Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

SummaryAbetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyce-ride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 μmol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients’ mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.

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The Activation of Human Factor X in Sodium Citrate: the Role of Factor VII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648014 ◽

1976 ◽

Vol 36 (01) ◽

pp. 104-114 ◽

Cited By ~ 3

Author(s):

D. L Aronson ◽

A. J Mustafa

Keyword(s):

Sodium Citrate ◽

Human Factor ◽

Deae Cellulose ◽

Factor Ix ◽

Factor Vii ◽

Factor X

SummaryHuman factor X was purified by several different procedures yielding products which had varying amounts of factor VII and factor IX. Treatment with CHC13 during the fractionation of the factor X removed 95% of the factor VII and factor IX activity and the resulting factor X activated more slowly when incubated in 25% sodium citrate. Removal of residual factor VII by DEAE cellulose chromatography yielded a factor X which activated still more slowly and less completely. When the factor VII, removed by chromatography, was added to the chromatographed factor X, the ability to be activated in 25% sodium citrate was restored. Confirmatory evidence for the role of factor VII in this reaction was the inhibition of the conversion of the factor X by both DFP and SBTI.

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Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽

10.1182/blood.v66.1.204.bloodjournal661204 ◽

1985 ◽

Vol 66 (1) ◽

pp. 204-212

Author(s):

NL Sanders ◽

SP Bajaj ◽

A Zivelin ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Additional Material ◽

Plasma Reaction ◽

Progress Curve ◽

Peptide Release ◽

Release Data

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

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Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽

10.1182/blood.v66.1.204.204 ◽

1985 ◽

Vol 66 (1) ◽

pp. 204-212 ◽

Cited By ~ 100

Author(s):

NL Sanders ◽

SP Bajaj ◽

A Zivelin ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Additional Material ◽

Plasma Reaction ◽

Progress Curve ◽

Peptide Release ◽

Release Data

Abstract A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

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Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽

10.1182/blood.v69.2.645.645 ◽

1987 ◽

Vol 69 (2) ◽

pp. 645-651 ◽

Cited By ~ 137

Author(s):

LV Rao ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Extrinsic Pathway ◽

Mechanism Of Inhibition ◽

Peptide Release ◽

Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

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Neonatal Polycythemia: A Disorder Associated With Hyperviscosity, Normal Coagulation Findings And Thrombocytopenia

10.1055/s-0038-1652910 ◽

1981 ◽

Author(s):

J Katz ◽

E Rodriguez ◽

C Madani ◽

D Hicks ◽

H E Branson

Keyword(s):

Plasma Exchange ◽

Gestational Age ◽

Degradation Products ◽

Factor Ix ◽

Factor Vii ◽

Large For Gestational Age ◽

Factor Xii ◽

Factor X ◽

Platelet Counts ◽

Before And After

Thirty-two newborns with elevated capillary hematocrits >65% were studied. Twenty-two newborns required plasmaexchange transfusion. All had central (venous) hematocrits >65% and had symptoms referrable to complications associated with this syndrome. Of the 22, 15 were appropriate-for-gestational age, 5 were small-for-gestational age, and 2 were large-for-gestational age. Viscosity measurements in the 10 newborns who did not require plasma-exchanges showed increased viscosity in 2 in the slow shear rates associated with bloodflow in the smaller vessels. Coagulation data before and after plasma exchange did not show a hypercoagulable state: PT-14.2±0.7 and 12.9±1.2 secs, PTT 49.9±3.6 and 42.2±3.2 secs, factor VII 73±5 and 78±5%, factor VIII 103±10 and 94±10%, AT III levels were low 14±1.2 and 17±1.3 mg/dl, fibrin degradation products were <10μg/ml, fibrin monomer was not detected, plasminogen levels were 5±0.8 and 7±0.9mg/dl, fibrinogen levels were 203±9.8 and 200±11.8 mg%. Vitamin K dependent factors were reduced factor V 44±6 and 49±11%, factor VII 77±5 and 86±5%, factor IX 28±2 and 42±3%, factor X 35±4 and 62±6%, factor XI 55±5 and 84±9%, factor XII 47±5 and 63±5%. Statistical significant differences were found only with factors IX, X, XI and XII. Thrombocytopenia was present in 6 patients (20% incidence) and post plasma exchange the platelet counts rose significantly and in 2 patients within 3 days reached normal levels. No statistical difference in the platelet counts were noted before and after the plasma-exchange and were similar to the levels determined in 10 newborn controls. Neonatal polycythemia with thrombocytopenia may indicate a more severe disorder, with hematocrits in the 6 patients >70%. It is suggested that the mechanism of the thrombocytopenia may be aggregates of platelets that deaggregate following plasmaexchange. The complications associated with neonatal polycythemia appear related to hyperviscosity, erythrocyte and platelet “sludging” in the smaller vessels.

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Prothrombin Activation Is Increased among Asymptomatic Carriers of the Prothrombin G20210A and Factor V Arg506Gln Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614034 ◽

2000 ◽

Vol 84 (09) ◽

pp. 396-400 ◽

Cited By ~ 15

Author(s):

Steve Humphries ◽

Belinda Smillie ◽

Lily Li ◽

Jacqueline Cooper ◽

Samad Barzegar ◽

...

Keyword(s):

Factor Viia ◽

Conclusive Evidence ◽

Factor Ix ◽

Factor Vii ◽

Prothrombin G20210a ◽

Factor V ◽

Factor X ◽

Coagulation Proteins ◽

Prothrombin Activation

SummaryThe risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50–61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G20210A and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment F1+2 [mean ± SD, 0.88 ± 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 ± 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 ± 0.24 among non-carriers for either mutation]. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.

