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The bispecific antibody emicizumab is increasingly used for hemophilia A treatment. However, its specificity for human factors IX and X (FIX and FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here, we describe a novel mouse model that allows emicizumab function to be examined. Briefly, FVIII-deficient mice received IV emicizumab 24 hours before tail-clip bleeding was performed. A second infusion with human FIX and FX, administered 5 minutes before bleeding, generated consistent levels of emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg doses) and of both FIX and FX (85 and 101 U/dL, respectively, after dosing at 100 U/kg). Plasma from these mice display FVIII-like activity in assays (diluted activated partial thromboplastin time and thrombin generation), similar to human samples containing emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail-clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared with mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted FVIII-like activity from emicizumab that corresponded to a dose of 4.5 U of FVIII per kilogram (ie, 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), emicizumab provided enough additive activity to allow complete bleeding arrest. This model could be useful for further in vivo analysis of emicizumab., (© 2020 by The American Society of Hematology.)

Back Ground: It has recently been reported that factor XIa can activate factor X directly and can bypass factors VIII-IX. We evaluated the consequences for factor analysis with the one-stage APTT., Methods: APTT was performed with the Actin FS reagent with ellagic acid as the standard. Silica, high lipid (PTT-A) or low lipid (PTT-LA) were also tested. Factor depleted and deficient plasma's were obtained from commercial sources., Results: The APTT clotting times in factor XII, XI, High Molecular Weight Kininogen, factor X and factor V deficient plasma's were all significantly longer (>100s) than the clotting times of factor VIII- and IX-depleted or deficient plasma's (<100s). That the shorter times for factor VIII and IX deficient plasmas were due to contact activation was supported by biphasic inhibition of the clotting times with addition of Corn Trypsin Inhibitor and Trasylol. The role of factor XI and the by-passing of factor VIII/IX was shown by the use of quenching antibodies towards factor XI and VIII. Enriching factor VIII or IX depleted plasma with purified factor XI and addition of factor XIa showed a strong dependence on factor XI level. Calibration curves for factor analysis were steeper for factors FXII, HMWK, FX and FV, compared to those of both factors VIII and IX. Curves for VIII/IX were found steeper by the use of APTT-A/silica-based, 50% diluted substrate plasma and low factor XI in the substrate plasma., Conclusions: In factors VIII and IX deficient plasmas, the APTT shows an activity which can be attributed to contact activation of factor X by factor XIa. This direct activity is lower with silica reagent compared to ellagic acid, dilution of plasma and low factor XI in substrate plasma., (Copyright © 2014. Published by Elsevier Ltd.)

Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

To investigate risk factors for thrombotic microangiopathy (TMA) after bone marrow transplantation (BMT), the levels of three clotting factors (7, 9 and 10) and hepatocyte growth factor (HGF) were measured. Among 46 consecutive patients who underwent BMT, six developed TMA and 40 did not. The levels of the clotting factors and HGF did not differ significantly between the six patients with TMA and the 40 patients without it. In two patients who developed TMA during the earlyperiod after BMT, however, the levels of the three clotting factors were significantly decreased even before BMT, along with a significant increase of HGF. These findings suggest that patients with severe hepatic dysfunction before BMT, especially those with impaired protein synthesis, had an increased risk of developing TMA soon after BMT. It was also suggested that measurement of clotting factors (7, 9 and 10) and HGF may be useful to predict the occurrence of TMA in the early period after BMT.

Antithrombin-III (AT) is a key inhibitor of blood coagulation that neutralizes activated serine esterases by forming covalent modified complexes (ATm). A new monoclonal antibody directed against short-lived AT-activated serine protease complexes provides a means of measuring subclinical coagulation activity during cardiopulmonary bypass (CPB). Twelve patients undergoing CPB for coronary artery bypass grafting were studied and AT, ATm, D-dimers (DD), and several other coagulation and fibrinolytic markers were measured during the surgical procedure. There were decreases in AT, factors V, II, X, IX, protein S (total and free), C4b-binding protein, thrombomodulin, and platelets counts, whereas heparin, ACT, thrombospondin, plasminogen activator inhibitor (PAI-1), and tissue plasminogen activator (tPA) increased. ATm and the percentage of ATm available (ATm/AT) showed a peak during CPB. These results demonstrate that during CPB, the use of heparin produces an equilibrium involving increased coagulation activation and consumption in association with increased fibrinolysis. The equilibrated consumption of both coagulation and fibrinolytic factors leads to low levels of all factors after cardiac surgery. The ATm assay allows assessment of the differential effects of CPB and surgical trauma on coagulation activation. It is speculated that ATm levels may be useful in monitoring the consumption of coagulation factors.

