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PubMed1984

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma.

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H-labeled factor X to the plasma resulted, after a short lag, in burst-like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.

Animals↗
PubMed1985

Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.

Factor IX is a vitamin K-dependent blood clotting zymogen that is functionally defective or absent in patients with hemophilia B. A method of immunoaffinity chromatography has been developed for a one-step high yield purification of factor IX directly from plasma. The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor IX conformer, but not to the conformer of factor IX found in the absence of metal ions. Anti-factor IX-Ca(II) antibodies were immobilized on an agarose matrix. Human plasma in the presence of 7.5 mM MgCl2 was applied to the antibody-agarose column. The factor IX that binds to these antibodies was specifically eluted by metal chelation with EDTA. This immunopurification resulted in a 10,000-fold one-step purification of the fully functional zymogen. Purified factor IX yielded a single band upon gel electrophoresis in Na-DodSO4 and had a specific activity of 120-150 units/mg. The purified factor IX was separated from other vitamin K-dependent blood clotting proteins and hepatitis virus; no activated factor IX was detected. This method has application for the large scale purification of factor IX for the treatment of hemophilia B.

Antibodies↗
PubMed1995

Enzymatic removal of sialic acid from human factor IX and factor X has no effect on their coagulant activity.

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full "tenase" activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.

Factor IX↗
PubMed1995

Measurement of activated factor IX in factor IX concentrates: correlation with in vivo thrombogenicity.

Current in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15-1.2 U/1000 in FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25-4. There was a significant positive correlation (r = 0.55, p < 0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

Animals↗
PubMed2004

The endogenous thrombin potential and high levels of coagulation factor VIII, factor IX and factor XI.

High plasma concentrations of factor VIII, factor IX and factor XI have been reported as thrombosis risk factors. Using the thrombin generation test in platelet-poor plasma, it was aimed to describe the mechanism for this increased thrombosis risk. Endogenous thrombin potential was measured in platelet-poor plasma in 180 patients with a history of thromboembolism, and results were compared with those of 180 age-matched and sex-matched controls. Subjects with major hereditary and acquired thrombophilia were excluded. Plasma concentrations of the clotting factor VIII, factor IX and factor XI were significantly elevated in patients compared with controls. The mean endogenous thrombin potential was significantly higher in patients than in controls: 191.3 +/- 3.1 (95% confidence interval, 185.3-197.4) arbitrary units versus 180.8 +/- 2.6 (95% confidence interval, 175.7-185.9) arbitrary units (P = 0.009). The endogenous thrombin potential was significantly higher in patients with elevated factor IX and factor XI, but elevated factor VIII was not associated with a significant increase in endogenous thrombin potential. In conclusion, the increased thrombosis risk associated with high plasma concentrations of factor IX and factor XI may be explained by the increase in endogenous thrombin potential. However, this did not help explain the association between elevated factor VIII and thrombosis risk.

Adult↗
PubMed1989

Comparative interactions of factor IX and factor IXa with human platelets.

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.

Binding, Competitive↗
PubMed1977

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

Amino Acid Sequence↗
PubMed1986

Heterogeneity of factor IX BM. Difference of cleavage sites by factor XIa and Ca2+ in factor IX Kashihara, factor IX Nagoya and factor IX Niigata.

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca2+ or by RVV-X/Ca2+. A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B+ or BM is a heterogeneous disorder.

Antigens, Heterophile↗
PubMed1979

Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

Amino Acid Sequence↗
PubMed1978

Activation of human factor IX (Christmas factor).

Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.

Amino Acid Sequence↗
PubMed1993

Comparison of the behavior of normal factor IX and the factor IX Bm variant Hilo in the prothrombin time test using tissue factors from bovine, human, and rabbit sources.

A subset of hemophilia B patients have a prolonged bovine-brain prothrombin time. These CRM+ patients are classified as having hemophilia Bm. The prolongation of the prothrombin time has been reported only with bovine brain (referred to as ox brain in some literature) as the source of thromboplastin; prothrombin times determined with thromboplastin from rabbit brain or human brain are not reported to be prolonged. Factor IX from a hemophilia Bm patient (factor IX Hilo) was isolated. The activity of factor IX Hilo was compared to that of normal factor IX in prothrombin time assays when the thromboplastin source was of bovine, rabbit, or human origin. Factor IX, either normal or Hilo, prolonged a prothrombin time regardless of the tissue factor source. However, unless thromboplastin was from a bovine source, this prolongation required high concentrations of factor IX. Further, factor IX normal was as effective as factor IX Hilo in prolonging the prothrombin time when rabbit or human thromboplastin was used. With bovine thromboplastin, factor IX Hilo was significantly better than factor IX normal at prolonging the prothrombin time. The amount of prolongation was dependent on the amount of factor IX Hilo added. In addition, the prolongation was dependent on the concentration of factor X present in the sample. The prothrombin time changed as much as 20 seconds when the factor X concentration was varied from 50% to 150% to normal (fixed concentration of factor IX Hilo). These results demonstrate the difficulty of classifying the severity of a hemophilia Bm patient based on the bovine brain prothrombin time unless both the factor IX and factor X concentrations are known.

Animals↗
PubMed1986

The effect of platelets in the activation of human blood coagulation factor IX by factor XIa.

We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose-dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.

Blood Platelets↗
PubMed1993

Expression of coagulation factor IX (Christmas factor) in human hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer.

PURPOSE: In this study, we compared production of recombinant human factor IX by HepG2 cells transduced with a cytomegalovirus (CMV) promoter-controlled factor IX vector to endogenous production of factor IX by non-transduced primary rat hepatocytes. METHODS AND RESULTS: Northern analysis showed 2.8 kb transcripts corresponding to the known size of factor IX mRNA in primary hepatocyte preparations and vector factor IX transcripts of the expected sizes in transduced HepG2 cell preparations. Factor IX produced by transduced HepG2 cells was completely inhibited by a monospecific antibody against human factor IX. Western analysis showed that recombinant factor IX migrated to the region of native plasma factor IX at 56 Kd. Production of biologically active factor IX by transduced HepG2 cells was 20-fold greater than that by nontransduced primary hepatocytes. CONCLUSION: These data indicate that transduced HepG2 cells transcribe, synthesize, and secrete authentic factor IX, and that these genetically engineered cells secrete significantly greater amounts of factor IX than do nontransduced primary hepatocytes. Studies are in progress to determine the effect of hepatocyte mitogens on production of factor IX in transduced HepG2 cells and primary hepatocytes.

Animals↗