Further Studies on the “Dynia” Clotting Abnormality

1967 ◽  
Vol 17 (03/04) ◽  
pp. 365-380 ◽  
Author(s):  
L Pechet ◽  
F Cochios ◽  
D Deykin
Keyword(s):  
Factor Viii ◽  
Intermediate Product ◽  
Coagulation Factor ◽  
Normal Serum ◽  
Factor Ix ◽  
Factor X ◽  
Coagulation Abnormality ◽  
Coagulation Defect ◽  
Coumarin Drugs

Summary1. One patient with multiple bleeding episodes and 4 asymptomatic relatives in 3 generations reveal a coagulation abnormality that cannot be attributed to any known coagulation factor deficiency or to a circulating inhibitor. The abnormality is characterized by deficient generations of intrisic thromboplastin and intermediate product I, and can be corrected by normal plasma and serum.2. The ability of normal blood to correct the coagulation defect in vitro is heat labile; it is not removed from plasma by adsorption with BaSO4 or Al(OH)3. Neonatal blood and blood taken from patients either receiving coumarin drugs or with severe liver disease sustain the ability to correct the described defect. Ox, rabbit and dog blood are also corrective.3. The finding of normal factors XII, XI and IX activities, as well as normal serum thrombosis accelerator (STA) activity, places this abnormality beyond the stage of factor IX activation.4. The data suggest that the familial coagulation abnormality is located in the clotting sequence either in the activation of factor VIII, or in the activation of factor X by factor VIII. Either interpretation is in agreement with the finding of abnormal intermediate product I generation, which reflects primarily the intrinsic activation of factor X.5. The frequency of this defect is unknown. Conceivably some patients considered to have factor XI (PTA) deficiency because of a plasma and serum correctable defect in their thromboplastin generation, may actually have a coagulation defect similar to that described in the Dynia family.6. It is suggested that the name “Dynia defect” be temporarily assigned to this clotting abnormality.

scholarly journals A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

Characterization Of The In Vitro “Factor VIII Bypassing Activity”

1981 ◽  
Author(s):  
D L Aronson ◽  
J Bagley
Keyword(s):  
Factor Viii ◽  
Early Stage ◽  
Deae Cellulose ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor V ◽  
Factor X ◽  
Hemostatic Effect ◽  
Prolonged Aptt

The in vitro correction of the prolonged APTT of hemophilic plasma has been ascribed to an uncharacterized entity “Factor VIII Bypassing Activity.” Such products also correct the prolonged APTT plasma deficient in Factor IX, Factor X and Factor XII, but not of Factor V deficient plasma. Correction of the APTT in Factor VIII deficient plasma by early stage coagulants such as Factor XIIa, Kallikrein and Factor IXa is minimal. These results indicate that this in vitro activity acts at the level of either the activation of Factor X or the activation of prothrombin.A coagulant has been prepared from serum by barium precipitation, heparin-agarose, DEAE cellulose and high pressure liquid chromatography (HPLC). The in vitro coagulant properties are similar to “activated” prothrombin complex (Autoplex) and the biologic and chemical properties are identical to activated Factor X.Infusion of the partially purified serum coagulant into normal dogs was well tolerated and, in contrast to Factor IX concentrates, gave no signs of DIC. Infusion into bleeding hemophilic dogs had no hemostatic effect. It is concluded that a major portion of the in vitro potency of activated prothrombin concentrates is due to activated Factor X, a material which when infused has no in vivo hemostatic effect.Acknowledgments - The authors gratefully acknowledge the studies of Dr. Henry Kingdon in hemophilic dogs.

Reduced Polyethylene Glycol-Conjugated B-Domain–Deleted Factor VIII (PEG-BDD-FVIII) Clearance: Selective Peg Steric Modulation without Affecting Potency

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1471-1471 ◽  
Author(s):  
Eric Blasko ◽  
Lilley Leong ◽  
Derek S Sim ◽  
Liang Tang ◽  
Elena Ho ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Factor Viii ◽  
Half Life ◽  
Thrombin Generation ◽  
Hepatic Clearance ◽  
Factor Ix ◽  
Life Extension ◽  
Factor X ◽  
Plasma Half Life

