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PubMed1978

[Current aspects on evaluation of assays of factor-VIII activity, factor-VIII associated protein and factor-VIII neutralizing antibody (author's transl)].

In evaluating factor-VIII activity it should be noted that regarding the remaining activity of deficient plasma a linear reference curve is achieved. In the standard population factor-VIII activity and factor-VIII associated protein are distributed approximately lognormally. Due to this distribution certain results have been gained for the optimal choice of localisation and dispersion measures. It is assumed that the proportions of neutralized factor-VIII activity in plasma are distributed according to Poisson. The applicability of the Poisson distribution was also proved for the free factor-VIII activity portion. Due to the Poisson distribution the antibody unit is clearly defined, thus eliminating a further discussion on the establishment of an arbitrary standardized antibody unit.

Antibodies↗
PubMed2002

The evidence behind inhibitor treatment with porcine factor VIII.

Factor VIII auto- and alloantibodies neutralise porcine factor VIII to a lesser extent than factor VIII of human origin. The reduced reactivity of the porcine molecule, predominantly due to sequence variation in the A2 and C2 domains, has been the rationale for using porcine factor VIII to secure haemostasis for patients with factor VIII inhibitors. Porcine factor VIII has been shown to provide effective haemostatic control particularly for patients with intermediate inhibitor titres with limited porcine cross-reactivity. Small studies have indicated porcine factor VIII can be associated with desensitisation of some factor VIII inhibitor patients. Porcine factor VIII has been shown to produce mild platelet agglutination, an effect that may enhance its efficacy. Adverse reactions are dose-related and do not preclude safe and effective long-term home use for the subgroup of inhibitor patients with modest or absent anamnestic response. Efforts to secure source plasma free of viral markers, particularly porcine parvovirus, have limited the supply of this therapeutic product.

Animals↗
PubMed1997

Biosynthesis, assembly and secretion of coagulation factor VIII.

Factor VIII is a large complex glycoprotein that is deficient in hemophilia A. It has a domain organization consisting of A1-A2-B-A3-C1-C2 where the B domain is a heavily glycosylated region that is dispensable for procoagulant activity. Factor VIII expression is 10-to 20-fold lower than the homologous coagulation factor V. Factor VIII expression is limited due to a low level of steady-state messenger RNA in the cytoplasm and inefficient transport of the primary translation product from the endoplasmic reticulum to the Golgi apparatus. Within the secretory pathway, factor VIII is processed to a heterodimer of the heavy chain (domains A1-A2-B) in a metal ion association with the light chain (domains A3-C1-C2). Upon secretion from the cell, von Willebrand factor binds the light chain of factor VIII and stabilizes the factor, preventing degradation. Protein folding within the mammalian secretory pathway is facilitated by molecular chaperones. Within the endoplasmic reticulum, factor VIII exhibits stable interaction with protein chaperones identified as the immunoglobulin-binding protein (BiP), calnexin and calreticulin. BiP is a peptide-dependent ATPase that interacts with exposed hydrophobic surfaces on unfolded proteins or unassembled protein subunits. A potential BiP binding site within factor VIII has been identified. Mutation of a single amino acid residue in the potential BiP binding site increased the secretion efficiency of factor VIII by threefold. Interestingly, the proposed BiP binding site is adjacent to a type-1 copper binding site within the A1 domain that is required for interaction between the factor VIII A1 domain and the A3 domain. We propose that Cu(I) binds the type-1 copper ion-binding site in the A1 domain and provides the essential requirement for a stable interaction between the heavy and light chains. Calnexin and calreticulin are transmembrane and lumenal proteins, respectively, localized to the endoplasmic reticulum, which associate transiently with many soluble and membrane glycoproteins during folding and subunit assembly. The calnexin and calreticulin interaction with factor VIII occurs primarily through amino-terminal linked oligosaccharides within the heavily glycosylated factor VIII B domain and this interaction appears to be required for factor VIII secretion. The findings suggest that factor VIII cycles through interactions with BiP, calnexin and calreticulin. Although the interaction with BiP does not appear to be required for factor VIII secretion, data suggest that the calnexin and/or calreticulin interaction is required for secretion. The observations suggest a unique requirement for carbohydrate processing and calnexin/calreticulin interaction that may limit the productive secretion of factor VIII and have implications for approaches towards somatic cell gene therapy for hemophilia A.

