IMMUNOASSAY FOR FACTOR VIII-HEAVY CHAIN. AN INDICATOR FOR IMMUNE COMPLEXES DURING HIGH DOSE FVIII INHIBITOR TREATMENT

1987 ◽  
Author(s):  
O Nordfang ◽  
M Ezban ◽  
J B Knudsen
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Test Sample ◽  
High Dose ◽  
Fviii Inhibitor ◽  
Normal Human

Specificity studies have shown that most hemophilia A inhibitor antibodies are directed towards the light chain of coagulation factor VIII (FVIII). Thus, conventional immunoassays for FVIII-antigen (FVIII:Ag) presumably have reactivity for FVIII-Light Chain (FVIII-LC) . Our sandwich FVIII :Ag assay has been shewn to be specific for only FVIII-LC. We have now developed a specific immunoassay for FVIII-Heavy Chain (FVIII-HC) . This has made it possible to investigate the FVIII-HC content in hemophilia A plasma, and to study the expression of FVIII-HC in culture medium frcm transfected cell lines.By adding purified FVIII-LC and FVIII-HC in coagulation inhibition assay, plasma frcm one of seven hemophilia A inhibitor patients was found to be reactive with both FVIII-LC and FVIII-HC. IgG frcm this plasma was used for a FVIII-HC specific inhibition radioimmunoassay. The polyspecific antibodies were coated to microplates with removable wells. The coated wells were incubated with test sample and with purified 125I-FVIII-HC. When normal human plasma pool contains 1 U/ml of FVIII-HC, the sensitivity of the assay was 0.004 U/ml.For normal plasma and plasma frcm non inhibitor hemophilia A patients, FVIII-HC measurements correlated with FVIII:C and FVIII-LC measurements. However, after FVIII injection hemophilia A inhibitor patients in high dose FVIII treatment showed a much higher FVIII-HC content (1-5 U/ml) than FVIII-LC and FVIII:C (< 0.05 U/ml). These patients have previously been shown to have antibodies towards FVIII-LC. Therefore the antigen measurements indicate that inhibitor patients in high dose FVIII treatment have FVIII/anti-FVIII-LC immune complexes. These circulating immune complexes may be the mediator of an antibody dependent immune tolerance, during the high dose FVIII treatment.

scholarly journals Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

F VIII Subunits: Purification and Antigenic Properties

1987 ◽  
Vol 58 (04) ◽  
pp. 1043-1048 ◽  
Author(s):  
Ole Nordfang ◽  
Mirella Ezban ◽  
Jan J Hansen
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Hemophilia A ◽  
Exchange Chromatography ◽  
Inhibition Assay ◽  
Cation Exchange Chromatography ◽  
Inhibitor Activity ◽  
Fviii Inhibitor ◽  
Antigenic Properties

SummaryFactor VIII-Light Chain (FVIII-LC) and FVIII-Heavy Chain (FVIII-HC) were purified from human plasma by the use of immunosorbents containing monoclonal antibodies or human inhibitor antibodies. FVIII-LC was subsequently isolated in essentially pure state by cation exchange chromatography. The preparations obtained contained 50 ng of protein for each unit of FVIII-LC antigen (FVIII-LC: Ag).Affinity purified FVIII-LC and FVIII-HC preparations containing less than 0.3% of the opposite subunit were added in FVIILC inhibition assay of hemophilia A inhibitor antibodies. FVIII-LC was able to fully block the inhibitor activity in 6 out of 7 hemophilia A plasmas and partially block the inhibitor activity of one plasma. FVIII-HC only blocked FVIILC inhibiting antibodies form the plasma that was not fully blocked by FVIII-LC. It is suggested that FVIII-LC can be used for immunotherapy of the patients whose FVIILC inhibiting antibodies are directed towards FVIII-LC.When FVIII-LC was coupled to Sepharose at a concentration of 4800 units of FVIII-LC: Ag per ml Sepharose, 0.2 ml of the immunosorbent was able to bind 900 Bethesda units from 100 ml hemophilia A inhibitor plasma. This opens the possibility to remove FVIII inhibitor antibodies from circulation by extracorporeal immunotherapy with FVIII-LC coupled to Sepharose.

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3787-3787
Author(s):  
Pete Lollar ◽  
Ernest T. Parker ◽  
John F. Healey ◽  
Christopher B. Doering
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Analytical Ultracentrifugation ◽  
Conflicts Of Interest ◽  
Sedimentation Coefficient ◽  
Coagulation Factor ◽  
Velocity Sedimentation ◽  
Molar Excess

Abstract Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C–mediated proteolysis

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 669-677 ◽  
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
John C. Giddings ◽  
Kazuya Hosokawa ◽  
Masanori Nagata ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Immune Complexes ◽  
Protein C ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Fviii Activity

Abstract Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with Kdvalues of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation.

