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PubMed1983

Studies on the stability of factor VIII coagulant antigen (VIII:CAg) in the presence of VIII:C antibodies.

The stability of factor VIII coagulant antigens (VIII:CAg) at 56 degrees C was investigated using an immunoradiometric assay for VIII:CAg. In normal or CRM+ haemophilic plasmas VIII:CAg was rapidly inactivated at 56 degrees C. VIII:CAg in spontaneous VIII:C inhibitor plasmas and in post-treatment samples from haemophiliacs with VIII:C inhibitor was resistant to inactivation at 56 degrees C, indicating the presence of heat stable VIII:CAg-anti VIII:CAg complexes. In vitro VIII:CAg-anti VIII:CAg complexes were formed by incubation of diluted VIII:C antibodies and normal plasma and the stability of these complexes at 56 degrees C was studied. Haemophilic VIII:CAg antibodies formed heat stable immune complexes over a narrow range of inhibitor dilutions whilst some spontaneous VIII:CAg antibodies formed these stable complexes over a much wider range of dilutions emphasizing the difference in the properties of these antibodies.

Antibodies↗

PubMed2000

Phage display technology: a tool to explore the diversity of inhibitors to blood coagulation factor VIII.

Hemophilia A is a X-linked bleeding disorder that is caused by the functional absence of blood coagulation factor VIII. The bleeding tendency in hemophilia A patients can be corrected by the administration of plasma-derived or recombinant factor VIII concentrates. A serious complication in hemophilia care is the development of factor VIII neutralizing antibodies (inhibitors) that arise as a consequence of factor VIII replacement therapy. The majority of factor VIII inhibitors are directed toward epitopes located within the A2, A3, and C2 domains of factor VIII. In this article, we summarize current knowledge on the epitope specificity of factor VIII inhibitors. In addition, we will discuss recent information on the molecular characteristics of human anti-factor VIII antibodies generated by phage display technology.

Base Sequence↗

PubMed2006

[Biological activities of the coagulation factor VIII, IX in platelet concentrates collected by platelet apheresis during preservation].

The study was to explore the change of coagulation factor VIII and IX activities in the platelet suspension collected by platelet apheresis during storage at 22 degrees C. 18 samples of platelet concentrates were collected by the cs-3000 plus and stored at 22 degrees C and then FVIII: C, FIX: C activities were detected at 0, 12, 24, 48, 72, 96, 120 hours respectively by SYSMEX CA-1500. The results showed that FVIII: C activity was (100.51 + 44.02)% at 0 hour, and then decreased dramatically to 10% - 40% of primary level from 12 to 120 hours, while FIX: C activity was (120.93 +/- 20.50)% at 0 hour and decreased to 10% - 35% of primary level from 24 to 120 hours. In conclusion, FVIII and FIX in the platelet concentrates stored at 22 degrees C could keep their biological activities at physiologically high levels.

Blood Platelets↗

PubMed1999

Role of activation of the coagulation factor VIII in interaction with vWf, phospholipid, and functioning within the factor Xase complex.

Blood coagulation factor VIII (fVIII) in its nonactivated form circulates in plasma in a complex with von Willebrand factor (vWf). Upon activation by thrombin- or factor Xa-mediated site-specific proteolysis, activated fVIII (fVIIIa) serves as a cofactor for factor IXa. This protein complex assembled on a phospholipid surface (factor Xase) activates factor X. This complex plays the key role in the intrinsic pathway of blood coagulation. We reviewed the molecular events triggered by fVIII activation, which are required for the assembly and functioning of the Xase complex, including fVIIIa dissociation from vWf and a significant increase of fVIII affinity for binding to the phospholipid surface. Both events are mediated by activation-related cleavage within fVIII light chain (LCh), releasing the 40 amino-acid N-terminal LCh peptide, which is followed by a conformational change within the C2 domain. The conformational change within LCh is also required for the optimal fVIII cofactor functioning within the factor Xase complex, exerted via fVIIIa interactions with phospholipid, factor IXa, and factor X. Since factor IXa not only stabilizes but also proteolytically inactivates fVIIIa within the factor Xase complex, the stability of the membrane-bound fVIIIa in the presence and absence of factor IXa is discussed. In conclusion, we outline some new possible directions of the research. One of them arises from the recently demonstrated ability of plasma lipoproteins to provide a phospholipid surface for the assembly of the factor Xase complex in vitro. This finding raises a possibility that lipoproteins participate in factor Xase functioning in vivo and suggests a direct link between elevated levels of lipoproteins associated with atherosclerosis and increased thrombogenicity associated with this disease.