Animals↗
PubMed1988

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII.

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.

Blood Platelets↗
PubMed1981

Acquired haemophilia: functional study of antibodies to Factor VIII.

Factor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR:Ag levels were found in all patients. VIII:WF levels were 50% of those of VIIIR:Ag, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers. Antibody function has been characterized by kinetics of VIII:C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed: 1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (greater than 1). 2) Antibodies from other patients, usually with lower titres, inactivated VIII:C according to complex order kinetics, were not saturable, and had a less steep affinity slope (less than 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

Aged↗
PubMed1991

Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII.

Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.

Binding Sites↗
PubMed1988

Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII).

Factor VIII functions in the intrinsic pathway of coagulation as the cofactor for factor IXa proteolytic activation of factor X. Proteolytic cleavage is required for activation and may be responsible for inactivation of cofactor activity. To identify which of the multiple cleavages are required for activation and inactivation of factor VIII, site-directed DNA-mediated mutagenesis of the factor VIII cDNA was performed and the altered forms of factor VIII were expressed in COS-1 monkey cells and characterized. Conversion of arginine residues to isoleucine residues at the aminoterminal side of the cleavage sites at positions 740, 1648, and 1721 resulted in cleavage resistance at the modified site with no alteration in the in vitro procoagulant activity and the susceptibility to thrombin activation. Similar modification of the thrombin cleavage sites at either position 372 or position 1689 resulted in molecules with residual factor VIII activity but resistant to thrombin cleavage at the modified site and not susceptible to thrombin activation. Modification of the arginine to either an isoleucine or a lysine at residue 336, the site postulated for proteolytic inactivation by activated protein C, resulted in a factor VIII molecule with increased procoagulant activity. This increased activity may result from greater resistance to proteolytic inactivation. A model for the activation and inactivation of factor VIII is proposed.

Blood Coagulation↗
PubMed2005

Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII.

Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of beta-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V forma phospholipid binding site, but residues in the A3 and C1 domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvs WR1 and WR16 (epitope : E2181-M2199) with half saturation values of 23 and 47 muM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factor VIII to C2-specific scFvs WR13 and EL14 (epitope : K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factor VIII to the A3-C1-specific scFvs KM36 (epitope : Q1778-D1840) and KM38 (epitope : S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 microM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.

Antibody Specificity↗
PubMed2005

High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors.

Hemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the down-modulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIII-specific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophilia A. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII.

Animals↗
PubMed1989

In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma.

We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.

Blood Coagulation↗
PubMed2004

Relationships between factor VIII:Ag and factor VIII in recombinant and plasma-derived factor VIII concentrates.

