For the type 2N VWD mutants, variable partial stabilization of endogenous FVIII:C was observed relative to baseline: R763A (4.7-fold increase, p=0.01), R816W (1.2-fold decrease, p=0.04), R816W/R854Q (4.8-fold increase, p<0.0001), R854Q (2.1-fold increase, p=0.06).The correlation coefficient between VWF:Ag and FVIII:C was assessed for samples with VWF:Ag between 0.5-10 U/mL. Correlation between wild type VWF expression and FVIII:C was highly positive (r2=0.85, slope=189.5 ± 15.7, p<0.0001). Correlation between VWF:Ag and FVIII:C for mice expressing type 2N VWD mutants was variable: R763A (r2=0.89, slope=235.3 ± 18.15, p<0.0001), R816W (r2=0.591, slope=0.96 ± 2.8, p=0.7433), R816W/854Q (r2=0.72, slope=91.32 ± 10.64, p<0.0001) and R854Q (r2=0.705, slope=156.7 ± 24.4, p=0.0002). The slopes for R816W (p<0.0001) and R816W/R854Q (p=0.009) mutants were significantly different from wild type, suggesting impaired FVIII-stabilization in vivo.Conclusion: Expression of the type 2N VWD severe mutant R816W or the compound heterozygous R816W/R854Q mutant can recapitulate type 2N VWD in a murine model. Type 2N VWD mutations are associated with impaired secretion of VWF and/or decreased binding and stabilization of endogenous FVIII. DisclosuresNo relevant conflicts of interest to declare.

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

AbstractPlasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D′D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf−/− mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D′D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf−/− mice. Although the VWF D′D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D′D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D′D3 and VWF D′D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D′D3/D′D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII

IntroductionrVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. Materials and MethodsBinding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate®) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200–250IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. ResultsrVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. ConclusionsrVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

Structural Basis of Regulation of von Willebrand Factor Binding to Glycoprotein Ib

Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.

Innovative coagulation factors: albumin fusion technology and recombinant single-chain factor VIII

Albumin fusion technology has been used to enhance the pharmacokinetic properties of recombinant coagulation factors. The goal of linking albumin to coagulation factors is to extend the half-life of the coagulation factor, thereby allowing for less frequent dosing for patients with bleeding disorders, such as hemophilia. The novel recombinant fusion proteins linking coagulation factors VIIa and IX with albumin (rVIIa-FP and rIX-FP, respectively) have a longer half-life and similar hemostatic efficacy compared with available recombinant coagulation factor products. Clinical evaluation of these fusion proteins is underway, and preliminary results with rIX-FP in patients with hemophilia B are encouraging. Other advances in coagulation factor therapy include a unique recombinant single-chain factor VIII (FVIII) protein, which has improved intrinsic stability and a higher affinity for von Willebrand factor (VWF), relative to other recombinant FVIIIs, and a recombinant VWF-albumin fusion protein (rVWF-FP), which has a significant longer half-life compared to available VWF products. Evaluation of these novel recombinant proteins continues and will help determine their potential to enhance the management of patients with bleeding disorders.

Reversible Association of 14-3-3 with Platelet Glycoprotein Ibα Regulates the Ability of Cells to Adhere, Roll and Spread On VWF