We report a case of haemophilia B, who developed disseminated intravascular coagulation (DIC) with massive bleeding following an administration of factor IX complex concentrates. A 22-year-old male with severe haemophilia B had a bone fracture of the fibula and haemorrhage in the ankle joint because of a traffic accident. Factor IX complex concentrates were given to keep the plasma factor IX level more than 80% for surgery. 60 h after the infusion he showed epistaxis, haematuria and swelling of the injured ankle with evidence of DIC. A small dose of monoclonal antibody (mAb)-purified factor IX concentrates with heparin and gabexate relieved the haemorrhage. A high dose of the mAb-purified product attained more than 90% levels of plasma factor IX without coagulopathy. This observation emphasizes that currently available factor IX concentrates have thrombogenicity to induce DIC. Highly purified factor IX concentrates are needed for the high-dose replacement in haemophilia B.

Thromboembolic complications associated with prothrombin complex concentrate treatment may be related to the high levels of factors II and X in these products. We report here results from preclinical safety studies with a human coagulation factor IX product (AlphaNine; Alpha Therapeutic Corp., Los Angeles, Calif.) that contains no detectable factor II or VII and less than 10 units of factor X/100 units of factor IX. This product was manufactured from virally inactivated factor IX complex with a barium citrate adsorption step followed by affinity chromatography yielding factor IX concentrate with a specific activity of about 86 factor IX units/mg protein. Electrophoresis and immunoblot analysis indicated that the factor IX represents about 65% of the protein in this product. The virus inactivation step incorporated into the manufacturing process (incubation with n-heptane at 60 degrees C for 20 hours) was shown to inactivate at least 8.6 logs of type 1 human immunodeficiency virus. The barium citrate adsorption and affinity chromatography steps were found to remove 2.0 logs of the marker virus, vaccinia, and the DEAE ion-exchange chromatography used to produce factor IX complex was found to remove 1.4 logs of the marker virus, Sindbis. Analysis of three separate manufacturing lots with the polymerase chain reaction revealed no evidence of hepatitis C virus. The purified factor IX was nonthrombogenic when tested at doses of 450 units/kilogram in a rabbit stasis (Wessler) model, whereas the prothrombin complex concentrates were found to be thrombogenic at doses of less than 50 units/kg. There was no evidence of DIC in a porcine model after infusion of 200 units/kg of coagulation factor IX, as manifested by negative fibrin monomer tests, the absence of fibrin in blood vessels at autopsy, little or no change in prothrombin times and partial thromboplastin times, and only moderate decreases in platelet levels after infusion.

Purer factor IX concentrates, containing very little or no factor II or X, have been developed in an attempt to avoid the thromboembolic complications that occur with prothrombin complex concentrates (PCC), which also contain factors II and X and variable amounts of factor VII. To evaluate ex vivo the thrombogenic potential of one of these purer concentrates, we studied whether large single doses produced signs of activation of the coagulation cascade in patients with haemophilia B, and compared the results with those obtained after infusion of a PCC. Seven patients were infused with 50 IU/kg of factor IX concentrate and seven additional patients were subsequently infused with 100 IU/kg of the same concentrate. After the infusions, factor IX levels rose in proportion to the administered dose while the concentrations of factor II and factor X did not rise at all. At both doses of concentrate, we did not observe significant post-infusion increments in the levels of the factor X activation peptide (a measure of the activity of the factor VIIa-tissue factor complex and/or the factor IXa-VIIIa-activated surface complex), prothrombin fragment 1 + 2 (a measure of factor Xa activity), and fibrinopeptide A (a measure of thrombin activity). We also infused 10 patients with a PCC (50 IU/kg). After the infusions, significant rises in the concentrations of the factor X activation peptide and prothrombin fragment were observed. Therefore, it appears that the infusion of a PCC to patients with haemophilia B can augment factor X activation and subsequently thrombin generation in vivo and that this process can be abrogated by the administration of more pure factor IX concentrate.