A variety of plasma-derived (pd) and recombinant (r) factor VIII (FVIII) concentrates are used to prevent and treat bleeding in severe hemophilia A patients. A significant side effect of FVIII replacement is the development of FVIII neutralizing antibodies (inhibitors) in up to 30% of patients receiving FVIII concentrates. The FVIII protein content (FVIII:Ag) per unit of FVIII:C in FVIII concentrates, and how effectively the FVIII:Ag in FVIII concentrates binds to von Willebrand factor (VWF) may provide information relevant for the survival of FVIII:C in vivo and for estimating the risk for inhibitor development. The FVIII:Ag content of nine r-FVIII and nine pd-FVIII concentrates were quantified in this study using two enzyme-linked immunosorbent assay (ELISA) platforms. The two ELISA platforms were based on the use of a monoclonal anti-(FVIII light chain)-IgG and polyclonal anti-FVIII antibodies as capture antibodies and both ELISAs were equally able to detect > or =0.005 IU of FVIII:Ag. Measured in international units, the r-FVIII concentrates contained significantly higher FVIII:Ag per unit of FVIII:C than the pd-FVIII concentrates. The VWF-binding profiles of the r-FVIII and pd-FVIII concentrates were also determined by gel filtration chromatography. Unlike the plasma-derived products, the r-FVIII concentrates invariably contained a fraction of FVIII:Ag molecules (approximately 20%) which was unable to associate with VWF. Given that VWF regulates both factor VIII proteolysis and survival of FVIII:Ag in vivo, the fraction of FVIII:Ag unable to bind to VWF may have a reduced survival and be more susceptible to proteolytic degradation in vivo. The extent to which the fractions of FVIII:Ag in concentrates able and unable to bind to VWF contribute to inhibitor development in severe FVIII-deficient patients is unknown.

Blotting, Western↗
PubMed1983

The relationship of biological and immunological activities of factor VIII.

Factor VIII is an essential blood clotting factor which consists of two protein moieties, each with distinct biological functions and antigenic determinants. The immunological markers were originally seen as indicators of the biological activities; however this view has been increasingly challenged. We have investigated the biological and immunological properties of Factor VIII to clarify these relationships. Plasma stored at room temperature for 21 days lost biological activity, but retained immunological activity: The procoagulant activity was reduced to 35% and the ristocetin cofactor activity to 75.4% of their original levels; but the reactivities of both procoagulant antigen and Factor VIII related antigen were maintained. A dissociation of activities was also demonstrated in serum, in which the procoagulant activity was 10% and the procoagulant antigen 72% of corresponding plasma values. These results indicate that the antigenic reactivities are not appropriate markers for Factor VIII biological activity.

Antigens↗
PubMed1992

Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa.

Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

Amino Acid Sequence↗
PubMed1991

Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity.

Human factor VIII and factor VIIIa were proteolytically inactivated by activated protein C. Cleavages occurred within the heavy chain (contiguous A1-A2-B domains) of factor VIII and in the heavy chain-derived A1 and A2 subunits of factor VIIIa, whereas no proteolysis was observed in the light chain or light chain-derived A3-C1-C2 subunit. Reactivity to an anti-A2 domain monoclonal antibody and NH2-terminal sequence analysis of three terminal digest fragments from factor VIII allowed ordering of fragments and identification of cleavage sites. Fragment A1 was derived from the NH2 terminus and resulted from cleavage at Arg336-Met337. The A2 domain was bisected following cleavage at Arg562-Gly563 and yielded fragments designated A2N and A2C. A third cleavage site is proposed at the A2-B junction (Arg740-Ser741) since fragment A2C was of equivalent size when derived either from factor VIII or factor VIIIa. The site at Arg562 was preferentially cleaved first in factor VIII(alpha) compared with the site at Arg336, and it was this initial cleavage that most closely correlated with the loss of cofactor activity. Factor VIIIa was inactivated 5-fold faster than factor VIII, possibly as a result of increased protease utilization of the site at Arg562 when the A2 subunit is not contiguous with the A1 domain. When initial cleavage occurred at Arg336, it appeared to preclude subsequent cleavage at Arg562, possibly by promoting dissociation of the A2 domain (subunit) from the A1/light chain dimer. This conclusion was supported by the failure of protease treated A1/A3-C1-C2 dimer to bind A2 subunit and gel filtration analysis that showed dissociation of the A2 domain-derived fragments, A2N and A2C, from the A1 fragment/light chain dimer. These results suggest a mechanism for activated protein C-catalyzed inactivation of factor VIII(alpha) involving both covalent alteration and fragment dissociation.

Amino Acid Sequence↗
PubMed1982

Role of human factor VIII in factor X activation.

The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.

Blood Coagulation↗