Cysteine Endopeptidases↗

PubMed2005

Inhibition of plasma coagulation through interaction between oxidized low-density lipoprotein and blood coagulation factor VIII.

Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. Thrombosis is the major mechanism underlying acute complications of atherosclerosis. In the present study, we analyzed the interaction between OxLDL and blood coagulation factors, which are involved in the blood coagulation pathway. We investigated the effect of OxLDL on plasma coagulation by measuring prothrombin time (PT) as a parameter of the extrinsic pathway of blood coagulation and activated partial thromboplastin time (APTT) as a parameter of the intrinsic pathway of blood coagulation following the addition of OxLDL to plasma. OxLDL, but not native LDL, caused prolongation of APTT in a dose- and oxidation time-dependent manner. In addition, the oxidatively modified product of acetylated LDL (AcLDL), but not AcLDL, also caused prolongation of APTT. The inhibition of lysophosphatidylcholine production in OxLDL by phenylmethylsulfonyl fluoride or Pefabloc pretreatment of LDL resulted in a prolongation of APTT, which was equivalent to the effect of OxLDL. Moreover, OxLDL significantly inhibited blood coagulation factor VIII, IX, and XI activity. Furthermore, we demonstrated that recombinant factor VIII binds to OxLDL and that factor VIII associated with OxLDL is detected in the incubation mixture of OxLDL and plasma. These results indicate that the binding of factor VIII to OxLDL affects the intrinsic pathway of the blood coagulation cascade. The present study suggests that the interaction between OxLDL and factor VIII may provide important information on the initiation and progression of atherosclerosis.

Blood Coagulation↗

PubMed1999

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA, an automated coagulation analyzer.

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p < 0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p < 0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.

Automation↗

PubMed2006

Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

Alanine↗

PubMed2003

Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII.

Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.

Alanine↗

PubMed1993

[Intragenic variable number tandom repeats in coagulation factor VIII gene of Chinese origin].

Using polymerase chain reaction, we analysed the variable number tandom repeats (VNTR) in intron 13 of coagulation factor VIII gene in the Chinese. Study of 115 X chromosomes from 77 normal Chinese revealed that the VNTR is composed of at least 4 different alleles in the Chinese, i.e. 19, 20, 21, and 23 repeats 53% of the females analysed were heterozygous of this VNTR. The discovery and application of this new molecular genetic marker is of significance in genetic consultation and prenatal diagnosis of hemophilia A.

Alleles↗

PubMed1995

[Measurements of the activity of the coagulation factors VIII:C, IX,XI, and XII in cats].

Using an optimized test, reference ranges were established for the activity of the coagulation factors VIII:C (72-124%), IX (81-130%), XI (69-134%) and XII (51-142%) in the cat (n = 58 cats, 2.5-97.5% quantile). Compared to a reference curve prepared by using a cat-pool plasma (n = 50), the factor VIII:C activity in humans (commercial human reference plasma) was 7.6%, i.e. the factor VIII:C activity in cats was 13 fold higher than in humans. The values for the factors IX, XI and XII depended distinctly on the dilution step of the human plasma. The activity of the cat in relation to humans was approximately 90% (factor IX), 170% (factor XI) and 135% (factor XII). Two clinically healthy cats who were conspicuous pre-operative because of a distinctly prolonged, activated partial thromboplastin time showed a decreased factor XII activity.

Animals↗

PubMed1993

Clinical evaluation of viral safety of coagulation factor VIII and IX concentrates.