Mice to which Factor IX concentrates are administered intraperitoneally at high doses (approximately 1000 u/kg) die between 4 and 18 hours thereafter. Histologic examination reveals hemorrhage in the lung and liver. Death is prevented by reduction of the concentrate with dithiothreitol but not by treatment of the preparation with enzyme inhibitors (diisopropyperosphorofluoridate, soybean trypsin inhibitor, heparin). Prothrombin, devoid of Factors VII, IX and X, also is lethal to mice. Activation of the Factor IX concentrate by insolubilized Russel's Viper Venom (NAPTT 1/5000 less than 20'') resulted in decreased mortality when compared to controls (4/30 vs. 11/20). These experiments indicate that some of the adverse effects of Factor IX concentrates may result from high levels of zymogens rather than the presence of small amounts of active enzymes. Intensive animal testing might be indicated for routine quality control of Factor IX concentrates.

Spectrophotometric methods were used to assay the clotting factors II, VII, IX and X in plasma of 33 subjectively healthy human donors and in plasma of 98 patients receiving long-term oral anticoagulant therapy. In 33 normal subjects the interindividual variations in the plasma activities of the clotting factors II, VII, IX and X are respectively 12.2, 21.4, 11.0 and 15.0%. After correction for the assay variations the remaining biological variations are respectively 11.7, 21.2, 9.7 and 14.8%. Plasma from 98 patients receiving long-term anticoagulant therapy was assayed with 'Thrombotest', a clotting test in whole blood introduced by Owren and in these plasmas the activity of each of the vitamin K-dependent factors was assayed with spectrophotometric methods. For the clotting factors IX and VII, novel spectrophotometric methods were applied and the plasma activities thus measured were compared to results obtained with factor IX and VII clotting assays. Chromogenic activities of the different factors were correlated among each other and with 1/Thrombotest values. When the therapeutic range for Thrombotest values is set between 5 and 12.5% the corresponding therapeutic ranges for the activity of the factors II, VII, IX and X are respectively 12.6-36.1, 27.0-52.3, 23.1-49.3 and 18.9-36.2% (expressed as a percentage of the activity in normal pool plasma). The chromogenic assays for the factors II, VII, IX and X provide the same information on the therapeutic state of the patients in respectively 86.7, 78.6, 81.6 and 89.8% of the cases. Finally we discuss the suitability of the different assays to monitor oral anticoagulant therapy.

Activation of Factor IX and Factor X was studied by adding 125I-Factor IX or 125I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor Xa produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added 125I-Factor IX or 125I-Factor X, tissue factor and Ca2+ ions, the percentage of total radioactivity in the heavy chain peak of 125I-IXa and the heavy chain peak of 125I-Xa increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of 125I-Factor IX and percent cleavage of 125I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Factor IX by the tissue factor-Factor VII reaction produce represents a physiologically significant step in normal haemostasis.

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca2+. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (TM) of 54.5 +/- 0.5 degrees C and a calorimetric enthalpy (delta Hc) of 105 +/- 15 kcal/mol, in the absence of Ca2+. In the presence of Ca2+ concentrations sufficient to saturate its sites on Factor IX, the Tm value is increased to 57.0 +/- 0.5 degrees C and the delta Hc is virtually unchanged. When the activation intermediate, Factor IX alpha, is similarly analyzed in the absence of Ca2+, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca2+, Factor IX alpha displayed thermograms characterized by a TM of 51.0 +/- 0.5 degrees C and a delta Hc of 109 +/- 10 kcal/mol. The activated product, Factor IXa alpha, in the absence of Ca2+ (the values in the presence of saturating Ca2+ are given in parentheses), undergoes thermal denaturation with a TM of 54.5 +/- 0.5 degrees C (57.0 +/- 0.5 degrees C) and a delta Hc of 158 +/- 10 kcal/mol (156 +/- 10 kcal/mol). Similarly, the terminal-activation product, Factor IXa beta, displays a TM of 51.5 +/- 0.5 degrees C (54.0 +/- 0.5 degrees C) and a delta Hc of 85 +/- 5 kcal/mol (126 +/- 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a TM of 54.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 102 +/- 10 kcal/mol (118 +/- 10 kcal/mol), whereas its activated form, Factor Xa beta, possesses a TM of 55.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 92.0 +/- 5 kcal/mol (136 +/- 10 kcal/mol). These studies indicate that, for many of these proteins, Ca2+ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.