Abstract Prophylactic factor VIII (FVIII) replacement therapy in hemophilia A requires intravenous administration up to every other day due to the short half-life of FVIII in plasma. Plasma half-life extension of FVIII by polyethylene glycol (PEG) conjugation is thought to be mediated by decreasing hepatic clearance of FVIII. BAY 94-9027 is a rationally designed B-domain–deleted (BDD) FVIII molecule, in which a single 60-kDa PEG molecule was attached to a specific amino acid (1804) to increase its circulating half-life and reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain while preserving full biological function. BAY 94-9027 is currently in clinical trials and has prolonged half-life and improved efficacy in animal models and humans. As a first step in determining whether the half-life extension with BAY 94-9027 is related to steric hindrance exerted by PEG, we investigated whether PEG impacts BAY 94-9027 binding interactions. Direct binding of HKB11-derived FVIII, BAY 94-9027 or BDD-FVIII, was assessed by measuring the ability of a panel of immobilized monoclonal antibodies directed toward different FVIII domains to capture FVIII. Interactions with more physiologic partners were indirectly assessed by thrombin generation assay (TGA) and by an in vitro hepatocyte clearance assay. TGA monitored FVIII-dependent thrombin generation, while the hepatocyte clearance assay assessed the ability of primary human hepatocytes to remove FVIII from the incubation medium. Our results indicate that the presence of the A3-directed PEG reduced BAY 94-9027 capture by immobilized antibodies directed toward the FVIII regions at or near the site of conjugation. Capture by antibodies directed toward the A3 and C2 domains were most impacted, while those directed toward A1 and A2 still bound BAY 94-9027. The A3-specific C7F7 antibody showed ~50% lower capture of BAY 94-9027 vs BDD-FVIII at 20 ng/mL of FVIII. C7F7 capture of PEG-BDD-FVIII was further reduced when a di-PEG conjugate of BDD-FVIII was subjected to the same assay, again confirming that PEG sterically modulates PEG-BDD-FVIII reactivity to the antibody. To determine whether the steric effects observed with PEG may impact FVIII function globally, TGA was performed with BAY 94-9027 spiked into FVIII-deficient plasma and subjected to 1 pM tissue factor initiation. By TGA, both BDD-FVIII and BAY 94-9027 generated comparable peak thrombin levels, with EC50 values of 3.9 and 3.2 nM for BDD-FVIII and BAY 94-9027, respectively. As thrombin generation is a consequence of activated FVIII amplification of factor X activation by activated factor IX, these results indicate that the PEG did not disrupt activated PEG-BDD-FVIII interactions with its partners in the factor Xase enzyme complex, consistent with published PEG-BDD-FVIII efficacy. By hepatocyte clearance assay, PEG-BDD-FVIII clearance was reduced ~30-40% compared with BDD-FVIII, regardless of whether von Willebrand factor was present. This reduction in hepatocyte clearance is likely to contribute to the prolonged plasma half-life reported for BAY 94-9027 (Mei B, et al. Blood. 2010;116(2):270-279; Coyle TE, et al. Journal of Thrombosis and Haemostasis. 2014;12(4):488-496). Disclosures Blasko: Bayer Healthcare: Employment. Leong:Bayer Healthcare: Employment. Sim:Bayer Healthcare: Employment. Tang:Bayer Healthcare: Employment. Ho:Bayer Healthcare: Employment. Wu:Bayer Healthcare: Employment. Kauser:Bayer Healthcare: Employment. Subramanyam:Bayer Healthcare: Employment.

A Factor VIII Minigene Comprising the Truncated Intron I of Factor IX Highly Improves the In Vitro Production of Factor VIII

2001 ◽  
Vol 86 (08) ◽  
pp. 596-603 ◽  
Author(s):  
Jean-Luc Plantier ◽  
Marie-Hélène Rodriguez ◽  
Nathalie Enjolras ◽  
Olivier Attali ◽  
Claude Négrier
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Haemophilia A ◽  
Intracellular Accumulation ◽  
In Vitro Production ◽  
Insertion Sites ◽  
Recombinant Fviii ◽  
Vitro Production

SummaryThe biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

scholarly journals Characterization of Factor VIII-Inhibitor By-Passing Activity (FEIBA)

