Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2267-2273 ◽  
Author(s):  
Kathelijne Peerlinck ◽  
Marc G. Jacquemin ◽  
Jef Arnout ◽  
Marc F. Hoylaerts ◽  
Jean Guy G. Gilles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Ii ◽  
Wild Type ◽  
Mutation Site ◽  
Fviii Activity ◽  
Polyclonal Igg ◽  
Reduced Rate ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2267-2273 ◽  
Author(s):  
Kathelijne Peerlinck ◽  
Marc G. Jacquemin ◽  
Jef Arnout ◽  
Marc F. Hoylaerts ◽  
Jean Guy G. Gilles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Ii ◽  
Wild Type ◽  
Mutation Site ◽  
Fviii Activity ◽  
Polyclonal Igg ◽  
Reduced Rate ◽  
Von Willebrand ◽  
Willebrand Factor

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

scholarly journals Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1151-1153 ◽  
Author(s):  
Shannon L. Meeks ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Rachel T. Barrow ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Xa ◽  
Enzyme Linked Immunosorbent Assay ◽  
C2 Domain ◽  
Type Ii ◽  
Fviii Inhibitor ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Murine Monoclonal Antibodies

Abstract The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa. Group BC antibodies are the most common and display high specific inhibitory activity and type II kinetics. The C2 epitope groups recognized by 26 polyclonal human anti-fVIII inhibitor plasmas were identified by a novel competition enzyme-linked immunosorbent assay using group-specific murine monoclonal antibodies. Most of the anti-C2 inhibitor plasmas inhibited the binding of both classical and nonclassical antibodies. These results suggest that nonclassical anti-C2 antibodies contribute significantly to the pathogenicity of fVIII inhibitors.

The Basis for the Increase in Factor VIII Procoagulant Activity During Exercise

1983 ◽  
Vol 49 (01) ◽  
pp. 053-057 ◽  
Author(s):  
Robert G Kopitsky ◽  
Mary Ellen P Switzer ◽  
R Sanders Williams ◽  
Patrick A McKee
Keyword(s):  
Factor Viii ◽  
Acute Exercise ◽  
Post Exercise ◽  
Fviii Activity ◽  
Plasma Factor ◽  
The Subject ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Healthy Males ◽  
Related Increase

SummaryWe studied the effect of acute exercise on the ability of thrombin to activate plasma factor VIII (FVIII) activity in 20 healthy males. The subject showed an average exercise-related increase in FVIII activity of 54.5±8.2% over pre-exercise FVIII activity (p<0.001). When exposed to the same concentration of thrombin, post-exercise FVIII activity showed greater enhancement than pre-exercise FVIII activity: 157.1±12.8% increase in activity versus 117.3±9.9%, respectively (p<0.01). The degree of the potentiated thrombin effect in post-exercise samples relative to pre-exercise samples was linearly correlated with the degree of the exercise-related increase in FVIII activity. Taken together with our previous observations that the extent of thrombin enhancement of FVIII activity varies inversely with the mole ratio of FVIII/von Willebrand factor subunits to thrombin, these findings imply that release of FVIII does not occur during exercise, and that the exercise-related increase in FVIII activity results primarily, if not completely, from activation of already circulating but inactive FVIII.

scholarly journals The mutation Arg (53)----Trp causes von Willebrand disease Normandy by abolishing binding to factor VIII. Studies with recombinant von Willebrand factor

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 563-567 ◽  
Author(s):  
S Jorieux ◽  
EA Tuley ◽  
C Gaucher ◽  
C Mazurier ◽  
JE Sadler
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Protein Complex ◽  
Von Willebrand Disease ◽  
Cho Cell ◽  
Fviii Activity ◽  
New Variant ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named “Normandy,” characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction.

