Factor IX Alloantibodies Shorten the Bovine Thromboplastin Coagulation Time of Normal Human Plasma

SummaryWe have previously demonstrated that neutralization of factor IX in normal plasma by heterologous antisera shortens the one stage prothrombin time determined with bovine thromboplastin. In this study, a similar effect of homologous antibodies was demonstrated. Addition of plasma from two patients with hemophilia B- and an acquired inhibitor to factor IX gave a shortening of the prothrombin time of plasma from normal persons, compared to the prothrombin time determined after addition of control plasma from a patient with hemophilia B- and no inhibitor. Addition of inhibitor plasma had a similar effect on the prothrombin time of plasma from four patients with hemophilia B+ and one patient with hemophilia Br, but had no effect on the prothrombin time of plasma from ten patients with hemophilia B-. Complexes between factor IX and the human inhibitor could be demonstrated both before and after the coagulation with bovine thromboplastin. These complexes were demonstrated as a factor IX antigen with a reduced electrophoretic mobility in crossed immunoelectrophoresis against a rabbit antiserum to factor IX. The results demonstrate that normal factor IX loses the ability to act as an inhibitor in the coagulation with bovine thromboplastin after having formed a soluble complex with a homologous antibody, although the factor IX molecules still have antigenic determinants which are free to react with the rabbit antibodies.

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Homologous Antibodies to Factor IX Shorten the Bovine Thromboplastin Coagulation Time of Human Plasma

10.1055/s-0039-1687271 ◽

1979 ◽

Author(s):

K.H. Örstavik

Keyword(s):

Prothrombin Time ◽

Rabbit Antiserum ◽

Factor Ix ◽

Hemophilia B ◽

Coagulation Time ◽

Crossed Immunoelectrophoresis ◽

Ability To Act ◽

Before And After ◽

The One ◽

Control Plasma

We have previously demonstrated that neutralization of factor IX in normal plasma by heterologous antisera shortens the one stage prothrombin time determined with bovine thromboplastin. In this study we demonstrate a similar effect of ahomologousantibody. Addition of 1/5 volume of plasma irom a patient with hemophilia B- and an acquired inhibitor to factor IX (titre 5 U/ml) gave a shortening of the prothrombin time of plasma from 7 normal persons [mean 34 sec) compared to the prothrombin time determined atter addition of 1/5 volume of control plasma from a patient with hemophilia B- and no acquired inhibitor [mean 39 sec). Addition of inhibitor plasma had no eftect on the prothrombin time of plasma from 6 patients with hemophilia B-. Complexes between factor IX and the human inhibitor could be demonstrated both before and after the coagulation with bovine thromboplastin. These complexes Were demonstrated as a factor IX antigen with a reduced electrophoretic mobility in crossed Immunoelectrophoresis against a rabbit antiserum to factor IX, The results indicate that normal tactor IX loses the ability to act as an inhibitor in the coagulation with bovine thromboplastin after having formed a soluble complex with a homologous antibody.

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Demonstration of Complexes Between Factor IX And IGG Antibodies from Hemophilia B Patients

10.1055/s-0039-1687397 ◽

1979 ◽

Author(s):

K.H. Örstavik

Keyword(s):

Rabbit Antiserum ◽

Factor Ix ◽

Hemophilia B ◽

Light Chains ◽

Igg Antibodies ◽

Human Igg ◽

Agarose Gels ◽

Crossed Immunoelectrophoresis ◽

Inhibitor Titre ◽

Control Plasma

Plasma from three patients with hemophilia B- and an acquired inhibitor to factor IX (titres: 5 U/ml, 0.73 U/ml and 0.32 U/ml) were incubated with purified factor IX and submitted to crossed Immunoelectrophoresis against a rabbit antiserum to factor IX. A heterogenous precipitin arc was seen instead of the homogenous precipitin arc seen when a control plasma from a patient with hemophilia B- and no acquired inhibitor was used for the incubation. Human IgG antibodies were demonstrated in the cathodal part of the precipitin aros. This was achieved by incubation of the agarose gels, after the electrophoresis, with a peroxidase conjugated rabbit antiserum to human IgG. The IgG antibodies from the two patients with the highest inhibitor titre contained both kappa and lambda light chains, and were thus of a polyclonal nature. It is concluded that IgG antibodies from low-tltred as well as high-titred inhibitor plasmas form complexes with factor IX. The detection of IgG in complex with factor IX may be a sensitive technique for the detection of low-titred inhibitors to factor IX.

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Performances of an Artificial Reagent for the One-stage Factor IX Assay

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647848 ◽

1975 ◽

Vol 33 (03) ◽

pp. 547-552 ◽

Cited By ~ 4

Author(s):

L Meunier ◽

J. P Allain ◽

D Frommel

Keyword(s):

Human Plasma ◽

Factor Ix ◽

Severe Haemophilia ◽

Normal Human Plasma ◽

Haemophilia B ◽

One Stage ◽

Normal Human ◽

The One

SummaryA mixture of adsorbed normal human plasma and chicken plasma was prepared as reagent for factor IX measurement using a one-stage method. The substrate was found to be specific for factor IX. Its performances tested on samples displaying factor IX activity ranging from <l%–2,500% compared favorably with those obtained when using the plasma of severe haemophilia B patients as substrate.

