scholarly journals Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1220-1227 ◽  
Author(s):  
D Menache ◽  
HE Behre ◽  
CL Orthner ◽  
H Nunez ◽  
HD Anderson ◽  
...  
Keyword(s):  
Animal Models ◽  
Specific Activity ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Coagulation Factor Ix

Abstract Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

scholarly journals Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1220-1227
Author(s):  
D Menache ◽  
HE Behre ◽  
CL Orthner ◽  
H Nunez ◽  
HD Anderson ◽  
...  
Keyword(s):  
Animal Models ◽  
Specific Activity ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Coagulation Factor Ix

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Abstract Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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.

Purification and Properties of an Abnormal Blood Coagulation Factor IX (Factor IXBm)/Kinetics of Its Inhibition of Factor X Activation by Factor VII and Bovine Tissue Factor

1981 ◽  
Vol 45 (01) ◽  
pp. 055-059 ◽  
Author(s):  
B Østerud ◽  
C K Kasper ◽  
K K Lavine ◽  
C Prodanos ◽  
S I Rapaport
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Limited Proteolysis ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Factor Xi ◽  
Coagulation Factor Ix ◽  
Bovine Tissue

SummaryAn abnormal blood coagulation factor IX has been isolated from the blood of a hemophilia B patient with a variant of the disease (hemophilia Bm) characterized by a normal concentration of factor IX antigen, negligible factor IX coagulant activity, and a prolonged prothrombin time with bovine tissue factor. The isolated protein (factor IXBm) had the same apparent molecular weight as normal factor IX (55,000) and the same mobility on two dimensional immunoelectrophoresis as normal factor IX. Factor IXBm underwent limited proteolysis induced by activated factor XI, in the presence of Ca2+ ions, or induced by the reaction product of tissue factor, factor VII and Ca2+ ions. A timecourse study showed that activated factor XI cleaved factor IXBm and factor IX at similar rates. However, in contrast to normal factor IX, the limited protelysis of factor IXBm did not generate procoagulant activity.In kinetic experiments purified factor IXBm behaved like a competitive inhibitor (Ki of 0.017 μM) of the activation of factor X by bovine tissue factor and factor VII. Normal factor IX was also found to inhibit the reaction but required a four-fold higher concentration to achieve the same inhibitory effects as factor IXBm.

A Distinction between the Role of Precursor and Activated Forms of Clotting Factors in the Genesis of Stasis Thrombi

1967 ◽  
Vol 18 (01/02) ◽  
pp. 012-023 ◽  
Author(s):  
S Wessler ◽  
E. T Yin ◽  
L. W Gaston ◽  
Iona Nicol
Keyword(s):  
Human Serum ◽  
Factor Ix ◽  
In Vivo Studies ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Insight Into

Summary1. In vivo studies in rabbits have shown that the liver diminishes the thrombo-genicity of infused human serum.2. In vitro rabbit liver perfusions with human serum have demonstrated the loss of serum thrombogenicity within 5 min after the onset of the perfusion. Associated with this loss of thrombotic capacity is a marked decrease in the activation product (AP) and labile factor IX (PPA) activity in the infused serum.3. The liver appears to have the capacity to discriminate between circulating activated clotting activities such as AP and PPA and inactive procoagulants such as stable or genuine factor IX, factor VII and factor X. The latter are not cleared from the circulation by the liver.4. These studies provide some insight into the mechanism whereby circulating activated clotting activities and retarded blood flow predispose to thrombosis.

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 Phosphatidylethanolamine-phosphatidylserine binding synergy of seven coagulation factors revealed using Nanodisc arrays on silicon photonic sensors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara M. Medfisch ◽  
Ellen M. Muehl ◽  
James H. Morrissey ◽  
Ryan C. Bailey
Keyword(s):  
Dissociation Constants ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Clotting Factors ◽  
Protein Z ◽  
Factor Binding ◽  
Silicon Photonic

Abstract Blood coagulation is regulated through protein–protein and protein–lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)—a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.

scholarly journals Determinants of plasma factor VIIa levels in humans

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3021-3025
Author(s):  
S Eichinger ◽  
PM Mannucci ◽  
F Tradati ◽  
AA Arbini ◽  
RD Rosenberg ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Functional Assay ◽  
Factor X ◽  
Clotting Factors ◽  
Plasma Factor

Several enzymes can activate factor VII in vitro, but the protease responsible for generating factor VIIa in vivo has not been determined. Using recombinant tissue factor that has undergone a COOH-terminal truncation, a sensitive functional assay has been established for measuring plasma factor VIIa levels. To evaluate the mechanism responsible for the generation of factor VIIa in vivo, we measured the levels of this enzyme after administering purified concentrates of factor IX and factor VIII to patients with severe deficiencies of these clotting factors. In patients with hemophilia B, factor VIIa levels were initially reduced to 0.5 +/- 0.1 ng/mL and gradually increased to normal after infusing 100 U/kg of body weight (BW) of factor IX. Despite these increases, there were no significant changes in the generation of factor Xa or thrombin. In patients with hemophilia A, only a slight reduction in factor VIIa levels (2.5 +/- 1.3 ng/mL) was observed as compared with controls (3.3 +/- 1.1 ng/mL) and no significant changes were observed after factor VIII levels were normalized. The administration of recombinant factor VIIa (10 micrograms/kg BW) to patients with factor VII deficiency increased the mean circulating level of the enzyme to 118 ng/mL, but this only resulted in normalization of the levels of the activation peptides of factor IX and factor X. The above data indicate that factor IXa is primarily responsible for the basal levels of free factor VIIa generated in vivo (ie, in the absence of thrombosis or provocative stimuli) and that changes in the plasma concentrations of free factor VIIa in the blood do not necessarily lead to alterations in the extent of factor X activation.

scholarly journals Determinants of plasma factor VIIa levels in humans

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3021-3025 ◽  
Author(s):  
S Eichinger ◽  
PM Mannucci ◽  
F Tradati ◽  
AA Arbini ◽  
RD Rosenberg ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Functional Assay ◽  
Factor X ◽  
Clotting Factors ◽  
Plasma Factor

Abstract Several enzymes can activate factor VII in vitro, but the protease responsible for generating factor VIIa in vivo has not been determined. Using recombinant tissue factor that has undergone a COOH-terminal truncation, a sensitive functional assay has been established for measuring plasma factor VIIa levels. To evaluate the mechanism responsible for the generation of factor VIIa in vivo, we measured the levels of this enzyme after administering purified concentrates of factor IX and factor VIII to patients with severe deficiencies of these clotting factors. In patients with hemophilia B, factor VIIa levels were initially reduced to 0.5 +/- 0.1 ng/mL and gradually increased to normal after infusing 100 U/kg of body weight (BW) of factor IX. Despite these increases, there were no significant changes in the generation of factor Xa or thrombin. In patients with hemophilia A, only a slight reduction in factor VIIa levels (2.5 +/- 1.3 ng/mL) was observed as compared with controls (3.3 +/- 1.1 ng/mL) and no significant changes were observed after factor VIII levels were normalized. The administration of recombinant factor VIIa (10 micrograms/kg BW) to patients with factor VII deficiency increased the mean circulating level of the enzyme to 118 ng/mL, but this only resulted in normalization of the levels of the activation peptides of factor IX and factor X. The above data indicate that factor IXa is primarily responsible for the basal levels of free factor VIIa generated in vivo (ie, in the absence of thrombosis or provocative stimuli) and that changes in the plasma concentrations of free factor VIIa in the blood do not necessarily lead to alterations in the extent of factor X activation.

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