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Model of a Ternary Complex between Activated Factor VII, Tissue Factor and Factor IX

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613157 ◽

2002 ◽

Vol 88 (07) ◽

pp. 74-82 ◽

Cited By ~ 24

Author(s):

Shu-wen Chen ◽

Jean-François Schved ◽

Jean-Luc Pellequer ◽

Muriel Giansily-Blaizot

Keyword(s):

Tissue Factor ◽

Ternary Complex ◽

Search Algorithm ◽

Factor Ix ◽

Site Directed Mutagenesis ◽

Coagulation Cascade ◽

Factor Vii ◽

Factor X ◽

Three Dimensional Model ◽

Inhibitory Peptide

SummaryUpon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

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Novel Insights and New Developments Regarding Coagulation Revealed by Studies of the Anti-Factor IXa (Activated Factor IX)/Factor X Bispecific Antibody, Emicizumab

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.312919 ◽

2020 ◽

Vol 40 (5) ◽

pp. 1148-1154

Author(s):

Koji Yada ◽

Keiji Nogami

Keyword(s):

Hemophilia A ◽

Activated Protein C ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

New Developments ◽

Activated Factor Vii ◽

Factor Ixa ◽

Thrombin Activation ◽

No Activation

Emicizumab is a humanized anti-FIXa/FX (factor IXa/X) bispecific monoclonal antibody that mimics FVIIIa (activated factor VIII) cofactor function. The hemostatic efficacy of emicizumab has been confirmed in clinical studies of patients with hemophilia A, irrespective of the presence of FVIII inhibitors. Emicizumab differs in some properties from FVIIIa molecule. Emicizumab requires no activation by thrombin and is not inactivated by activated protein C, but emicizumab-mediated coagulation is regulatable and maintains hemostasis. A small amount of FIXa (activated factor IX) is required to initiate emicizumab-mediated hemostasis, whereas tissue factor/FVIIa (activated factor VII)-mediated FXa (activated factor X) and thrombin activation initiates FVIIIa-mediated hemostasis. Fibrin formation, followed by fibrinolysis, appears to be similar between emicizumab- and FVIIIa-mediated hemostasis. These results suggest possible future uses of emicizumab for treating hemorrhagic diseases other than hemophilia A and reveal previously unobservable behaviors of procoagulation and anticoagulation factors in conventional hemostasis. Here, we have reviewed novel insights and new developments regarding coagulation highlighted by emicizumab.

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Regular Prophylactic Treatment with Factor IX P® in a Patient with Severe Factor X Deficiency.

Blood ◽

10.1182/blood.v108.11.4050.4050 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4050-4050

Author(s):

Andrea Gerhardt ◽

Fatima Araba ◽

Rainer B. Zotz ◽

Rudiger E. Scharf

Keyword(s):

Joint Pain ◽

Prophylactic Treatment ◽

Factor Ix ◽

Factor Vii ◽

Coagulation Disorder ◽

Factor X ◽

Dental Surgery ◽

Thrombotic Risk ◽

Factor X Deficiency ◽

Patient Reported

Abstract Background: Congenital factor X deficiency, a rare coagulation disorder with variable severity, is an inherited autosomal recessive disorder. The incidence of homozygous factor X deficiency is ~ 1 in 1 million of the general population. The gene encoding for factor X is found adjacent to that encoding for factor VII on chromosome 13q34. Bleeding sites vary according to the severity of the deficiency. Mucocutaneous soft tissue hemorrhages, including menorrhagia in women, are common. Hemarthros, exsanguinating postoperative hemorrhage, pseudotumors, and hemorrhages of the central nervous system have been reported in severely affected patients. Mildly affected patients experience easy bruising and excessive bleeding after trauma or surgery. Treatment options consist of fresh frozen plasma (FFP), prothrombin complex concentrates (PCC) containing factor X or pasteurized Factor IX P® (ZLB Behring). Disadvantage of FFP is the large infusion volume, potential viral transmission, and no standardized factor X content. These aspects, in addition to the thrombotic risk, also need to be addressed for the PCCs. Factor IX P®, which is virus inactivated, contains almost equal amounts of factor IX (1200 IU) and X (800 IU) and suits therefore well for the treatment of factor X deficiency. Case report: We report on our experience of prophylactic treatment with Factor IX P® in a 31-year-old male with severe factor X deficiency (< 1%) associated with a homozygous Cys350Phe mutation in exon 8 on chromosome 13. After birth the patient experienced severe mucosal bleedings and haematomas and later on various joint bleedings with consecutive hemophilic arthropathy. Initially he received FFP on demand and later regular prophylaxis with PCC (containing 600 IU factor X) 2 to 3 times a week (~ 20–25 IU/kg/bw), age at onset of prophylaxis ~ 7 years. The patient is positive for HIV, HCV, and HBV (known since 1984). He is now on regular prophylaxis with Factor IX P® since 7 months. The prophylaxis is given 2 times a week in doses of ~ 20 IU/kg bw. The trough level after 72 hours was 12% using PCC and 20% using Factor IX P®. The patient reported on joint pain when factor X activities were below 20%. The rate of joint pain episodes is lower when using Factor IX P® two times a week as compared to PCC two to three times a week. Orthopedic and dental surgery were performed using Factor IX P® concentrate with excellent hemostatic effect, no thromboembolic complications, and no adverse drug reactions. In conclusion, prophylactic treatment with Factor IX P® in severe factor X deficient patients appears to be an effective and safe therapeutic option.

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