Removal of Potentially Thrombogenic Material from a Concentrate of Factors II, IX & X by Polyethylene Glycol Precipitation

Factor Xa ◽

In Vitro Tests ◽

Factor Ii ◽

Polyethylene Glycol Precipitation

Polyethylene glycol(PEG) precipitation has beer used for the removal of hepatitis B surface antigen (HBsAg) from a concentrate of factors II, IX & X of intermediate purity. HBsAg is precipitated into a fraction PI at 20% (w/v) PEG and pH 6.5 while factors II, IX & X remaining in the supernatant are subsequently precipitated at 30% (w/v) PEG and pH. 5.2These various fractions have been assessed for the presence of potentially thrombogenic materials by in vitro tests. In comparison with the starting material the precipitate rich In factors II, IX & X has a significantly increased non-activated partial thromboplastin time (NAPTT) and a decreased rate of factor Xa generation. By contrast the waste fraction (PI) exhibits a significantly decreased NAPTT and an increased rate of factor Xa generation suggesting that potentially thrombogenic material has been selectively precipitated. This procedure has been used to prepare a factor II, IX & X concentrate containing 100 U FIX/ml with reduced contamination by both HBsAg and thrombogenic materials.

In Vitro Thrombogenicity Tests of Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657035 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1355-1367 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A Chirnside ◽

R A Elton

Keyword(s):

Factor Viii ◽

Generation Time ◽

Factor Ix ◽

In Vitro Tests ◽

Factor Viii Inhibitor ◽

Factor Ixa ◽

Viii Inhibitor

SummaryVarious factor IX concentrates have been examined in a number of in vitro tests of thrombogenicity. The results suggest that some tests are superfluous as in concentrates with activity in any of these tests activation is revealed by a combination of the non-activated partial thromboplastin time, the thrombin (or Xa) generation time and factor VIII inhibitor bypassing activity tests. Assay of individual coagulant enzymes revealed that most concentrates contained more factor IXa than Xa. However only a small number of concentrates, chiefly those that had been purposefully activated, contained appreciable amounts of either enzyme.

Large-scale purification of hepatitis B surface antigen

Journal of Clinical Microbiology ◽

10.1128/jcm.3.6.626-631.1976 ◽

1976 ◽

Vol 3 (6) ◽

pp. 626-631

Author(s):

J Vnek ◽

A M Prince

Keyword(s):

Polyethylene Glycol ◽

Hepatitis B ◽

Surface Antigen ◽

Phosphate Buffer ◽

Sodium Phosphate ◽

Large Scale ◽

Sodium Phosphate Buffer ◽

Subsequent Elution ◽

Polyethylene Glycol Precipitation

Hepatitis B surface antigen was concentrated and purified from plasma by two simple steps of purification. In the first step the antigen was purified 24-fold by polyethylene glycol precipitation. An additional 10-fold purification was achieved by batchwise adsorption to hydroxylapatite and subsequent elution with 0.02 M sodium phosphate buffer.

Modified Tests for Measuring the Thrombogenic Potential of Factor IX Concentrates.

10.1055/s-0039-1684742 ◽

1979 ◽

Author(s):

Sarah M. Middleton ◽

Jessie T. Douglas ◽

C.D. Forbes ◽

C.R.M. Prentice

Keyword(s):

Factor Xa ◽

Factor Ix ◽

Procoagulant Activity ◽

In Vitro Tests ◽

Factor V ◽

Thrombogenic Potential ◽

Sepharose 4B ◽

Generation Test

In vitro tests for screening potential thrombogenicity of factor IX concentrates are unsatisfactory as it has been shown that the non-activated partial thromboplastin time (NAPTT) and the TGt50 (reflecting thrombin generation by the concentrate after recalcification} do not correlate with each other. We have modified the TGt50 by addition of optimum concentrations of factor V and phospholipid, and compared it with the NAPTT and factor Xa generation test using the chromogenic substrate S2222. The modified TGtSO correlates with both the NAPTT and the factor Xa generation test suggesting that these tests are measuring the same entity. Chromatography of concentrates on sepharose 4B indicates that the high MW void volume material has procoagulant activity as measured by the unmodified TGt50 whilst the retained volume he procoagulant activity as measured by the TGt50 and the NAPTT. When, however, phospholipid is added to the unmodified TG150 the activity of the high MW component is lost and only that of the retained volume is still present. The data suggests that the modified TGt50 and the NAPTT measure the same procoagulant activity in these concentrates.

