Plasma-derived Factor VIII Research Articles

Clinical efficacy of a novel VWF-containing FVIII concentrate, Wilate®, in the prophylaxis and treatment of bleeding episodes in previously treated haemophilia A patients

The new-generation coagulation factor VIII (FVIII)/von Willebrand factor (VWF) concentrate, Wilate®, is effective in the therapy of von Willebrand disease for which it was originally developed. The ratio of haemostatic components (FVIII to VWF) of this high-purity plasma-derived concentrate is 1:1, with VWF naturally stabilising FVIII. Bleeding episode control in patients with haemophilia A (HA) is generally managed by replacement therapy with recombinant or plasma-derived FVIII. When complexed with VWF as in the physiological situation, the product may also show a lower incidence of inhibitor development in patients with HA. The clinical efficacy, safety and tolerability of Wilate® were therefore evaluated in 81 previously treated individual patients with severe HA. Data from 5 good clinical practice (GCP) prospective clinical studies were pooled and assessed with regards to the prevention and treatment of bleeding and during surgical procedures. Haemostatic efficacy was excellent or good in 96.7% of 1495 rated treatments of bleeding episodes. The average dose per treatment was approximately 30IU FVIII/kg. In surgical settings, the global haemostatic efficacy of Wilate® was generally rated excellent or good for use during and after operations with a mean total dose of FVIII of 848.6IU/kg±578.5IU/kg administered peri-operatively. Overall, tolerability of Wilate® was rated very good or good by patients and physicians. Rarely occurring adverse events were mild in intensity and no anti-FVIII inhibitors were detected. Wilate® also displayed a good viral safety profile with no seroconversions occurring in response to treatment. These data show that Wilate® is an efficacious treatment option in the management of patients with severe HA.

Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors

Antibodies (inhibitors and non-neutralising antibodies [NNA]) directed against factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Inhibitors reduce FVIII pro-coagulant properties, whereas NNA are directed against non-functional epitopes. NNA are poorly studied and their prevalence, epitope specificity and physiopathology inadequately defined. The aim of this study was first to evaluate NNA prevalence in a French retrospective multicentric series of 210 patients without inhibitors, then to determine their epitope specificity (against the heavy chain [HC] or the light chain [LC] of FVIII) and particularly to assess the prevalence of anti-B domain NNA using specifically designed x-MAP assays. NNA occurred in 18.1% of patients (38/210) and their prevalence was not influenced by the severity of the disease. Among the 38 patients with NNA, 73.7% had anti-FVIII Abs against the HC, 13.2% against the LC and 13.2% had anti-FVIII Abs against both chains. There is thus a clear immuno-dominance of the HC of FVIII in the epitope profile of NNA, whatever the severity of HA. The proportion of NNA that recognised the B domain was 18.4% (n=7/38). A multivariate analysis did not highlight differences in NNA occurrence between patients treated with recombinant FVIII or with plasma- derived FVIII (19.6% vs. 14.9%, p=0.53).

Hemophilia A: Patient Impact and Economic Burden of the Disease

AbstractAbstract 4763 Introduction:Hemophilia A is a rare but serious bleeding disorder caused by a deficiency in blood clotting factor VIII (FVIII). To examine the impact of hemophilia A and replacement FVIII therapies from a humanistic and economic perspective, a targeted literature review was conducted. Methods:Searches were conducted in MEDLINE® and the National Health Service Economic Evaluation Database, using combinations of disease, patient-reported outcome and economic-related key words, limited to articles published in English between January 2000 and January 2010 (inclusive). A total of 34 full-text articles (from 653 abstracts retrieved) were selected for detailed consideration. Results:Findings revealed increased mortality rates and decreased life expectancy among people with hemophilia A, compared with the general population. This is largely a result of the transmission of blood-borne viruses (e.g. HIV and Hepatitis-C) due to use of plasma-derived FVIII (pdFVIII) concentrates. Improvements in viral attenuation processes plus donor screening practices for pdFVIII products have reduced the risks of viral transmission, but risk of transmission still exists in relation to non-enveloped viruses and other unknown pathogens. Newly developed recombinant FVIII therapies minimizes these risks; however, such therapies are not currently widely available globally. All available FVIII therapies use demanding regimens requiring regular time-consuming injections, which can be detrimental to patients' health-related quality of life and limit adherence to replacement FVIII therapy. Non-adherence to factor replacement therapy is associated with diminished product efficacy, poorer health outcomes and increasing economic expenditure. Treating hemophilia A is costly, primarily due to the high acquisition cost of replacement FVIII products. However, indirect costs related to patient disability and level of care also contribute to economic burden. Prophylactic treatment initially may appear to be more expensive than on-demand treatment but is associated with greater clinical efficacy and improved long-term outcomes, which may lead to cost savings over the course of a patient's lifetime. Conclusions:Hemophilia A is associated with considerable humanistic and economic burden. There remain substantial unmet needs with regard to the safety, convenience, global access to treatment incl. reliable product supply and total care costs associated with factor replacement therapies. Disclosures:Strandberg-Larsen:Novo Nordisk A/S: Employment. Gater:Novo Nordisk A/S: Research Funding. Marlow:Novo Nordisk A/S: Research Funding. Thomson:Novo Nordisk A/S: Research Funding.

