Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII

2011 ◽  
Vol 105 (06) ◽  
pp. 989-998 ◽  
Author(s):  
Koji Yada ◽  
Kenichi Ogiwara ◽  
Masaru Shibata ◽  
Midori Shima ◽  
Keiji Nogami
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Polyclonal Antibodies ◽  
Factor Viii Inhibitor ◽  
Therapeutic Implications ◽  
Fviii Inhibitor ◽  
American Society

SummaryFactor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TFcatalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40–60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose-dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.Note: An account of this work was presented at the 51st annual meeting of the American Society of Hematology, 2009, New Orleans, LA, USA. This work was supported by grants for MEXT KAKENHI 21591370 in Japan and Bayer Hemophilia Award program.

Effects of Anti-FVIII Inhibitors On Factor VIIa/Tissue Factor-Catalyzed Activation and Inactivation of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3169-3169
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Normal Individuals ◽  
The Difference ◽  
Functional Inactivation

Abstract Abstract 3169 Poster Board III-110 Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. We have recently demonstrated that FVIIa/TF rapidly activated FVIII by proteolysis of the heavy chain (HCh), and served physiologically as a potent activator for up-regulation of FVIII activity in very early-timed phase (ASH #1036, 2008). FVIII inhibitors develop as alloantibodies in multi-transfused patients with hemophilia A and also arise as autoantibodies in normal individuals. FVIII inactivation by inhibitors is associated with impairment of FVIII(a) cofactor function through the binding to functional crucial epitopes in FVIII. Anti-C2 inhibitors prevent FVIII binding to phospholipid, von Willebrand factor, and FXa. Anti-A2 inhibitors prevent FVIII binding to FIXa and thrombin. However, effects of these inhibitors on FVIIa action for FVIII have remained to be studied. In this study, we prepared 13 of anti-FVIII inhibitor IgGs (2 of anti-A2, 7 of anti-C2 with type 1 behavior, and 4 of anti-C2 with type 2). We first examined FVIIa/TF-catalyzed FVIII activation in the presence of anti-FVIII inhibitors in one-stage clotting assay. The levels of FVIII activity (10 nM) elevated rapidly by ∼2.0-fold within 30 sec after adding of FVIIa/TF (1 nM), and subsequently decreased to the initial level within 20 min. The presence of anti-FVIII inhibitors did not significantly affect FVIIa/TF-catalyzed FVIII activation (by 1.7∼2.2-fold) compared to normal IgG. This action was independent of the difference of inhibitor epitopes. In addition, FVIIa-catalyzed FVIIIa inactivation with anti-A2 or anti-C2 with type 2 inhibitors was little affected, similar to that with normal IgG. However, of note, all of anti-C2 with type 1 significantly inhibited FVIIa-catalyzed inactivation of FVIIIa. Inactivation rates of FVIIa with anti-C2 with type 1 (k ∼0.15) was ∼40% less than that with control IgG (k ∼0.24), supporting that the presence of anti-C2 with type 1 might persist the activity of FVIIIa generated by FVIIa. To clarify this inhibitory mechanism of anti-C2 with type 1, we performed FVIIa-catalyzed FVIII cleavage in Western blotting. FVIIa/TF (1 nM) proteolyzed the HCh of FVIII (10 nM) rapidly by cleavages at Arg372 (and Arg740), whilst cleavage at Arg336 in the A1 domain was appeared at ∼2.5 min, supporting that cleavages at Arg372 and Arg336 by FVIIa contribute to the up- and down-regulation of FVIII(a) activity, respectively. All inhibitors, independent of recognizing epitopes, did not affect FVIIa-catalyzed cleavage at Arg372. However, the presence of anti-C2 type 1 delayed the cleavage at Arg336 in timed- and dose-dependent manners, whilst either anti-A2 or anti-C2 type 2 did not affect, consistent with the functional inactivation results. FVIIa binds to the A2, A3, and C2 domains in FVIII. Based on our findings, FVIIa-interactive sites on FVIII unlikely overlapped with anti-A2 and -C2 inhibitor epitopes, and inhibition of Arg336 cleavage may be due to conformational change caused by antibody binding. Furthermore, FVIIa indeed activates FVIII even in the presence of anti-FVIII inhibitors, different from thrombin, FXa, etc, and it would be important to predict the effect of FVIIa for FVIII to determine the characteristics of anti-FVIII inhibitors. Disclosures No relevant conflicts of interest to declare.