The In Vitro Effects of Pentamidine Isethionate on Coagulation and Fibrinolysis

Molecules ◽

10.3390/molecules24112146 ◽

2019 ◽

Vol 24 (11) ◽

pp. 2146 ◽

Cited By ~ 1

Author(s):

Rami A. Al-Horani ◽

Daytriona Clemons ◽

Madhusoodanan Mottamal

Keyword(s):

Factor Xa ◽

Factor Xiiia ◽

Thrombin Time ◽

Normal Plasma ◽

Pentamidine Isethionate ◽

In Vitro Effects ◽

Coagulation And Fibrinolysis

Pentamidine is bis-oxybenzamidine-based antiprotozoal drug. The parenteral use of pentamidine appears to affect the processes of blood coagulation and/or fibrinolysis resulting in rare but potentially life-threatening blood clot formation. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. To investigate the potential underlying molecular mechanism(s) of pentamidine’s effects on coagulation and fibrinolysis, we studied its effects on clotting times in normal and deficient human plasmas. Using normal plasma, pentamidine isethionate doubled the activated partial thromboplastin time at 27.5 µM, doubled the prothrombin time at 45.7 µM, and weakly doubled the thrombin time at 158.17 µM. Using plasmas deficient of factors VIIa, IXa, XIa, or XIIa, the concentrations to double the activated partial thromboplastin time were similar to that obtained using normal plasma. Pentamidine also inhibited plasmin-mediated clot lysis with half-maximal inhibitory concentration (IC50) value of ~3.6 μM. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4 µM and 8.4 µM, respectively. Interestingly, it did not significantly inhibit thrombin, factor XIa, factor XIIIa, neutrophil elastase, or chymotrypsin at the highest concentrations tested. Michaelis-Menten kinetics and molecular modeling studies revealed that pentamidine inhibits factor Xa and plasmin in a competitive fashion. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on coagulation and fibrinolysis via the disruption of the proteolytic activity of factor Xa and plasmin.

Hepatitis B virus vaccine production from hepatitis B surface antigen: purification by polyethylene glycol precipitation isopyknic flotation and gel chromatography

Vaccine ◽

10.1016/s0264-410x(98)90041-x ◽

1984 ◽

Vol 2 (1) ◽

pp. 107

Keyword(s):

Polyethylene Glycol ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Gel Chromatography ◽

Vaccine Production ◽

B Virus ◽

Polyethylene Glycol Precipitation ◽

Antigen Purification ◽

Hepatitis B Virus Vaccine

Monitoring Heparin Therapy: Relationships between the Activated Partial Thromboplastin Time and Heparin Assays Based on Ex-Vivo Heparin Samples

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645678 ◽

1990 ◽

Vol 63 (01) ◽

pp. 016-023 ◽

Cited By ~ 41

Author(s):

A M H P van den Bessekaar ◽

J Meeuwisse-Braun ◽

R M Bertina

Keyword(s):

Plasma Sample ◽

Ex Vivo ◽

Correlation Coefficients ◽

Heparin Therapy ◽

Thromboembolic Disease ◽

Venous Thromboembolic Disease ◽

Venous Thromboembolic

SummaryFive different APTT reagents, two amidolytic anti-ITa assays, one amidoiytic anti-Xa assay, and one coagulometric anti-Xa/ anti-IIa assay were used to assess the effect of heparin in patients treated for venous thromboembolic disease. Good correlations were observed between lug-transformed APYE> determined with the various reagents (correlation coefficients: 0.92-0.96).Nevertheless there were important differences in the slopes of the lines of relationship between the APTT reagents.Good correlations were observed between the anti-Xa and anti-IIa assay results (correlation coefficients: 0.92-0.97). However, the amidolytic anti-Xa activity was significantly higher (p <0.001) than the two amidolytic anti-IIa activities. Less good correlations were observed between the log-transformed APTTs and the anti-Xa or anti-IIa activities (correlation coefficients: 0.64-0.78). The correlations were improved by transforming the APTT into APTT-ratio, i.e. the ratio of the patient’s APTT to the same patient’s APTT after removal of heparin from the plasma sample by means of ECTEOLA-cellulose treatment. The correlation coefficients of log (AFTT-ratio) with anti-Xa or anti-IIa ranged from 0.76 to 0.87.For both APTT and amidolytic heparin assay, the response to in vitro heparin was different from the response to ex vivo heparin.Therefore, equivalent therapeutic ranges should be assessed by using ex vivo samples rather than in vitro heparin. Because of the response differences between the APTT reagents, it is not adequate to define a therapeutic range for heparin therapy without specification of the reagent.