Functional characteristics of N8, a new recombinant FVIII

The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one-stage clot assay was 9300±400 IU mg(-1) based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97-1.03). N8 bound to von Willebrand factor with Kd values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa-catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as Km and kcat for FX activation was in the same range as those observed for Advate®. The rate of activated protein C (APC)-catalysed inactivation was similar for activated N8 and Advate®. N8 improved thrombin generation in a dose-dependent manner and induced similar rates of thrombin generation as Advate® and the plasma-derived FVIII product Haemate®. Using thromboelastography (TEG®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate® was found based on TEG® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor-related protein (LRP) of N8 and Advate® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.

Continuous infusion during total joint arthroplasty in Japanese haemophilia A patients: comparison study among two recombinants and one plasma‐derived factor VIII

As for the available factor VIII (FVIII) concentrates in Japan, there are two recombinant FVIII concentrates (Kogenate-FS and Advate) and one highly purified plasma-derived FVIII concentrate (Cross-Eight M). To evaluate the inter-product variability, the differences in the continuous infusion rates and total consumption of the above three concentrates were compared when continuous infusion was used as the administration mode to control bleeding during 28 total joint arthroplasties (TJAs) for 17 patients. There were no significant differences among the FVIII plasma levels during surgery, except day 0. Advate needed to be given at a significantly higher infusion rate (4.2-2.1 IU kg(-1) h(-1)) than the other two concentrates (Kogenate-FS: 1.0-2.9 IU kg(-1) h(-1), P < 0.01 and P < 0.05; Cross-Eight M: 3.2-1.8 IU kg(-1 )h(-1), P < 0.01); however, their infusion rates were within the rates which were previously reported. The total consumption of Advate (652.1 IU kg(-1)) was also significantly greater than either of the other concentrates (Kogenate-FS: 395.1 IU kg(-1), P < 0.01; Cross-Eight M: 519.1 IU kg(-1), P < 0.05). The results of this study showed that the continuous infusion of three FVIII concentrates is effective and safe during TJA, and also showed the differences in the continuous infusion rates and total consumption among concentrates when continuous infusion was used to control bleeding during surgery. These two results suggested that the continuous infusion of FVIII concentrate is a good administration mode, but there is still room for further investigation to use it as a more cost-effective and safer administration mode.

The prevalence of factor VIII inhibitors and genetic aspects of inhibitor development in Chinese patients with haemophilia A

The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma-derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long-range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.

Comparison among plasma-derived clotting factor VIII by using monodimensional gel electrophoresis and mass spectrometry.

Deficiency or dysfunction of coagulation factor VIII (FVIII) is the underlying cause of haemophilia A. Haemophilic patients are at present treated with plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) in order to correct their clotting deficiency. pdFVIII concentrates are exclusively produced from human plasma upon pooling from multiple donors. It is not know whether the presence of excess of other plasma proteins, in addition to von Willebrand factor, could stimulate untoward immune responses in the recipient. Thus, information regarding the presence of contaminants in commercial products is of concern. Two commercially available pdFVIII concentrates were characterized through SDS-PAGE and mass spectrometry Emoclot and Beriate. The components of two pdFVIII products considered in this study were well identified by mass spectrometry analysis, in both cases we found abundant components coming from blood plasma, and some other contaminants. Only in Beriate we also found truncated form of pdFVIII. The two pdFVIII examined showed the presence of vWF, Fibrinogen in excess, and other substances that could be considered as contaminants or impurities.

Asymptomatic factor VII deficiency: gene analysis and structure–function relationships

Factor VII Padua is a variant form of factor VII deficiency characterized by a prolongation of the prothrombin time (PT), when the assay is performed using rabbit brain thromboplastin. The PT is normal when performed using either human or ox brain thromboplastin reagents, or a recombinant human tissue factor-based thromboplastin. We report a case of an African-American woman with asymptomatic factor VII deficiency, who had a prolonged PT and factor VII activity levels of 5-8% using rabbit brain thromboplastin, but a normal PT and factor VII activity levels when measured using recombinant human brain thromboplastin or tissue factor. The amino acid substitution (R304Q), which gives rise to factor VII Padua, was found in our patient, making this only the fourth African-American case described to date with this mutation. Our report emphasizes the importance of identifying this benign form of factor VII deficiency in order to avoid unnecessary exposure of patients to treatment with either plasma-derived products or recombinant activated factor VII.