Molecular analysis of FVIII gene in severe HA patients of Costa Rica

2010 ◽  
Vol 30 (S 01) ◽  
pp. S150-S152
Author(s):  
G. Jiménez-Cruz ◽  
M. Mendez ◽  
P. Chaverri ◽  
P. Alvarado ◽  
W. Schröder ◽  
...  
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Costa Rican ◽  
Factor Viii Gene ◽  
Intron 22 Inversion ◽  
Intron 1 ◽  
Polymerase Chain ◽  
Inversion Analysis

SummaryHaemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

Human Factor VIII Inhibitor Alloantibodies with a C2 Epitope Inhibit Factor Xa-catalyzed Factor VIII Activation: A new Anti-factor VIII Inhibitory Mechanism

2002 ◽  
Vol 87 (03) ◽  
pp. 459-465 ◽  
Author(s):  
Keiji Nogami ◽  
Katsumi Nishiya ◽  
Yoshihiko Sakurai ◽  
Ichiro Tanaka ◽  
John Giddings ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Inhibitory Mechanism ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Human Factor Viii ◽  
Sds Page ◽  
Viii Inhibitor ◽  
C1 Domains

SummaryFactor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-C1 domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in onestage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)’2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

scholarly journals Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1811-1819 ◽  
Author(s):  
D Scandella ◽  
GE Gilbert ◽  
M Shima ◽  
H Nakai ◽  
C Eagleson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Intrinsic Factor ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Fviii Inhibitor ◽  
Human Factor Viii

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1330-1335 ◽  
Author(s):  
Cornelis van 't Veer ◽  
Neal J. Golden ◽  
Kenneth G. Mann
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Factor Vii ◽  
Lag Phase ◽  
Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

scholarly journals A putative inhibitory mechanism in the tenase complex responsible for loss of coagulation function in acquired haemophilia A patients with anti-C2 autoantibodies

2012 ◽  
Vol 107 (02) ◽  
pp. 288-301 ◽  
Author(s):  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Midori Shima ◽  
Keiji Nogami
Keyword(s):  
Waveform Analysis ◽  
Haemophilia A ◽  
Inhibitory Mechanism ◽  
Abstract Form ◽  
Phospholipid Binding ◽  
Coagulation Function ◽  
Acquired Haemophilia ◽  
American Society

SummaryAcquired haemophilia A (AHA) is caused by the development of factor (F)VIII autoantibodies, demonstrating type 1 or type 2 inhibitory behaviour, and results in more serious haemorrhagic symptoms than in congenital severe HA. The reason(s) for this remains unknown, however. The global coagulation assays, thrombin generation tests and clot waveform analysis, demonstrated that coagulation parameters in patients with AHA-type 2 inhibitor were more significantly depressed than those in patients with moderate HA with similar FVIII activities. Thrombin and intrinsic FXa generation tests were significantly depressed in AHA-type 1 and AHA-type 2 compared to severe HA, and more defective in AHA-type 1 than in AHA-type 2. To investigate these inhibitory mechanism(s), anti-FVIII autoantibodies were purified from AHA plasmas. AHA-type 1 autoantibodies, containing an anti-C2 ESH4-epitope, blocked FVIII(a)-phospholipid binding, whilst AHA-type 2, containing an anti-C2 ESH8-epitope, inhibited thrombin-catalysed FVIII activation. The coagulation function in a reconstituted AHA-model containing exogenous ESH4 or ESH8 was more abnormal than in severe HA. The addition of anti-FIX antibody to FVIII-deficient plasma resulted in lower coagulation function than its absence. These results support the concept that global coagulation might be more suppressed in AHA than in severe HA due to the inhibition of FIXa-dependent FX activation by steric hindrance in the presence of FVIII-anti-C2 autoantibodies. Additionally, AHA-type 1 inhibitors prevented FVIIIa-phospholipid binding, essential for the tenase complex, whilst AHA-type 2 antibodies decreased FXa generation by inhibiting thrombin-catalysed FVIII activation. These two distinct mechanisms might, in part, contribute to and exacerbate the serious haemorrhagic symptoms in AHA.Presented in abstract form at the 52nd annual meeting of the American Society of Hematology, Orlando, Florida, USA, December 6, 2010.

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