INFUSION OF MONOCLONAL ANTIBODY IMMUNOAFFINITY PURIFIED FACTOR IX IN RABBITS: COMPARISON WITH COMMERCIAL CONCENTRATES

1987 ◽  
Author(s):  
K J Smith
Keyword(s):  
Monoclonal Antibody ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor V ◽  
At Iii ◽  
Patients At Risk ◽  
Post Infusion

Commercial concentrates (CC) of vitamin K dependent coagulation factors may cause thrombosis or coagulation factor consumption while more highly purified (17 U/mg) factor IX (IX) concentrates do not seem to be thrombogenic (Menache et al, Blood 64:1220, 1984). Monoclonal antibody (MAb) immunoaffinity purified IX of high specific activity from CC or recombinant factor IX sources may also improve therapy. In this report, 400 mg of Affigel-10 linked A-7 MAb was used to bind factor IX in the presence of metal ions (20 mM MgCl2). Elution of IX was with 20 mM EDTA. Thrombogenicity of CC and IX prepared from CC by MAb immunoaffinity was tested. A CC which was thrombogenic in the stasis thrombosis assay (CC #1) produced large thrombi at doses of 50, 50, and 100 U/kg while none were seen with the IX produced from this CC at doses of 106 and 234 U/kg. A heparin treated CC (CC #2) which was not thrombogenic in the stasis-thrombosis assay at doses of 100 U/kg was infused in 4 rabbits at 100 U/kg and platelets, fibrinogen, AT-III antigen, and factors IX, V, and VIII were monitored for 5 hours post-infusion. Immunoaffinity IX from this CC was infused in 4 rabbits at 214-243 U/kg for comparison. Mean platelet count decrease was 20% in CC group and 8% for the IX group. Mean factor V and VIII decreased 26 and 36% respectively with CC while no decrease was seen in the IX rabbits (p < .05). Fibrinogen values and AT-III did not differ for IX or CC groups. Mean factor IX activity at 1 hour increased 1.6 fold for CC and 3 fold for IX. Yields for IX purification were 80 and 85%. Clotting activity was 143 and 101 U/mg and antigen was approximately 200 U/mg. Purification was over 90 fold by MAb immunoaf f inity. There was no detectable factor II, VII or X activity in MAb purified IX. Non-activated PTT was greater than 200 seconds for CC #1 and 158 seconds for CC #2. Column capacity was at least 150 mg. These results demonstrate that factor IX is not the thrombogenic component of some CC. Also, IX prepared by MAb immunoaffinity may have therapeutic advantage for patients at risk for thrombosis and adverse effects of contaminating proteins in commercial concentrates.

scholarly journals МЕТОДОЛОГІЧНІ ПІДХОДИ ОДЕРЖАННЯ ФАКТОРА VIII ЗСІДАННЯ КРОВІ

2020 ◽  
Vol 79 (1-2) ◽  
pp. 102-108
Author(s):  
N. O. Shurko ◽  
V. L. Novak
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Modern Technology ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Protein Therapy ◽  
Biotechnological Approach

The article deals with basic methods used by modern technology to obtain coagulation factor VIII (FVIII). The blood plasma fractionation remains the only biotechnological approach to make life-saving protein therapy to treat human diseases. The biological medicines from human plasma play a vital role in the treatment of patients with different diseases. These products include a range of coagulation factors (FVIII, FIX, the prothrombin complex, Von Willebrand factor, fibrinogen etc.), immunoglobulins, protease inhibitors, anticoagulants and albumin. Four plasma proteins are commercially important for production: albumin, IgG, factor VIII, and factor IX. VIII is a coagulation factor in the blood, which is missing or defective in patients with Hemophilia A. Replacement therapy with FVIII concentrates constitutes the basis for hemophilia care. Cryoprecipitate was described in the mid 60's of the XX century as a first concentrate of antihemophilic FVIII.The main indications for the clinical use of cryoprecipitate were hypofibrinogenemia or disfibrinogenemia. Previously, cryoprecipitate was used for treatment of hemophilia A and von Willebrand’s disease. Traditional FVIII production methods included deposition steps, which were aimed at elimination of protein impurities such as fibrinogen, fibronectin and immunoglobulins. These technologies could use the combination of methods at low temperatures or the addition of protein precipitating substances (PEG, polyvinylpyrrolidone, dextran, ficol, percol etc.). Using chromatographic methods in FVIII production technology allowed receiving high purity and specific activity concentrate of FVIII. Ion exchange chromatography techniques are often used in order to isolate coagulation FVIII. These techniques include methods of affinity chromatography as well as the use of monoclonal antibodies to bind of FVIII. Nowadays, production of plasma concentrate of FVIII is used in combination with different chromatographic techniques.