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Thromboplastin as a Reagent

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654189 ◽

1970 ◽

Vol 23 (03) ◽

pp. 585-592 ◽

Cited By ~ 2

Author(s):

A .J Quick

Keyword(s):

Prothrombin Time ◽

Oral Anticoagulant Therapy ◽

Quantitative Measure ◽

Rabbit Brain ◽

Bacterial Action ◽

Normal Human ◽

Tissue Thromboplastin ◽

Time Test ◽

The One ◽

Evacuated Tube

SummaryTissue thromboplastin is the key reagent in the one-stage prothrombin time test. To obtain reliable results, a potent thromboplastin with constant activity and stability is required. This need is met by acetone-dehydrated rabbit brain. This reagent, when protected against oxidation by sealing in an evacuated tube, retains its full activity indefinitely. The basic prothrombin time serves as a screening test for depletion of prothrombin, factors V, VII and X. Thromboplastin is slowly inactivated by oxidation and also by bacterial action. Phenol prevents the latter reaction and therefore a phenolized extract of either human brain or acetone-dehydrated rabbit brain serves as a satisfactory reagent for the prothrombin time when employed to control oral anticoagulant therapy.The constant 12 sec prothrombin time of fresh normal human plasma is fixed by the concentration of active prothrombin. By modifying the basic prothrombin time by adding excess of factors V, VII or X, the test is made a specific quantitative measure of each of these factors.

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Factor IX Deventer-Evidence for the Heterogeneity of Hemophilia BM

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657148 ◽

1982 ◽

Vol 47 (02) ◽

pp. 136-140 ◽

Cited By ~ 8

Author(s):

R M Bertina ◽

I K van der Linden

Keyword(s):

Prothrombin Time ◽

Factor Ix ◽

Procoagulant Activity ◽

Hemophilia B ◽

Effective Inhibitor ◽

Severe Hemophilia ◽

Peptide Bonds ◽

Proteolytic Activation

SummaryFactor IX Deventer was isolated from the plasma of a patient with severe hemophilia B. The patient was classified as BM because of an abnormal prolongation (2.1 times) of the ox-brain prothrombin time, that could be corrected by addition of antifactor IX serum. Experiments with the isolated factor IX Deventer showed that one of the two peptide bonds involved in the proteolytic activation of factor IX cannot be cleaved by physiological or non-physiological activators (XIa and RVV-X, respectively). Such a defect can explain why the molecule has no procoagulant activity. At present it is not clear why this defect makes factor IX Deventer such an effective inhibitor of the ox-brain prothrombin time. It is proposed that hemophilia BM is a heterogeneous disorder.

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Thrombin generation is not increased in the blood of hemophilia B patients after the infusion of a purified factor IX concentrate

Blood ◽

10.1182/blood.v76.12.2540.2540 ◽

1990 ◽

Vol 76 (12) ◽

pp. 2540-2545 ◽

Cited By ~ 41

Author(s):

PM Mannucci ◽

KA Bauer ◽

A Gringeri ◽

S Barzegar ◽

B Bottasso ◽

...

Keyword(s):

Significant Risk ◽

Factor Xa ◽

Factor Ix ◽

Hemophilia B ◽

Coagulation Cascade ◽

Platelet Factor ◽

Sensitive Index ◽

Before And After ◽

Fibrin Monomers

Abstract Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15–42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.

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Factor IX Related Antigen in Hemophllia B

10.1055/s-0039-1682505 ◽

1977 ◽

Author(s):

H.C. Yang ◽

P.H. Levine

Keyword(s):

Hemophilia A ◽

Rabbit Antiserum ◽

Factor Ix ◽

The Other ◽

Hemophilia B ◽

Precipitin Line

The presence of factor IX related antigen (FIXRA) was studied in 15 hemophilia B patients, 20 normals, 2 obligate carriers of hemophilia B, 10 hemophilia A patients and 2 patients on Coumadin therapy. A monospecific rabbit antiserum for factor IX was used in a counterimmuno-electrophoresis (CIEP) system. Only 3 out of the 15 hemophilia B patients, representing 3 of 10 pedigrees, had a factor IX related antigen as demonstrated by a precipitin line in CIEP; the other 34 subjects all had demonstrable FIXRA. The presence of FIXRA in hemophilia B did not correlate with the factor IX procoagulant level. The hemophilia B group with FIXRA could partially neutralize a human inhibitor to factor IX. Hemophilia B is a heterogeneous disease in which a minority have a factor IX related antigen.