Virucidal methods to inactivate infectious agents are based on various methods of heating or chemically treating plasma concentrates of coagulation factors VIII and IX used in the treatment of hemophilia A and B. This clinical evaluation of the viral safety of such 'treated' concentrates is mainly based on the prospective study of previously untreated hemophiliacs by means of clinical and serological markers of viral infection. Although there have been a few focal episodes of human immunodeficiency virus (HIV) transmission by clotting factors, these have been traced to ineffective virucidal methods that are no longer used or to clerical errors during the manufacturing process. Viral inactivation by pasteurization, vapor heating, heating in the lyophilized state at 80 degrees C and addition of solvent/detergent definitely decreases the risk of infection with hepatitis B and C. The current screening of plasma units for antibody to hepatitis C virus prior to inclusion in pools for concentrate production should further decrease the risk of hepatitis C infection. Other viruses, such as parvovirus and the hepatitis A virus, may still cause infections because they are quite resistant to virucidal methods. On the whole, virucidal methods have greatly reduced the risk of new HIV infections and, to a lesser degree, hepatitis.

Blood↗

PubMedDate not supplied

Lack of von Willebrand factor, factor VIII related antigen and factor VIII coagulant response to human growth hormone infusion in type 2 diabetes mellitus.

Excessive growth hormone (GH) secretion and platelet hyperaggregation have been considered to be involved in the development of the vascular complications of diabetes mellitus (DM). Trying to find a common link between GH and platelet hyperaggregation, we measured von Willebrand or ristocetin cofactor activity (VIII R:Rcof), factor VIII-related antigen (VIII R:Ag) and factor VIII coagulant activity (VIII:C) in ten type 2 DM (NIDDM) and seven normal control (C) human subjects. These three parameters were measured before (time 0), and after a one-hour intravenous infusion of 0.1 U/kg bw of GH, (times 1, 4, 6, and 24 hours). The NIDDM subjects were nonobese and without clinical evidence of diabetic vascular complications. Despite remarkably high levels of GH reached during the infusion (average 280 ng/ml), there were no significant changes in the measured parameters in either NIDDM or C group. The baseline levels of factors VIII R:Rcof, VIII R:Ag and VIII:C were also not significantly different in the two groups. Changes in GH serum levels seem to have no effect on the factors VIII:C, VIII R:Ag or VIII R:Rcof levels in normals (C) or in NIDDM subjects without evidence of vascular complications. These results do not preclude the possibility that there may be a different response to GH in DM patients with advanced vascular complications and probable endothelial cell abnormalities.

Adult↗

PubMed2003

Recognition of coagulation factor VIII by CD4+ T cells of healthy humans.

Hemophilia A patients treated with coagulation factor (F)VIII may develop an anti-FVIII immune response. Anti-FVIII antibodies may occur also in healthy subjects. To understand the extent to which an immune response to FVIII occurs in healthy subjects, we investigated the proliferative response of blood CD4+ T cells from 90 blood donors to FVIII and to pools of overlapping synthetic peptides spanning the sequences of individual FVIII domains (A1-A3, C1-C2). Most subjects responded to FVIII and several FVIII domains. Men had stronger responses to FVIII than women, and older subjects than younger subjects. The domain-induced responses were weaker than the FVIII-induced responses, yet their intensity in individual subjects correlated with that of the response to FVIII. We examined whether Th1 and/or Th2 cells responded to FVIII in 68 subjects, by determining the CD4+ T cells that secreted interferon-gamma (IFN-gamma) or interleukin (IL)-5 after stimulation with FVIII: 25 subjects had FVIII-specific IFN-gamma-secreting cells, and seven of them had also FVIII-specific IL-5-secreting cells. None had only IL-5-secreting cells. Thus, a CD4+ T cell response to FVIII, which first involves Th1 cells, is common among subjects with a normal procoagulant function.

Adult↗

PubMed1982

Inactivation of factor VIII coagulant activity by two different types of human antibodies.

