scholarly journals The role of emicizumab, a bispecific factor IXa- and factor X-directed antibody, for the prevention of bleeding episodes in patients with hemophilia A

2018 ◽  
Vol 9 (10) ◽  
pp. 319-334 ◽  
Author(s):  
Tristan Knight ◽  
Michael U. Callaghan

Hemophilia A, characterized by impaired or absent expression of factor VIII, has long been managed via direct factor replacement. Functionally, factor VIII acts as a cofactor for factor IXa and allows activation of factor X, which, in combination with factor V, generates thrombin. Bispecific antibodies such as emicizumab are recombinant, monoclonal antibodies capable of recognizing and binding to two distinct antigenic targets simultaneously; emicizumab binds factors IXa and X, resulting in spatial approximation and activation of factor X, thereby mimicking the actions of factor VIII. Critically, the presence of antifactor VIII antibodies, for example, inhibitors, impacts neither the mechanism nor the efficacy by which emicizumab functions. The results and interim analyses of the emicizumab clinical trials, HAVEN 1, 2, 3, and 4, are additionally reviewed and discussed.

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman

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.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.


2000 ◽  
Vol 83 (03) ◽  
pp. 387-391 ◽  
Author(s):  
I.R. Walker ◽  
J. Teitel ◽  
M.-C. Poon ◽  
B. Ritchie ◽  
J. Akabutu ◽  
...  

SummaryTo determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.


1981 ◽  
Author(s):  
T J Frost ◽  
R S Lau

The cascade mechanism for the coagulation pathway has prevailed since its original proposal, and subsequent observations have supported the role of thrombin on coagulation reactions other than in Fibrinogen - Fibrin conversion. In very small quantities, thrombin has been reported as increasing the activity of Factor VIII: C. We report here, observations which support a reverse proposal, namely that Factor VIII: C increases activity of thrombin in Fibrinogen - Fibrin conversion.Thrombin clotting times were performed employing human Fibrinogen solutions, fresh citrated normal platelet - poor human plasma and fresh citrated platelet - poor plasma from severely affected Hemophilia A patients, in whom Factor VIII : C assays were less than 1% in a one stage, activated partial thromboplastin time assay. A platelet aggregometer was used to assess the thrombin time, and the rate of clot formation (fibrin polymerization) was indicated by optical density change in the form of a curve, the tangent of the maximal slope of curve being equated with this clot formation rate.Results indicate that with very small concentrations of thrombin, Factor VIII: C enhances the rate of fibrin clot formation and that Factor VIII: C restores the abnormal rate of clot formation observed in severe Hemophilia A; the test system was known to be calcium-free, which ensured that activation of Factor X did not ensue. The concentration of Fibrinogen remained constant in all experiments. The observed phenomena are confirmed when employing purified fibrinogen solutions, using decreasing amounts of Factor VIII: C.


1985 ◽  
Vol 54 (03) ◽  
pp. 654-660 ◽  
Author(s):  
K Mertens ◽  
A van Wijngaarden ◽  
R M Bertina

SummaryThe role of factor VIII in the activation of human factor X by factor IXa, Ca2+ and phospholipid has been investigated. Factor VIII stimulated the factor Xa formation after activation by factor Xa or thrombin; the activity of thrombin-activated factor VIII was about 4-fold that of factor Xa-activated factor VIII. The isolated procoagulant moiety of the factor VIII complex behaved identically to the complete complex, whereas the von Willebrand factor moiety did not participate in the factor Xa formation. Thrombin-activated factor VIII complex (factor Villa) was used to study the effect of factor Villa in kinetic experiments. The results revealed a complex kinetic behaviour, including substrate inhibition and non-linearity of the reaction rate with the enzyme concentration. Using previously obtained insight into the kinetics of factor X activation in the absence of factor VIII, the results were found to support the hypothesis that factor Villa participates in the factor Xa formation in a complex with phospholipid-bound factor IXa; the formation of the factor VUIa-factor IXa complex then increases the catalytic efficiency of the factor IXa by 500-fold.


1977 ◽  
Author(s):  
Earl W. Davie ◽  
Gordon Vehar ◽  
Kazuo Fujikawa ◽  
Richard Di Scipio

Factor IXa and factor VIII participate in the middle phase of blood coagulation. These two proteins convert factor X to factor Xa in the presence of calcium ions and phospholipid. The coagulant activity of factor VIII is increased 50-100 fold by the addition of thrombin, and this activity is stabilized in the presence of CaCl2. The activated product (tentatively identified as activated factor VIII) was readily inhibited by diisopropyl phosphorofluoridate or antithrombin III, suggesting that it is a serine enzyme. The exact role of this enzyme in the conversion of factor X to factor Xa, however, is not known. When factor X (bovine or human) is converted to factor Xa, an activation peptide is cleaved from the amino-terminal end of the heavy chain. This gives rise to a new amino-terminal sequence of Ile-Val-Gly-Gly-in the heavy chain. No change occurs in the light chain during the activation reaction. These data indicate that the basic mechanism involved in the conversion of human and bovine factor X to factor Xa appears to be essentially identical and probably involves the formation of a charge relay system characteristic of the pancreatic serine proteases.


Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 928-940
Author(s):  
MB Hultin ◽  
Y Nemerson

We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.


Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 928-940 ◽  
Author(s):  
MB Hultin ◽  
Y Nemerson

Abstract We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.


1967 ◽  
Vol 18 (01/02) ◽  
pp. 211-222 ◽  
Author(s):  
C Hougie ◽  
K. W. E Denson ◽  
Rosemary Biggs

SummaryThe products of the reaction of activated factor IX with factor VIII, phospholipid and calcium have been studied using gel filtration on Sephadex G-200. Both factor VIII and phospholipid are completely excluded from the gel (distribution coefficient or KD of zero) while factor IXa behaves like a protein similar in size to albumin (KD approx. 0.4). Factor IXa complexes with phospholipid when calcium ions are added to the eluting buffer. When factor VIII, factor IXa, phospholipid and calcium are applied to the column and eluted with added Ca++ in the eluting buffer, a labile product with a KD of 0 is formed. The product apparently dissociates in the absence of added calcium ions in the eluting buffer. In the presence of calcium ions, the distribution coefficient of factor IX, when applied alone to the column, increased. The above findings are analogous to those reported between activated factor X, thrombin-activated factor V, phospholipid and calcium occurring later in the coagulation process.


1982 ◽  
Vol 47 (02) ◽  
pp. 096-100 ◽  
Author(s):  
K Mertens ◽  
R M Bertina

SummaryThe 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.


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