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Detection of Acquired Inhibitors of Factor IX with Precipitating Rabbit Antisera Against Factor IX

10.1055/s-0039-1680588 ◽

1977 ◽

Author(s):

K.H. Ørstavik ◽

I.M. Nilsson

Keyword(s):

Simple Technique ◽

Rabbit Antiserum ◽

Factor Ix ◽

Hemophilia B ◽

Slight Reduction ◽

Agarose Gel ◽

High Concentration ◽

The Third ◽

Normal Plasma ◽

Inhibitor Titre

The effect of acquired inhibitors of factor IX on factor IX antigen was tested in a modified electroimmunoassay with a rabbit antiserum against factor IX. Plasma from three patients with hemophilia B and inhibitor against factor IX was mixed with agarose and casted in a one cm high intermediate gel between the sample wells and the agarose gel containing the rabbit antiserum. The precipitation of factor IX antigen by the rabbit antiserum was prevented when factor IX antigen migrated through the intermediate gel containing plasma from two of the patients with inhibitor titre 7.9 and 0.8 U/ml. The concentration of inhibitor plasma necessary to reduce by 50% the amount of precipitated factor IX antigen from 10 μl normal plasma could be determined for the two patients and was approximately 2% for the patient witli titre 7.9 and 15% for the patient with titre 0.8. The third patient had a titre of 0.13 U/ml and only a slight reduction in precipitated antigen was obtained with a high concentration of plasma in the intermediate gel (30%). The precipitation of factor IX antigen from 7 patients with hemophilia B+ (factor IX antigen 17-109%)and one patient with hemophilia BM (factor IX antigen 98%) was also inhibited by plasma from the two patients with inhibitor titre 7.9 and 0.8. This study suggests that absorption in intermediate gel may be a simple technique for the detection of inhibitors with titre 0.8 and higher and also for the study of the specificity of inhibitors.

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Carrier Detection in Hemophilia B.

10.1055/s-0039-1682597 ◽

1977 ◽

Author(s):

E. Briët ◽

R.M. Bertina ◽

N.H. van Tilburg ◽

J.J. Veltkamp

Keyword(s):

Oral Contraceptives ◽

Neutralization Test ◽

A Priori ◽

Rabbit Antiserum ◽

Neutralization Assay ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Detection ◽

Activity Levels ◽

Normal Women

We examined 37 obligatory carriers of hemophilia B and 40 normal women. The levels of both factor IX activity and factor IX antigen were determined. The factor IX antigen levels were assayed in an inhibitor neutralization test in which a rabbit antiserum was used. 12 out of the 37 carriers had oral contraceptives and so did 20 out of the 40 normals.Higher levels of both factor IX activity and factor IX antigen were found in the women, carriers as well as normals, using oral contraceptives. In carrier detection programs, therefore, a distinction should be made between women using the pill and those wo do not. The attempt to discriminate between carriers and normals on basis of factor IX activity and antigen levels proved to be as unsatisfactory as it was on the basis of factor IX activity levels only. The overlapping area of normals and carriers was smaller in the group of women on oral contraceptive medication. On the basis of our findings we expect that we can detect with a 90% confidence 17 carriers and 17 normals in a group of 100 potential carriers with an a priori chance on carriership of 50%. The detection rate is somewhat higher for women using oral contraception. The assay of factor IX antigen by means of the inhibitor neutralization assay does not appear to improve carrier detection.Results with an immuno-electrophoretic assay of factor IX antigen will be discussed.

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Hemophilia B: Genetic Variants and Carrier Detection

10.1055/s-0039-1682645 ◽

1977 ◽

Author(s):

C.K. Kasper ◽

B. Østerud ◽

S.I. Rapaport

Keyword(s):

Prothrombin Time ◽

Genetic Variants ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Detection ◽

Severe Hemophilia ◽

Human Factor Ix ◽

Normal Women

In 92 males with hemophilia B from 71 kindreds, we measured factor IX activity, prothrombin time using bovine thromboplastin (bovine tpln time), and factor IX antigen both by inhibitor neutralization using a human factor IX inhibitor and by electroimmunoassay using a precipitating rabbit anti-human-factor IX antiserum. Eighty patients with 3% or less factor IX activity could be divided into 4 groups:(1) 7 patients with greatly prolonged bovine tpln times and normal levels of factor IX antigen;(2) 17 patients with mildly prolonged bovine tpln times and factor IX antigen levels between about 25% and normal;(3) 8 patients with normal bovine tpln times and antigen levels between about 25% and normal; (4) 48 patients with normal bovine tpln times and no measureable antigen excess. Some of the latter group were also tested with a chicken anti-human-factor IX antiserum and no antigen was found. None of 12 patients with mild hemophilia B (factor IX activity of 4 to 22%) had a prolonged bovine tpln time although 4 patients had excess factor IX antigen over activity. Thus, about 1/3 of these 92 hemophilia B patients had evidence of an abnormal factor IX molecule. Factor IX activity was also measured in 48 normal women and in 51 definite carriers of severe hemophilia B. Probability curves were derived to estimate the chance of a woman being a carrier based upon her factor IX level and her degree of kinship to a definite carrier. The relation between factor IX activity and antigen was also delineated for normal women and for carriers. In kindreds in which affected males had excess antigen, some carriers could be distinguished from normal women on the basis of excess antigen over activity. In appropriate kindreds, prolonged bovine tpln times helped distinguish some carriers.

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