1977 ◽  
Author(s):  
K. Hess ◽  
N. Shih ◽  
G. Tishkoff
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor Viii Inhibitor ◽  
Factor X ◽  
Clotting Factors ◽  
Human Factor Ix ◽  
Viii Inhibitor

In an attempt to identify the thrombogenic factor in human factor IX concentrates, we have studied the role of trace quantities of activated clotting factors employing an assay that compares the Factor VIII-like activity of IX concentrates with the ability of these products to restore to normal the abnormal activated partial thromboplastin time (APTT) of Factor VIII inhibitor plasma after 1 minute and 40 minute incubation. A coagulant activity (FEIBA) was evident when partially purified Factors X and II were combined in vitro. Factor Xa (4 × 10-4 u) plus Factor II gave negative results. Factor IIa (5.5 × 10-2u), when combined with Factor X, generated FEIBA. Activated clotting factors (Xa, IIa) when tested alone, at comparable levels, were devoid of FEIBA. Our results suggest a mechanism, distinct from activated clotting factors, that can effectively by-pass the Factor VIII defect in the coagulation cascade. The proposed mechanism appears to also by-pass the normal inhibitory properties (i.e., antithrombin III) of human blood.

The Role of Factor VIII in the Activation of Human Blood Coagulation Factor X by Activated Factor IX

1985 ◽  
Vol 54 (03) ◽  
pp. 654-660 ◽  
Author(s):  
K Mertens ◽  
A van Wijngaarden ◽  
R M Bertina
Keyword(s):  
Factor Viii ◽  
Substrate Inhibition ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Catalytic Efficiency ◽  
Enzyme Concentration ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa

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.

Cloning and Characterization of the Murine Coagulation Factor X Gene

2000 ◽  
Vol 83 (05) ◽  
pp. 732-735 ◽  
Author(s):  
Adrian Cooper ◽  
Zhong Liang ◽  
Francis Castellino ◽  
Elliot Rosen
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Factor Viii ◽  
Start Codon ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Coagulation Factor Vii ◽  
Coagulation Factor V

SummaryThe gene encoding murine coagulation factor X (fX) was isolated and characterized from a λFIX II library generated from murine genomic DNA. The 20130 bp sequence contains 18049 nucleotides that extend from the initiating methionine to the polyadenylation site. 1056 nucleotides 5’ of the start codon were determined and contain putative start sites for the FX mRNA as well as sites for binding of putative transcription factors. The sequence extends 1024 3’ of the polyadenylattion site.The gene contains 8 exons and 7 introns which were determined by comparing the mouse FX cDNA and gene sequences. The exonic structure of the gene is similar to that of the other mammalian vitamin K-dependent serine proteases of the coagulation system. These include an exon encoding the prepropepetide, the gladomain, a short helical stack, two exons for the two EGF domains, the activation pepetide, and two exons encoding the serine protease domain. The 5’ sequence of the mouse FX gene overlaps with the 3’ region of the FVII gene indicating that the murine FVII and FX gene are arranged in a head to tail arrangement as they are in humans. Abbreviations: fVII, coagulation factor VII; fIX, coagulation factor IX; fX, coagulation factor X; PC, Protein C; fV, coagulation factor V; fVa, activated coagulation factor V; fVIII, coagulation factor VIII; fVIIIa, activated coagulation factor VIII.

Proteolytic processing of human coagulation factor IX by plasmin

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 943-951 ◽  
Author(s):  
John A. Samis ◽  
Gillian D. Ramsey ◽  
John B. Walker ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coagulation Factor ◽  
Proteolytic Processing ◽  
Factor Ix ◽  
Stable Complex ◽  
Factor X ◽  
Active Site Probe

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 μM) by 80% and the activity of F.IXa (4 μM) by 50% after 30 minutes at 37°C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated–F.IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator–catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

1966 ◽  
Vol 15 (03/04) ◽  
pp. 349-364 ◽  
Author(s):  
A.H Özge ◽  
H.C Rowsell ◽  
H.G Downie ◽  
J.F Mustard
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Factor Ix ◽  
Clotting Factor ◽  
Blood Ph ◽  
Order Of Magnitude ◽  
Dose Dependent ◽  
Generation Test ◽  
Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

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