The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2038-2038
Author(s):  
Qizhao Wang ◽  
Jenni Firrman ◽  
Katie A Pokiniewski ◽  
Wenjing Cao ◽  
Hongying Wei ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Defect ◽  
High Dose ◽  
Coagulation Activity ◽  
Fviii Activity ◽  
Cofactor Activity

Abstract Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies. In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. Disclosures No relevant conflicts of interest to declare.

scholarly journals A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1767-1775 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
R Prescott
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Coagulation Factor ◽  
Neutralization Assay ◽  
Recombinant Polypeptide ◽  
C2 Domains ◽  
Fviii Inhibitor ◽  
Immunoblotting Assay ◽  
A2 Domain ◽  
Light Chain Antibody

Human antibodies that inactivate coagulation factor VIII (fVIII), known as inhibitors, have been shown by immunoblotting or immunoprecipitation assays to bind predominantly to epitopes within the A2 and/or C2 domains of the fVIII protein. Because these assays simply measure antibody binding, a soluble recombinant polypeptide containing the fVIII A2 domain was used to develop a quantitative inhibitor neutralization assay for antibodies that bound only to A2 by immunoblotting assay. Antibodies from six of eight inhibitor plasmas were fully neutralized by A2 (> or = 90%), whereas two were only partially neutralized. These results established the fVIII inhibitor properties of anti-A2 antibodies. In immunoprecipitation assays, five of the eight inhibitors also had significant levels of anti-light-chain antibody. In one case, this light-chain antibody was shown to have inhibitor activity. Because it did not bind to the C2 domain, this antibody appears to define a new inhibitor epitope within the fVIII light chain. Another inhibitor, which was partially neutralized by A2, was not neutralized by the light chain, even though it contained anti- light-chain antibodies by immunoprecipitation assay. Our results show additional complexities of the immune response to fVIII.

scholarly journals A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1767-1775 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
R Prescott
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Coagulation Factor ◽  
Neutralization Assay ◽  
Recombinant Polypeptide ◽  
C2 Domains ◽  
Fviii Inhibitor ◽  
Immunoblotting Assay ◽  
A2 Domain ◽  
Light Chain Antibody

Abstract Human antibodies that inactivate coagulation factor VIII (fVIII), known as inhibitors, have been shown by immunoblotting or immunoprecipitation assays to bind predominantly to epitopes within the A2 and/or C2 domains of the fVIII protein. Because these assays simply measure antibody binding, a soluble recombinant polypeptide containing the fVIII A2 domain was used to develop a quantitative inhibitor neutralization assay for antibodies that bound only to A2 by immunoblotting assay. Antibodies from six of eight inhibitor plasmas were fully neutralized by A2 (> or = 90%), whereas two were only partially neutralized. These results established the fVIII inhibitor properties of anti-A2 antibodies. In immunoprecipitation assays, five of the eight inhibitors also had significant levels of anti-light-chain antibody. In one case, this light-chain antibody was shown to have inhibitor activity. Because it did not bind to the C2 domain, this antibody appears to define a new inhibitor epitope within the fVIII light chain. Another inhibitor, which was partially neutralized by A2, was not neutralized by the light chain, even though it contained anti- light-chain antibodies by immunoprecipitation assay. Our results show additional complexities of the immune response to fVIII.

scholarly journals Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain

1999 ◽  
Vol 81 (01) ◽  
pp. 39-44 ◽  
Author(s):  
R. Laub ◽  
Di Giambattista ◽  
P. Fondu ◽  
H.-H. Brackmann ◽  
H. Lenk ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hepatitis A ◽  
Hemophilia A ◽  
Hepatitis A Virus ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Inhibitor Titer

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Induction of Human Factor VIII Inhibitors in Rats by Immunization with Human Recombinant Factor VIII: a Small Animal Model for Humans with High Responder Inhibitor Phenotype

1996 ◽  
Vol 75 (02) ◽  
pp. 318-325 ◽  
Author(s):  
M A Jarvis ◽  
L G Levin ◽  
J A Harrison ◽  
D J DePianto ◽  
C M Suzuki ◽  
...  
Keyword(s):  
Antibody Response ◽  
Factor Viii ◽  
Rat Model ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Small Animal ◽  
Response Characteristic ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Human Factor Viii

SummaryHemophilia A is a clotting disorder that is due to reduced or absent coagulation factor VIII (FVIII) activity. In approximately 25% of people with severe hemophilia A, standard treatment with intravenous plasma-derived or recombinant FVIII (rFVIII) induces anti-FVIII antibodies that inhibit FVIII activity (inhibitors). We describe the development of a rat model to study the formation of inhibitors. Immunization of rats with human rFVIII in adjuvant induced an anti-human rFVIII antibody response characteristic of an anti-FVIII inhibitor response in hemophilia A patients. The rats exhibited a rapid, polyclonal secondary antibody response to human rFVIII. These antibodies were reactive against epitopes located in the heavy and light chains. All the rFVIII-immunized rats developed antibodies against the FVIII C2 domain, a region of major reactivity in hemophilia A patients with inhibitors. Furthermore, competition ELISAs demonstrated that rat and human anti-FVIII antibodies recognized identical or overlapping epitopes of the FVIII molecule. The rat anti-FVIII antibodies also functioned as human FVIII inhibitors with titers ranging from 120 to 2048 Bethesda Units (B.U.). We propose that this rat model may be useful to investigate immune responses to FVIII and may lead to better therapies for FVIII inhibitors.

scholarly journals Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

1989 ◽  
Vol 86 (17) ◽  
pp. 6508-6512 ◽  
Author(s):  
D C Hill-Eubanks ◽  
C G Parker ◽  
P Lollar
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Coagulation Factor ◽  
Stable Form ◽  
Proteolytic Activation ◽  
Chain Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

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