Von Willebrand factor-containing factor VIII concentrates and inhibitors in haemophilia A

The development of inhibitors that neutralise the function of factor VIII (FVIII) is currently not only the most challenging complication associated with the treatment of haemophilia A but it also increases the disease-related morbidity as bleeding episodes do not respond to standard therapy. The main short-term goal of the treatment of inhibitor patients is to control bleeding episodes while the long-term one is to permanently eradicate the inhibitor by immune tolerance induction, particularly in the case of high-titer antibodies. Due to some in vitro studies and clinical observations, some investigators have suggested that FVIII concentrates containing von Willebrand factor (VWF) may be less immunogenic than high-purity or recombinant FVIII products. It has also been suggested that success rates for immune tolerance induction are higher when plasma-derived FVIII products are used. The currently available data from laboratory and clinical studies on the role of VWF in inhibitor development and eradication in haemophilia A is critically analysed in this review. As a result, we have not found definitive evidence supporting a role for product type on inhibitor incidence and inhibitor eradication in haemophilia A patients.

A case of intracranial hemorrhage in a neonate with congenital factor VII deficiency

Congenital factor VII deficiency is a rare autosomal-recessive bleeding disorder. Bleeding manifestations and clinical findings vary widely, ranging from asymptomatic subjects to patients with hemorrhages that may cause significant handicaps. Treatment has traditionally involved factor VII(FVII) replacement therapy using fresh frozen plasma, prothrombin complex concentrates or plasma-derived FVII concentrates. Recombinant activated FVII (NovoSeven®) is currently considered the first-line treatment for replacement therapy of FVII deficiency. Here we present a case of severe intracerebral and intraventricular hemorrhage in a neonate with congenital FVII deficiency.

Prevalence and Epitope Specificity of Non-Neutralizing Antibodies in Haemophilia A Patients without Inhibitors Using a Multiplex x-MAP Technology.

Abstract 3493Poster Board III-430 Background and ObjectivesAntibodies (Abs) directed against Factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Anti-FVIII Abs neutralize procoagulant activity of FVIII. They are called inhibitors. Non-neutralizing antibodies (NNA) have also been described in hemophiliacs treated by FVIII concentrates. Their role and prevalence are still under debate. NNA could form immune complexes with FVIII and be responsible of an increased FVIII clearance inducing a shortened half-life. The aim of this retrospective study was to evaluate the prevalence of NNA in a large cohort of HA patients without inhibitors and to determine their epitope specificity against the two chains of FVIII or the B domain, using a x-MAP technology. Indeed, this approach has many advantages: it is fast, requires small volumes of plasma, and is suitable for multiplexing immuno-assay. MethodsSamples of 187 patients (mean age 29 years, range [1-86]) with severe (n= 133), moderate (n= 31) or mild (n= 23) HA from three haemophilia centers were studied. All patients have been previously treated and were inhibitor-free at the time of the test (<0.6 Bethesda Units/mL). Among them 33% (n=61) were treated by plasma-derived FVIII while 67% (n=126) had received recombinant FVIII (rFVIII). Two assays based on x-MAP technology were used: (i) the first determined the NNA reactivity with the heavy chain (HC) or the light chain (LC) of FVIII (Lavigne-Lissalde et al. 2008); (ii) the second identified the NNA directed towards the B-domain, by differential binding to full-length rFVIII or B-domain deleted rFVIII. ResultsThe observed NNA prevalence in the 187 patients under study was 24% (n=45): 13.3% (n=6) of NNA were directed against the LC, 62.2% (n= 28) were directed against the HC and 24.5% (n=11) against both chains. In the NNA positive population, the proportion of NNA directed toward the B-domain was of 9% (n=4). No difference was observed in the prevalence between the 3 centers. The prevalence of NNA in patients grouped into four categories of age [1-15 yrs] (n=51, NNA prevalence: 27%), [16-30 yrs] (n=65; 20%), [31-45 yrs] (n=35; 20%) and [>45 yrs] (n=33; 27%) was not statistically different. ConclusionThis retrospective study, using the x-MAP technology, found a prevalence of 24% NNA in HA patients, comparable to other reports in the literature. Most of the NNA were directed against the HC of FVIII. In 9% of the cases, NNA recognized the B domain. Further prospective studies will allow to better explain the role of NNA, overall on the FVIII pharmacokinetics and particularly those binding to the B domain. Other prospective studies could highlight a difference according to the type of treatment.Lavigne-Lissalde G. et al. (2008). “Simultaneous detection and epitope mapping of anti-factor VIII antibodies.” Thromb Haemost99(6): 1090-6 Disclosures:No relevant conflicts of interest to declare.