Identification of Three Non-Overlapping Epitopes in the C2 Domain of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1007-1007
Author(s):  
Shannon Meeks ◽  
John F. Healey ◽  
John (Pete) S. Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Structural Complexity ◽  
Basis Set ◽  
Type I ◽  
C2 Domain ◽  
Type Ii ◽  
Secondary Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. The repertoire of antibodies to the C2 domain is functionally complex, including antibodies that inhibit fVIII binding to phospholipid and von Willebrand factor and display either type I or type II kinetics. The goal of this study was to investigate the diversity of the immune response to the C2 domain of human fVIII in a murine hemophilia A model and to identify a set of non-overlapping epitopes recognized by these fVIII inhibitors. A panel of 55 murine anti-C2 antibodies was obtained, which included 53 antibodies from anti-fVIII hybridomas produced in our laboratory and previously described antibodies ESH-4 and ESH-8. Nine of the hybridomas were cloned by limiting dilution and the corresponding monoclonal IgG antibodies were purified. IgG from the remaining 44 hybridomas was isolated directly from the hybridoma supernatants and biotinylated. The 9 monoclonal antibodies along with ESH-4 and ESH-8 were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The panel of 55 biotinylated antibodies was used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively. A basis set of 3 antibodies, 1B5-1B, 3E6-1B, and (2)117-1B, was defined, which consisted of a set of non-overlapping antibodies that as a group competed for binding of human fVIII with all other antibodies. 1B5-1B and 3E6-1B exhibited type I kinetics with Bethesda titers of 950 and 30, respectively. The Bethesda titer of (2)117-1B could not be determined due to its type II behavior. ESH-4 and ESH-8 had the same epitope profiles as 3E6-1B and (2)117-1B, respectively. Of the 52 non-basis set antibodies, 85% overlapped with 1B5-1B, 29% with 3E6-1B, and 56% with (2)117-1B. The majority of the antibodies overlapped with more than one member of the basis set, leading to the identification of five classes of antibodies (see figure). Because of the large number of antibodies characterized, it is unlikely that the frequency of antibodies in any additional classes is high. The elucidation of the structural complexity of the anti-fVIII C2 repertoire should be useful in the characterization of the pathogenicity of C2 inhibitors. Figure Figure

The Relationship between ABO Groups, Factor VIII, von Willebrand Factor Levels and Platelet Function Analysis Using Cone and Plate(let) Analyzer.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4024-4024
Author(s):  
Maria Lourdes Barjas Castro ◽  
Aline Crucello ◽  
Heloise P. Fernandes ◽  
Norma C. Sousa ◽  
Joyce M. Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Blood Group ◽  
Platelet Function ◽  
Von Willebrand Factor ◽  
Function Analysis ◽  
World Health ◽  
Enzyme Linked Immunosorbent Assay ◽  
Abo Blood Group ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ABO blood group has been described to influence levels of von Willebrand factor (VWF), as well as factor VIII. Individuals carrying O allele have significant lower plasma levels of these factors. Indeed, recently non-O individuals have been described to have increased risk for both, arterial and venous thrombotic disease. VWF mediate platelet interaction with areas of damage blood vessel wall. Thus, it could be interesting to evaluate the possible influence of the ABO group in this interaction, particularly in situations in which low levels of VWF are close to those found in VW disease (such in O group). Cone and plate(let) analyzer (CPA) represent a simple and fast method, that allow the evaluation of platelet function (adhesion as well aggregation) in whole blood under shear conditions, closer to physiological conditions. In this method, no platelet agonists are needed and interaction with fibrinogen and VWF is particularly evaluated. The aim of the present study was to evaluate the influence of ABO group in platelet function using CPA. Samples from 15 male blood donors with no history of drug intake, were submitted to ABO serology and molecular analysis, VWF:Ag, FVIII dosages, and CPA analysis using Impact-R (Diamed - Switzerland), according to manufacturer’s instructions. ABO phenotypes were determined by agglutination test using monoclonal and polyclonal anti-A, B and AB antibodies (Asem-NPBI, São Paulo Brazil; DiaMed SA, Suisse; DiaMed Latino América, Brazil). H antigen was determined using anti-H lectin from Ulex europaeus (DiaMed Latino América, Brazil). ABO genotyping was performed by polymerase chain reaction (PCR) amplification of exons 6 and 7 of the ABO gene, followed by diagnostic restriction enzyme digestion. Factor VIII coagulant was measured by a one stage clothing method using a factor-VIII deficient substrate. VWF:Ag was measured by an enzyme linked immunosorbent assay (ELISA) using polyclonal antiserum (Dako, Denmark). Lyophilised commercial reference preparations of VWF:Ag, and FVIII, standardized against the World Health Organization standard, were used as the standards in this study. The age of the donors ranged from 27–65 years (median = 42 years). The donors were distributed according to ABO groups: 5 = OO; 5 = AB; 5 = AO. Median levels of factor VIII, according to blood group were: OO= 79% (70–142%); AO= 87% (80–140%); AB= 112% (98–200%). Median levels of VWF, according to blood group were: OO= 79% (50–99%); AO= 82% (73–120%); AB= 169% (92–250%). CPA analysis presented the following results: median AS in μm2 (average size) - OO= 24 (23–42); AO= 33 (24–42); AB= 23 (21–24) - median SC in % (surface coverage) - OO= 7.1 (4–13); AO= 8 (5–8); AB= 6.9 (4.8–8). No significant differences using Wilcoxon’s rank sum test were found among groups, when platelet function was analyzed. In conclusion, our results suggest that, although O allele carriers present lower levels of both factor VIII and VWF, the use of platelet function analysis does not seem to predict the risk for bleeding or thrombosis, according to individual ABO blood group.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4663-4670 ◽  
Author(s):  
S. Jorieux ◽  
C. Gaucher ◽  
J. Goudemand ◽  
C. Mazurier
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Stop Codon ◽  
Novel Mutation ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequencing Analysis ◽  
Normal Platelet ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient’s vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient’s vWF. The monoclonal antibody 31H3 against D’ domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D’ domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4663-4670 ◽  
Author(s):  
S. Jorieux ◽  
C. Gaucher ◽  
J. Goudemand ◽  
C. Mazurier
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Stop Codon ◽  
Novel Mutation ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequencing Analysis ◽  
Normal Platelet ◽  
Von Willebrand ◽  
Willebrand Factor