Human antibodies that inactivate factor VIII procoagulant activity (VIII:C) are heterogeneous in their kinetic properties. We report there here the properties of four type I and four type II antibodies classified according to Biggs et al. Type I antibodies have second-order inactivation kinetics and completely destroy VIII:C when present in high concentration; type II antibodies have more complex kinetics and do not completely inactivate VIII:C even when tested undiluted. The latter properties correspond to the in vivo finding in some patients that there is detectable VIII:C, even though there is also a significant inhibitor titer. It has been suggested that the antibody-antigen complex in these patients retains some VIII:C activity. This is unlikely, however, since protein-A-Sepharose (PAS) did not adsorb any VIII:C activity from mixtures of type II antibodies with normal human plasma. An alternate possibility, reduced VIII:C inactivation due to a steric effect the factor-VIII-related protein (VIIIR, von Willebrand factor), appears to be a more important factor, since three of four type II antibodies had inactivating properties like type I antibodies when they were tested with separated VIII:C instead of plasma. Although the fourth type II antibody did not completely inactivate separated VIII:C the residual coagulant activity was adsorbed from this mixture by PAS. These data indicate that type II anti-VIII:C react with different antigenic determinants than type I antibodies and that these determinants are partially blocked in the factor VIII complex by VIIIR.

Adsorption↗

PubMed1999

Expression of human F8B, a gene nested within the coagulation factor VIII gene, produces multiple eye defects and developmental alterations in chimeric and transgenic mice.

Factor VIII-associated gene B ( F8B ) is a small human gene of unknown function which is nested within the gene encoding coagulation factor VIII ( FVIII ) in chromosome band Xq28. The sequence of F8B includes the C2 cell adhesion motif of factor VIII, which has also been identified in numerous proteins known to play important roles during development. Here we have constructed both chimeric and transgenic mice expressing normal human F8B to investigate its possible developmental effects. The chimeras produced from embryonic stem cells transfected with normal F8B under control of a cytomegalovirus promoter and selected for neomycin resistance expressed readily detectable levels of F8B mRNA in multiple tissues. They showed growth retardation, microcephaly, reduced longevity and severe ocular defects, and although they were fertile, gave birth to no F8B heterozygous pups. Seven transgenic mouse lines, produced by injection of the transgene into fertilized oocytes, were viable and of normal size but expressed lower levels of F8B mRNA. Strikingly, they showed the same severe eye abnormalities as the chimeras. These defects included anterior segment dysgenesis, absent or abnormal lens, persistence of the primary vitreous, Harderian gland tumors and ectopic pigmented cells, suggesting that migration of neural crest cells might have been perturbed during eye development. In addition, dysplastic retinas and the absence of photoreceptors were observed, providing a mouse model for retinal degeneration.

Animals↗

PubMed1982

Activation of human coagulation factor VIII by activated factor X, the common product of the intrinsic and the extrinsic pathway of blood coagulation.

The intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII. This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.

Blood Coagulation↗

PubMed2002

Use of a non-depleting anti-CD4 antibody to modulate the immune response to coagulation factors VIII and IX.

The generation of antibodies to therapeutic factors VIII or IX is a major problem in the management of haemophilia and places potential limitations on the application of gene therapy. We have investigated the administration of a non-depleting anti-CD4 antibody for modulation of the immune response to human recombinant coagulation factors VIII and IX. In mice given these clotting factors, co-administration of anti-CD4 antibody significantly reduced the appearance of factor-specific antibodies. These data provide evidence that the neutralizing antibody response to exogenous coagulation factors may be controllable if non-depleting anti-CD4 antibody is co-administered at the time of initial replacement therapy.

Animals↗

PubMed1982

Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.

Partially purified preparations of porcine factor VIII:C were used to immunize mice and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabelled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines have been obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The third antibody does not inhibit VIII:C, but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/micrograms based on absorbance measurements. When analyzed on SDS gels, the unactivated VIII:C contains 3 bands of apparent molecular weight 166,000, 130,000 and 76,000. Thrombin treatment results in a 40 fold increase in activity and cleavage to products of 76,000, 67,000 an 50,000 and small amounts of lower molecular weight peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca++ dependent noncovalent interaction among the chains.

Animals↗