DEVELOPMENT OF A COAGULATION FACTOR X CONCENTRATE AS A BY-PRODUCT OF COAGULATION FACTOR IX PRODUCTION

1987 ◽  
Author(s):  
Shirley I Miekka ◽  
David B Clark ◽  
Doris Menache
Keyword(s):  
Native Species ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Pilot Scale ◽  
Factor Ix ◽  
Red Cross ◽  
Factor X ◽  
Coagulation Factor Ix ◽  
The Usa

The American Red Cross is developing a Coagu.lation Factor X (FX) concentrate to provide a safer alternative for replacement therapy in Factor X deficient patients, who can experience thromboembolic complications with current treatments. Based on a survey of hemophilia treatment centers, we estimate the frequency of the homozygous disorder to be approximately 1/150th that of hemophilia A, or about 65 patients in the USA. We have devised a method for producing FX as a by-product of our Coagulation Factor IX concentrate (FIX). The method starts with adsorption of cryoprecipitate supernatant plasma with DEAE-Sephadex resin followed by elution of Vitamin K-dependent coagulation factors. This material is adsorbed to sulfated dejctran resin and Factors II and X are eluted by increasing the salt concentration. At 0.45 M NaCl, FII elutes quickly while FX is retarded and can be recovered essentially free of FIX by collecting the slower eluting material. FIX is then recovered at still higher ionic strength. The pooled FX is concentrated, diafiltered and treated to inactivate viruses. Approximately 30% of plasma FX was recovered in pilot scale experiments (600 liters plasma). Specific activity was > 51 FX units / mg protein corresponding to a purity of around 50% and 3000-fold purification over plasma. The ratios of Factors "X : II : IX : Protein C were 1.0 : <0.03 : <0.03 : 0.2. The major contaminant, conprising nearly 50% of the protein, was found to be inter-alpha trypsin inhibitor (IaI), a serine protease inhibitor whose function in plasma has not yet been determined. This inhibitor is also present in the DEAE-Sephadex eluate and. in the FIX concentrate. However, Western blot and HPLC analyses have shown that IaI is present in two different forms.In FX it behaves as expected for the IaI monomer (Mr = 160 kDa), while in the DEAE-eluate and in FIX it exists also in r higher molecular weight form (≥400 kDa) corresponding either to aggregates, complexes or larger native species not previously described. The nature of the possible interaction of Ial w.vdi these coagulation factors is unknown and is currently boinn evaluated.

scholarly journals Evolutionary insights into coagulation factor IX Padua and other high-specific-activity variants

2021 ◽  
Vol 5 (5) ◽  
pp. 1324-1332
Author(s):  
Benjamin J. Samelson-Jones ◽  
Jonathan D. Finn ◽  
Leslie J. Raffini ◽  
Elizabeth P. Merricks ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Activity Factor ◽  
New Variant ◽  
Active Variant

Abstract The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.

Changes Occurring During Coagulation in Glass. I. Normal Human Blood

1960 ◽  
Vol 4 (01) ◽  
pp. 001-016
Author(s):  
Jessica H. Lewis ◽  
Paul Didisheim ◽  
John H. Ferguson ◽  
Kenichi Hattori
Keyword(s):  
Coagulation Factors ◽  
Factor Ix ◽  
Factor Vii ◽  
Sodium Oxalate ◽  
Factor V ◽  
Rapid Rise ◽  
Rapid Fall ◽  
Glass Tubes ◽  
Normal Human ◽  
The Individual

SummaryNormal whole blood was allowed to stand in glass tubes at 37° C, and the clotting process stopped at various intervals by the addition of sodium oxalate. During the first 15 minutes a marked acceleration of clotting activity was found. Study of the individual coagulation factors showed the following changes: a sustained and rapid fall in platelet count, a sustained and rapid rise in PTC (factor IX), a steady fall in fibrinogen, a more gradual fall in AHF (factor VIII), a rapid rise and subsequent fall in proaccelerin (factor V) activity, a somewhat lesser and slower rise and fall in proconvertin (factor VII) activity, and a slow fall in prothrombin concentration. No changes were noted in Hageman factor or PTA activities.