To localize beta-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing beta-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of beta-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.

To determine the functional properties of factor IX isolated from the plasma of CRM+ hemophilia B patients, an assay system using proteins isolated from human plasma had to be developed which would be amenable to kinetic studies under a variety of experimental conditions. The present study describes the activation of factor X by factor IXa, isolated from normal human plasma, in an assay system which allows manipulation of calcium, phospholipid and factor VIIIa concentrations. Initial rate measurements with factor VIIIa present in the system were made by incubating factor VIII with factor Xa immediately before factor IXa assay. With this approach, the initial rate of factor X activation was constant, with little evidence for a lag period. Within the framework of the assay system described in the present study, it should be possible to examine not only genetic variants of factor IX, but also variants of factor VIII, as well as providing a means of routine factor IXa and factor VIII(a) assays.

Adverse effects possibly due to the active clotting factors are sometimes reported upon administration of some preparations of Factor IX complex having conformed to the specifications in the Japanese Minimum Requirements for Biological Products. The author, therefore, proposed the revision of the test method for the absence of active clotting factors in the Minimum Requirements by introducing calcium ion into the test medium.

Previous work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3--30 minutes of incubation (Sas el al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor =X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into "activated" and "non activated" groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This aggreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

Factors II, IX, and X in the plasmas of 10 patients with 'hemorrhagic disease of the newborn' were investigated by means of electroimmunoassay and crossed immunoelectrophoresis (CIE). The biological activity was within 4.2-30 U/dl for factor II, 5.1-20 U/dl for factor IX, and 5.2-24 U/dl for factor X, whereas the immunological antigen was within 33-58, 25-50, and 35-60 U/dl, respectively. Thus, for all factors, 1.7 times more antigen than activity was present. The CIE pattern of factors II and IX in the presence of Ca++ ions clearly showed a biphasic precipitin arc, and in the absence of Ca++ ions, only one precipitin arc was observed. These results implied the presence of precursor proteins PIVKA-II and -IX in the plasma. However, even in the presence of Ca++ ions the CIE pattern of factor X antigen in the patient plasmas only showed a single precipitin arc with a slightly faster than normal electrophoretic mobility. PIVKA-II, -IX, and the abnormal factor X antigen (PIVKA-X) disappeared within 24 h after the patients were treated with vitamin K.

Three patients with Christmas disease whose plasma was shown to have a prolonged one-stage prothrombin time with ox brain thromboplastin have been investigated. These patients have an inhibitor for the reaction between factor X, factor VII, and ox brain extract. The abnormal constituent responsible for this inhibitor appears to be factor IX whuch is functionally inactive but antigenically indistinguishable from normal factor IX. It is proposed that patients might be classified into haemophilia B(+) for patients with this defect (Christmas disease(+)) and haemophilia B(-) (Christmas disease(-)) for patients who have classical Christmas disease.

A comparison has been made of the electrophoretic behavior, chemical composition, amino-terminal sequence, and immunological properties of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor). Some immunological crossreactivity was found between the antibody to prothrombin and factor X although prothrombin and factor X differ substantially in amino-acid and carbohydrate composition. Considerable amino-acid sequence homology was found in the amino-terminal portion of prothrombin, factor IX, and the light chain of factor X. These data provide further evidence to support the hypothesis that at least three of the vitamin K-dependent clotting factors have evolved from a common ancestral gene.