Monitoring Anticoagulant Therapy by Activated Partial Thromboplastin Time: Hirudin Assessment

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648943 ◽

1994 ◽

Vol 72 (05) ◽

pp. 685-692 ◽

Cited By ~ 43

Author(s):

Michael T Nurmohamed ◽

René J Berckmans ◽

Willy M Morriën-Salomons ◽

Fenny Berends ◽

Daan W Hommes ◽

...

Keyword(s):

Whole Blood ◽

Ex Vivo ◽

Fold Increase ◽

Heparin Therapy ◽

Recombinant Hirudin ◽

Interindividual Differences ◽

The Relationship

SummaryBackground. Recombinant hirudin (RH) is a new anticoagulant for prophylaxis and treatment of venous and arterial thrombosis. To which extent the activated partial thromboplastin time (APTT) is suitable for monitoring of RH has not been properly evaluated. Recently, a capillary whole blood device was developed for bed-side monitoring of the APTT and it was demonstrated that this device was suitable to monitor heparin therapy. However, monitoring of RH was not evaluated.Study Objectives. To evaluate in vitro and ex vivo the responsiveness and reproducibility for hirudin monitoring of the whole blood monitor and of plasma APTT assays, which were performed with several reagents and two conventional coagulometers.Results. Large interindividual differences in hirudin responsiveness were noted in both the in vitro and the ex vivo experiments. The relationship between the APTT, expressed as clotting time or ratio of initial and prolonged APTT, and the hirudin concentration was nonlinear. A 1.5-fold increase of the clotting times was obtained at 150-200 ng/ml plasma. However, only a 2-fold increase was obtained at hirudin levels varying from 300 ng to more than 750 ng RH/ml plasma regardless of the assays. The relationship linearized upon logarithmic conversion of the ratio and the hirudin concentration. Disregarding the interindividual differences, and presuming full linearity of the relationship, all combinations were equally responsive to hirudin.Conclusions. All assays were equally responsive to hirudin. Levels up to 300 ng/ml plasma can be reliably estimated with each assay. The manual device may be preferable in situations where rapid availability of test results is necessary.

Hepatitis D virus in 2021: virology, immunology and new treatment approaches for a difficult-to-treat disease

Gut ◽

10.1136/gutjnl-2020-323888 ◽

2021 ◽

pp. gutjnl-2020-323888

Author(s):

Stephan Urban ◽

Christoph Neumann-Haefelin ◽

Pietro Lampertico

Keyword(s):

Hepatitis B ◽

Treatment Strategies ◽

Phase Iii ◽

Interferon Α ◽

T Cell Epitopes ◽

The European Union ◽

Hepatitis D ◽

Hepatitis D Virus

Approximately 5% of individuals infected with hepatitis B virus (HBV) are coinfected with hepatitis D virus (HDV). Chronic HBV/HDV coinfection is associated with an unfavourable outcome, with many patients developing liver cirrhosis, liver failure and eventually hepatocellular carcinoma within 5–10 years. The identification of the HBV/HDV receptor and the development of novel in vitro and animal infection models allowed a more detailed study of the HDV life cycle in recent years, facilitating the development of specific antiviral drugs. The characterisation of HDV-specific CD4+ and CD8+T cell epitopes in untreated and treated patients also permitted a more precise understanding of HDV immunobiology and possibly paves the way for immunotherapeutic strategies to support upcoming specific therapies targeting viral or host factors. Pegylated interferon-α has been used for treating HDV patients for the last 30 years with only limited sustained responses. Here we describe novel treatment options with regard to their mode of action and their clinical effectiveness. Of those, the entry-inhibitor bulevirtide (formerly known as myrcludex B) received conditional marketing authorisation in the European Union (EU) in 2020 (Hepcludex). One additional drug, the prenylation inhibitor lonafarnib, is currently under investigation in phase III clinical trials. Other treatment strategies aim at targeting hepatitis B surface antigen, including the nucleic acid polymer REP2139Ca. These recent advances in HDV virology, immunology and treatment are important steps to make HDV a less difficult-to-treat virus and will be discussed.