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient’s vWF gene showed, at heterozygous state, a G→A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C→T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient’s vWF. The monoclonal antibody 31H3 against D’ domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D’ domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

Some Factor VIII (FVIII) Inhibitors Recognise a FVIII Epitope(s) that Is Present only on FVIII-vWF Complexes

1999 ◽  
Vol 82 (07) ◽  
pp. 40-45 ◽  
Author(s):  
Renaud Lavend’homme ◽  
Kathelijne Peerlinck ◽  
Marc Jacquemin ◽  
Marc Hoylaerts ◽  
Sylvie Jorieux ◽  
...  
Keyword(s):  
Factor Viii ◽  
B Cell ◽  
Cell Epitope ◽  
Dissociation Rate ◽  
Wild Type ◽  
B Cell Epitope ◽  
C1 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wild-type from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

Decreased factor VIII levels during acetaminophen-induced murine fulminant hepatic failure

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1743-1744 ◽  
Author(s):  
Christopher B. Doering ◽  
Ernest T. Parker ◽  
Christopher E. Nichols ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Fulminant Hepatic Failure ◽  
Hepatic Failure ◽  
Elevated Plasma ◽  
Fviii Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Circulating Levels ◽  
Factor Antigen

Abstract During human fulminant hepatic failure (FHF) circulating levels of most hemostatic proteins fall dramatically. Concurrently, factor VIII (fVIII) procoagulant activity rises despite destruction of the hepatocytes considered responsible for fVIII synthesis. This observation suggests a role for cells other than hepatocytes in fVIII biosynthesis during FHF. We have attempted to identify nonhepatocytic sites of fVIII biosynthesis by inducing FHF in mice using acetaminophen overdose, a common cause of human FHF. Acetaminophen-treated mice consistently displayed signs characteristic of FHF, including elevated plasma aminotransferase activity. However, acetaminophen-treated mice demonstrated markedly reduced fVIII activity, contrary to the observation in human FHF. von Willebrand factor antigen levels were only mildly reduced, suggesting that the decrease in fVIII is not secondary to loss of von Willebrand factor. These results imply that there are fundamental differences in the regulation of plasma fVIII levels between humans and mice.

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