EXPRESSION IN ONTOGENESIS OF HUMAN BLOOD COAGULATION FACTORS

1987 ◽  
Author(s):  
H J Hassan ◽  
A Leonardi ◽  
C Chelucci ◽  
R Guerriero ◽  
P M Mannucci ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Protein C ◽  
Antithrombin Iii ◽  
Liver Homogenate ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Adult Liver ◽  
Blood Coagulation Factors ◽  
Guanidine Isothiocyanate

We have analyzed the expression of several blood coagulation factors (IX, VIII, X, fibrinogen chains) and inhibitors (antithrombin III, protein C) in human embryonic and fetal livers, obtained from legal abortions at 6-11 week post-conception. The age was established by morphologic staging and particularly crown-rump lenght measurement.Total cellular RNA was isolated from partially purified hepatocytes or total liver homogenate using the guanidine isothiocyanate method. Poly(A)+ RNA was selected by oligodT cellulose chromatography. The size and the number of the embryonic and fetal transcripts are equivalent to those observed in adult liver, as evaluated by Northern blot analysis of total or poly(A)+ RNA hybridized to human cDNA probes.The level of coagulation factor transcripts in embryonic and fetal liver was evaluated by dot hybridization of total RNA (0.5-10 ug), as compared to RNA extracted from normal adult liver biopsies. The expression of blood coagulation factors in embryos is generally reduced for all factors, but at a different degree. In 5-11 wk liver, the level of factor IX is 5-10% of that observed in adults, while fibrinogen, protein C, antithrombin III RNA level rises from 25 to 50% and factor X is expressed at a level comparable to that observed in adult liver.We conclude that during these stages of development blood coagulation factors are expressed according to three different time, curves, possibly due to the effect of different types of regulatory mechanisms.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

scholarly journals Biological Rationale for New Drugs in the Bleeding Disorders Pipeline

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 397-404 ◽  
Author(s):  
Patrick F. Fogarty
Keyword(s):  
Coagulation Factor ◽  
Coagulation Factors ◽  
Developed Countries ◽  
Factor Ix ◽  
New Drugs ◽  
Joint Disease ◽  
Novel Agents ◽  
Clotting Factor ◽  
Alternative Pathways ◽  
Long Acting

AbstractSince the introduction of replacement coagulation factor infusions for the treatment of hemophilia in the 1970s and subsequent improvements in the safety profile of available factor VIII (FVIII) and factor IX (FIX) concentrates, mortality among patients with hemophilia has improved considerably and now parallels that of the noncoagulopathic population in developed countries. Substantial morbidity, however, continues from the development of inhibitory antibodies, a recognized complication of clotting factor replacement; from infections and thrombosis complicating placement of central venous catheters, which are required in children with hemophilia due to frequent prophylactic infusions of coagulation factors with defined half-lives; and from disabling joint disease in individuals without access to costly prophylaxis regimens. In response to the need for long-acting, more potent, less immunogenic, and more easily administered therapies, an impressive array of novel agents is nearly ready for use in the clinical setting. These therapeutics derive from rational bioengineering of recombinant coagulation factors or from the discovery of nonpeptide molecules that have the potential to support hemostasis through alternative pathways. The number of novel agents in clinical trials is increasing, and many of the initial results are promising. In addition to advancing treatment of bleeding episodes or enabling adherence to prophylactic infusions of clotting factor concentrate, newer therapeutics may also lead to improvements in joint health, quality of life, and tolerability of iatrogenic or comorbidity-associated bleeding challenges.

Titrating haemophilia B phenotypes using siRNA strategy: evidence that antithrombotic activity is separated from bleeding liability

2015 ◽  
Vol 113 (06) ◽  
pp. 1300-1311 ◽  
Author(s):  
Anil Thankappan ◽  
Walter R. Strapps ◽  
Marti DiPietro ◽  
Karen Leander ◽  
Zuo Zhang ◽  
...  
Keyword(s):  
Target Gene ◽  
Gene Knockout ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Antithrombotic Activity ◽  
Relative Contribution ◽  
Treatment And Prevention ◽  
Novel Approach ◽  
Gene Mrna

SummaryHaemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60–95 %) translating into a 50–99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.

scholarly journals Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant

2017 ◽  
Vol 377 (23) ◽  
pp. 2215-2227 ◽  
Author(s):  
Lindsey A. George ◽  
Spencer K. Sullivan ◽  
Adam Giermasz ◽  
John E.J. Rasko ◽  
Benjamin J. Samelson-Jones ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Activity Factor
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