Combined Factor V and Factor VIII Deficiency

Combined deficiency of factor V (FV) and factor VIII (FVIII) (F5F8D, or FV+FVIII) is a autosomal recessive bleeding disorder caused by mutations in genes encoding two components of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC-53), that is, lectin mannose binding protein ( LMAN1) and multiple coagulation factor deficiency 2 ( MCFD2), involved in the FV and FVIII intracellular transport rather than by DNA defects in the genes that encode the corresponding coagulation factors. F5F8D is estimated to be extremely rare (1:1,000,000) in the general population, but an increased frequency is observed in regions where consanguineous marriages are practiced. F5F8D is characterized by concomitantly low levels (usually between 5% and 20%) of both FV and FVIII and is associated with a mild to moderate bleeding tendency. Treatment of bleeding episodes requires a source of both FV and FVIII; replacement of FV is achieved only through use of fresh-frozen plasma (FFP) and replacement of FVIII by FFP and desmopressin or specific FVIII concentrates (plasma-derived or recombinant FVIII products).

Factor VII Deficiency

The complex formed between the procoagulant serine protease activated factor VII (FVII) and the membrane protein tissue factor, exposed on the vascular lumen upon injury, triggers the initiation of blood clotting. This review describes the clinical picture of FVII deficiency and provides information on diagnosis and management of the disease. FVII deficiency, the most common among the rare congenital coagulation disorders, is transmitted with autosomal recessive inheritance. Clinical phenotypes range from asymptomatic condition, even in homozygotes, to severe disease characterized by life-threatening and disabling symptoms (central nervous system and gastrointestinal bleeding and hemarthrosis), with early age of presentation and the need for prophylaxis. In females, menorrhagia is prevalent and affects two thirds of the patients of fertile age. Although FVII gene mutations are extremely heterogeneous, several recurrent mutations have been reported, a few of them relatively frequent. The study of genotype-phenotype relationships indicates that modifier (environmental and/or inherited) components modulate expressivity of FVII deficiency, as reflected by patients with identical FVII mutations and discordant clinical phenotypes. Several treatment options are available for FVII deficiency: the most effective are plasma-derived FVII concentrates and recombinant activated FVII (rFVIIa). Treatment-related side effects are rare.

Von Willebrand factor/factor VIII concentrates in the treatment of von Willebrand disease

Therapy for von Willebrand disease (VWD) aims to restore the hemostatic function conferred by von Willebrand factor (VWF), which facilitates platelet adhesion and aggregation, and serves to increase potentially low coagulation factor VIII (FVIII) in plasma. In patients unresponsive to desmopressin (DDAVP), the preferred treatment is with plasma-derived VWF-containing FVIII concentrates. Only a few of the available VWF/FVIII concentrates have been licensed for use in VWD based on prospective studies. The efficacy of VWF/FVIII concentrates depends on the content and quality of VWF and FVIII. Several studies have demonstrated the variability of the VWF contents, as well as the differences in the VWF multimer patterns (including the high molecular weight VWF multimers that are most effective in restoring hemostasis), among these concentrates. Treating physicians should be aware of these disparities and the potential clinical implications for patients with different VWD subtypes. Dosing has traditionally been calculated based on the FVIII content of the products, although dosing based on VWF functional activity [e.g., VWF ristocetin cofactor activity (VWF:RCo)] addresses the primary protein deficiency in VWD patients. Several clinical studies have demonstrated the efficacy of concentrates dosed according to VWF:RCo. Dosing is generally consistent across VWD subtypes, although patients with severe phenotypes or undergoing major procedures may require more infusions or longer treatment duration. Other considerations for the use of VWF-containing concentrates include laboratory monitoring of efficacy and safety issues such as thrombosis risk and thromboprophylaxis.

Anti-factor VIII Antibodies (Inhibitors) in Hemophilia A: In Dire Need of Basic and Therapeutic Research

Hemophilia A, a congenital, severe bleeding disorder, is caused by the absence or functional abnormality of factor VIII (FVIII) that occurs with a frequency of about one in 5,000 boys. FVIII is produced in the liver, and minute amounts of which (0.2 μg/ml plasma) ensure an efficient coagulation. Abnormalities in the gene encoding FVIII lead to the deficiency of FVIII or production of a functionally defective molecule. Therefore, substituting the FVIII molecule represents the treatment of choice during bleeding incidences in hemophilia patients. Thus, most hemophilia patients require regular supplementation with intravenously administered recombinant or plasma-derived FVIII. About 25–40% of the hemophilia A patients who receive FVIII develop antibodies against the infused FVIII. These antiFVIII alloantibodies are called “inhibitors.” FVIII inhibitors neutralize the infused FVIII, thus exposing the patients to an extremely precarious situation and a serious challenge to the clinicians, as subsequent

Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.

The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII

Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies