scholarly journals Production of a High-Titred Sheep Antiserum Against Human Factor IX and Immunological Demonstration of Factor IX Aggregates

1977 ◽  
Author(s):  
K.H. Ørstavik ◽  
A.M. Vennerød
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Rabbit Antiserum ◽  
Factor Ix ◽  
Factor X ◽  
Weak Band ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Plasma Factor

Plasma factor IX was purified from a factor IX concentrate by a five step procedure including absorption onto aluminium hydroxide, affinity chromatography on heparin-coupled Sepharose 4B, preparative disc gel electrophoresis, affinity chromatography on an immunosorbent column with rabbit antiserum against factor X and chromatography on DE-52 cellulose. The pooled fractions had a specific activity of approximately 250 U/mg protein. A sheep was immunized with pooled and concentrated fractions. An antiserum was produced which gave one main precipitin band and occasionally an additional weak band against normal plasma in double immunodiffusion. At a dilution of 1/100-1/200 the antiserum neutralized 90% of the factor IX activity in an equal volume of normal plasma.Polyacrylamide disc gel electrophoresis of the fractions from DE-52 cellulose revealed one major and three minor bands with lower electrophoretic mobility and intensity. The three minor bands disappeared on disc gel electrophoresis in the presence of 10 M urea. When the disc electrophoresis gel was submitted to electrophoresis into anagarose gel containing the sheep antiserum or a previously characterized rabbit antiserum against factor IX, four precipitin arcs corresponding to the four bands were seen. A reaction of identity was seen between the four arcs. This study demonstrates that a highly potent antiserum may be produced against factor IX in sheep.

scholarly journals Development of Monospecific Antibodies to Human Factor IX

1977 ◽  
Author(s):  
Cheryl Y. Tiarks ◽  
Chin-Hai Chang ◽  
Liberto Pechet
Keyword(s):  
Neutralizing Antibodies ◽  
Human Factor ◽  
Human Albumin ◽  
Factor Ix ◽  
Hemophilia B ◽  
Red Cross ◽  
Factor X ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

Isolation of carboxypeptidase N by affinity chromatography on column of CNBr-activated Sepharose with immobilized antibody

1979 ◽  
Vol 44 (2) ◽  
pp. 626-630 ◽  
Author(s):  
Eva Simonianová ◽  
Marie Petáková
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Serum Analysis ◽  
Polyacrylamide Gel ◽  
Rat Serum ◽  
Carboxypeptidase N

The isolation of rat serum carboxypeptidase N (EC 3.4.2.2) by affinity chromatography on a column of CNBr-activated Sepharose with immobilized antibody is described. The ligands used were either rabbit antiserum to rat carboxypeptidase N or the IgG fraction prepared from this serum. The coupling of the isolated antibodies to CNBr-activated Sepharose increased the capacity of the column approximately three times. The specific activity of the enzyme prepared by this method was 109-times higher than the activity of the serum. Analysis of the final product by polyacrylamide gel electrophoresis showed carboxypeptidase N and traces of albumin.

scholarly journals Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1081-1081
Author(s):  
Oblaise Mercury ◽  
Lucy Liu ◽  
Ayman Ismail ◽  
Ming Zhang ◽  
Qi Lu ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Culture Medium ◽  
Specific Activity ◽  
Protein S ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Hek293 Cells ◽  
Affinity Column ◽  
Factor X

Abstract Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity. Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content. Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column. Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible). Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. Disclosures Mercury: Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Purification Of Factors IX And X From Clinical Concentrate

1981 ◽  
Author(s):  
G C Russell ◽  
G Kemble ◽  
E G D Tuddenham
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Factor Ix ◽  
Factor X ◽  
Clinical Factor

Human factors IX and X have been purified to homogeneity from clinical factor IX concentrate that had been rejected for therapeutic use due to particulate contamination. (It was necessary to start with this material since in the UK, plasma is not commercially available). The procedure involved barium citrate adsorption followed by ammonium sulphate elution, DEAE- cellulose chromatography, gel filtration on Sephacryl S-200 and affinity chromatography on heparin sepharose gel. The preparation of factor IX at this stage showed a single band on SDS-polyacrylamide gel electrophoresis, of molecular weight 58,000. No change in molecular weight was observed in the presence of 2-mercaptoethanol. A further affinity chromatography column - poly (homoarginine) Sepharose or dextran sulphate sepharose - was necessary to obtain homogeneous factor X. The preparation obtained showed a single band on SDS-polyacrylamide gel electrophoresis of molecular weight 67,000. In the presence of 2-mercaptoethanol, two bands were obtained of molecular weights 49000 and 17000 representing the heavy and light chains respectively of factor X. The purified coagulation proteins contained no activated species detectable by nonactivated partial thromboplastin time or by chromogenic substrate (S2222) assay. Prothrombin protein Sand protein C are by-products of this purification procedure.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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.

THE MODE OF ACTION OF PENTOSAN POLYSULPHATE IN PLASMA

1987 ◽  
Author(s):  
S Béguin ◽  
H C Hemker
Keyword(s):  
Factor Viii ◽  
Linear Increase ◽  
Factor Ix ◽  
Factor X ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Viii And Ix ◽  
Pentosan Polysulphate ◽  
Lag Times ◽  
Prothrombinase Activity

We developed a method which enables as to compute the course of prothrombinase activity in clotting plasma (H.C. Hemker, G.M. Willems, S. Béguin: Thromb. Haemostas. 56, 9-17, 1986) and used this for a study of the effect of pentosan polysulphate (PPS) on thrombin generation.When added to normal plasma in the concentration range of 0-8 μg/ml PPS induces a linear increase of the pseudo first order decay constant of endogenous thrombin like heparin does, 1 ug of PPS being equivalent to 0.045 Aig of heparin. Contrary to heparin this action is partly (∼ 65%) dependent upon AT III and partly (∼ 35%) upon heparin cofactor II.In normal plasma PPS causes an inhibition of both extrinsic and intrinsic prothrombinase formation. Only in the intrinsic system an increase of the lag time of prothrombinase appearance is observed. Unlike heparin, PPS does not inhibit factor IXa induced thrombin formation neither does it inhibit prothrombinase formation in the presence of preactivated factor VIII. The prolongation of the lag times must therefore be ascribed to inhibition by PPS of the activation of factor VIII.The inhibition of extrinsic prothrombinase formation by PPS increases with progressive dilution of thromboplastin and is not seen in haemophilia A or B plasma. This demonstrates the existance of a factor VIII and IX dependent process in extrinsic coagulation that gains in importance when the potency of factgr VII-tissue factor complex decreases, i.e. the Josso pathway.PPS, but also heparin causes an unexplained increase of prothrombinase action in haemophIIic plasma. The same phenomenon may be expected to exist in normal plasma, be it obscured by a concomitant inhibition. This, together with the incomplete inhibition of factor VIII activation by PPS makes that we cannot use this inhibitor as a means to quantitate the Josso pathway. The best estimate that we can obtain is that, in the presence of 2% thromboplastin, the factor IX dependent activation of factor X contributes more then 20% to prothrombinase generation.

Binding of Myeloma Monoclonal Immunoglobulin with Fibrinogen and Fibrin

1979 ◽  
Author(s):  
Andrei Z. Budzynski ◽  
Stephanie A. Olexa ◽  
Bharat V. Pandya
Keyword(s):  
Multiple Myeloma ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Clotting Time ◽  
Clot Retraction ◽  
Monoclonal Immunoglobulin ◽  
Normal Plasma ◽  
Antigen Complex ◽  
Polypeptide Chains

Blood and plasma clots are frequently abnormal in patients with multiple myeloma a evidenced by the prolongation of clotting time, formation of bulky and gelatinous cloes and inhibition of clos retraction. It has been demonstrated in several multiple myeloma cases that the isolated immunoglobulin inhibited clot formation in normal plasma and decreased the rate of fibrin polymerization. In this work a hypothesis was tested whether these phenomena originate from binding of multiple myeloma immunoglobulin with fibrinogen and fibrin. Blood was obtained from a patient (J.I.) having IgAH gammopath; with prolonged clotting time and abnormal clot retraction. Washed plasma clots contaii ed largo amount of monoclonal IgAλ demonstrated by precipitation with anti-α or anti-λ antibodies and by the presence of heavy (65,000) and light (25,000) polypeptide chains. This myeloma IgA was incorporated into clots regardless of crosslinking and was extract able. Affinity chromatography of serum or heated plasma on insolubilized fibrin monoment resulted in recovery of large amount of myeloma monoclonal IgA, but not IgG, IgH or Igl. The isolated fibrin-specific IgA was taken up by clots obtained from normal plasma or purified fibrinogen, lnso]ubilized fibrinogen bound myeloma monoclonal IgA From plasma. Two-dimensional agarose/polyacrylamide slab gel electrophoresis demonstrated the presen of a fibrinogen-IgA complex in patient’s plasma. The results indicate that a myeloma monoclonal immunoglobulin binds with fibrinogen and fibrin, probably forming an antibot antigen complex, implying the Formation of autoantibodies against these proteins.

Sign in / Sign up

Export Citation Format

Share Document