Abstract 1061Platelet adhesion to sites of vascular injury is required for the arrest of bleeding. Initial platelet adhesion is mediated by binding of von Willebrand factor (VWF) to the platelet glycoprotein (GP) Ib-IX-V complex, leading to the activation of integrin α IIbβ3 and other molecules that mediate firm adhesion, spreading and thrombus formation. The GPIb-IX-V complex comprises 4 polypeptides: GPIbα, GPIbβ, GPIX and GPV, in a 2:4:2:1 stoichiometry. Only the first three polypeptides are required for full VWF binding function. GPIbα is a 610 amino acid polypeptide that binds every known complex ligand within its N-terminal 300 amino acids. The cytoplasmic domain comprises 96 amino acids and contains binding sites for filamin, PI 3-kinase and the scaffolding protein 14-3-3. The association of 14-3-3 with the GPIbα cytoplasmic domain regulates the affinity for VWF. Typically, 14-3-3 requires phosphoserine- or phosphothreonine-containing motifs to bind target proteins. One such motif is in the GPIbα cytoplasmic domain surrounding Ser609, which is phosphorylated and known to bind 14-3-3. Mutation of Ser609 to Ala abrogates 14-3-3 association, which has been proposed to reduce the ability of GPIbα to bind VWF. Platelet aggregation results in the dissociation of 14-3-3 from a subpopulation of GPIbα. Ser609 also becomes dephosphorylated upon platelet spreading. To dissect further the functional roles of 14-3-3 association with GPIbα, we expressed in Chinese hamster ovary (CHO) cells GPIb-IX complexes (GPIbα, GPIbβ, and GPIX) containing either wild type GPIbα, or GPIbα mutants S609A or S609E. In other proteins, mutation of Ser to Glu at the 14-3-3 binding site mimics phosphoserine, recapitulates 14-3-3 binding and often prevents 14-3-3 dissociation. We first assessed the ability of the WT and mutant GPIbα to associate with 14-3-3. As expected, we detected little 14-3-3 binding to GPIbα S609A. GPIbα S609E bound 14-3-3 to the same extent as did WT GPIbα, indicating that the Glu substitution was able to mimic Ser phosphorylation at residue 609. We then assessed the ability of the CHO cells to attach to and roll on VWF under flow over a wide range of shear rates. At 3.26 and 10 dyne/cm2 the α 609A and α 609E cells rolled twice as fast as the WT cells. Both CHO cells and platelets display a characteristic velocity nadir as shear rates increase. The α 609A and α 609E cell showed defective shear-enhanced adhesion; their slowest velocity was ∼3-fold faster than the WT cells. Because GPIbα is dephosphorylated upon platelet spreading, we also assessed the effect of the mutations on cell spreading on VWF. All three cell lines adhered similarly to VWF but a higher percentage of α 609A cells spread (67% vs 58% for WT and α 609E). Of the spread cells, the α S609E cells spread less well; their spread area was 15% less than the WT and α S609A cells. The morphology of the adherent, spread cells was dramatically different among the different cell lines. WT cells displayed a few filopodial extensions along with punctate phalloidin staining indicative of focal adhesions. In some cases the cells displayed stress fibers. The α S609A cells extended more and longer filopodia than the WT cells but had fewer focal adhesions and more stress fibers. The CHO α S609E cells extended thin filopodia that tended to be polarized at two sides of the cell body, and had fewer focal adhesions and no stress fibers. We also examined the effect of the mutations on localization of the GPIb-IX complex to lipid raft membrane microdomains, which is required for platelet adhesion to VWF. Raft GPIbα was reduced by 40% in the S609A cells but increased 1.6-fold in the S609E cells.In summary, lack of 14-3-3 association decreased raft localization of the complex, reduced shear-induced cell adhesion, but increased cell spreading. Stable 14-3-3 association increased raft localization, but decreased shear-induced cell adhesion and decreased the ability of cells to fully spread. Together, our results demonstrate that regulated 14-3-3 association mediated by the phosphorylation status of S609 is required for coordinated adhesion, and cell spreading. Together, our results demonstrate that the functions of the GPIb-IX complex are regulated by the ability of GPIbα Ser609 to both bind and release 14-3-3 and suggest that it is not 14-3-3 binding per se that regulates GPIbα function. Disclosures:No relevant conflicts of interest to declare.

Validation of the first commercial ELISA for type 2N von Willebrand’s disease diagnosis

Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.

Pioneering designs for recombinant coagulation factors

Coagulation factor replacement therapy has become an established part of the prevention and treatment of bleeding episodes in patients with bleeding disorders, such as hemophilia A, hemophilia B, and von Willebrand disease. Advances in recombinant DNA technology have made it possible to modify recombinant coagulation factor products to enhance their pharmacokinetic properties, biological activity, or other characteristics. CSL Behring has created two novel albumin fusion proteins with the goal of extending the half-life of activated recombinant factor VII (rFVIIa) and recombinant factor IX (rFIX) to allow for less frequent dosing. Preclinical evaluation indicates that the resulting recombinant albumin fusion proteins (rVIIa-FP and rIX-FP) have improved pharmacokinetic properties, including an extended half-life, with preservation of hemostatic efficacy. Clinical evaluation of rIX-FP is now underway in patients with hemophilia B. In addition, we have designed a unique recombinant single-chain factor VIII protein (CSL627) that has improved stability during the manufacturing process and a high affinity for von Willebrand factor, relative to other recombinant factor VIII products. Ongoing studies of rVIIa-FP, rIX-FP, and CSL627 will help further define the potential clinical utility of these novel recombinant proteins in the management of patients with bleeding disorders.