Photometric Assay of Factor VIIIC With a Chromogenic Substrate

1979 ◽  
Author(s):  
H. Vinazzer
Keyword(s):  
Factor Viii ◽  
Serine Proteases ◽  
Hemophilia A ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Chromogenic Substrate ◽  
Normal Individuals ◽  
Factor Viii Concentrates ◽  
Viii Inhibitor ◽  
The Difference

By the aid of chromogenic substrates, highly specific assays of some serine proteases can be carried out. The substrate used for factor VIIIC-assays was Bz-Ile-Glu-Gly-Arg-pNA [S-2222 KABI) which measures factor Xa. When all components necessary for factor Xa activation except factor VIIIC are kept at constant levels, the resulting Xa-activity is in direct relation to the concentration of factor VIIIC. The substrate plasma was a mixture of hemophilia A plasma with factor VIII inhibitor plasma with a remaining inhibitor activity of between 0.1 and 0.5 units per ml. This substrate was defibrinated by ancrod. For assays of factor VIIIC, this plasma was mixed with the diluted test plasma, cepheloplastin, and calcium chloride at 37°C After a constant activation time, the chromogenic substrate was added and the difference in OD/min was measured at 405 nm. The calibration curve was linear between 1% and over 200% factor VIIIC activity, and the average CV was 7.9%. This method was compared to a standard one-stage method for factor VIIIC. Identical results were obtained in plasma samples of normal individuals, in samples of high factor VIIIC, activity, in plasma from hemophilia A patients, and in factor VIII concentrates. The advantages of this method over the clotting method are: independence of the results from variations of factors V,II, and I in the reaction mixture, stability of the reagents, a better discrimination of factor VIIIC levels in the range between 30% and over 100%, and the possibility of automatization of the method.

scholarly journals Factor VIII inhibitor development in Egyptian hemophilia patients: does intron 22 inversion mutation play a role?

2020 ◽  
Vol 46 (1) ◽  
Author(s):  
Laila M. Sherief ◽  
Osama A. Gaber ◽  
Hala Mosaad Youssef ◽  
Hanan S. Sherbiny ◽  
Wesam a Mokhtar ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viii Inhibitor ◽  
Inhibitor Development ◽  
Fviii Inhibitor ◽  
Intron 22 Inversion ◽  
Egyptian Patients ◽  
Factor Viii Concentrates ◽  
Viii Inhibitor ◽  
Almost All

Abstract Background Hemophilia A (HA) is an X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII (FVIII). The development of inhibitor antibodies against FVIII is the most challenging complication of treatment. Mutations in the FVIII gene is one of the genetic factors that leads to development of FVIII inhibitors especially intron 22 inversion (Inv22). Objectives This study was carried out to assess the frequency of Inv22 of FVIII gene in Egyptian patients with hemophilia A and its role as a risk factor for developing inhibitors. Patients and methods Seventy-two patients with severe HA and 48 patients with moderate HA were enrolled in the current study. All patients were treated on demand with either plasma-derived factor VIII or recombinant factor VIII concentrates. Genotyping of FVIII Inv22 was performed by LD-PCR while the presence and magnitude of inhibitor activity in blood was determined by the Bethesda assay. Results Around 23% of all hemophilia cases had positive Inv22. Intron 22 inversion mutation was detected in 6 and 33% of patients with moderate and severe HA respectively. Twenty-one cases (18%) of all hemophilic patients developed inhibitors. Thirty-7% of patients with Inv22 had inhibitor in their blood, almost all, but one, had severe HA. The risk of an inhibitor development during replacement therapy was four folds higher among Inv22 positive cases as compared with mutation negative peers (OR 4.3, 95% CI 1.6–11.9, P = 0.003). Conclusions The prevalence of Inv22 of F VIII in Egyptian hemophiliacs is nearly like that of other population. This mutation was more frequently detected among severe hemophilic patients as compared with moderately affected peers. The presence of Inv22 mutation significantly predispose to FVIII inhibitor development.

A Higher than Expected Incidence of Factor VIII Inhibitors in Multitransfused Haemophilia A Patients Treated with an Intermediate Purity Pasteurized Factor VIII Concentrate

1993 ◽  
Vol 69 (02) ◽  
pp. 115-118 ◽  
Author(s):  
Kathelijne Peerlinck ◽  
Jef Arnout ◽  
Jean Guy Gilles ◽  
Jean-Marie Saint-Remy ◽  
Jos Vermylen
Keyword(s):  
Factor Viii ◽  
Randomized Trials ◽  
Specific Factor ◽  
Factor Viii Inhibitor ◽  
Haemophilia A ◽  
Viral Inactivation ◽  
Gradual Decline ◽  
Inhibitor Level ◽  
Factor Viii Concentrates ◽  
Viii Inhibitor

SummaryIn May 1990, 218 patients with haemophilia A regularly attending the Leuven Haemophilia Center were randomly assigned to a group receiving either of two newly introduced factor VIII concentrates: factor VIII-P, an intermediate purity pasteurized concentrate, or factor VIII-SD, a high purity concentrate treated with solvent-detergent for viral inactivation.Patients were followed from May 1990 until October 1991. Between August 1991 and October 1991 a clinically important factor VIII inhibitor was detected in five out of the 109 patients receiving factor VIII-P while none of the 109 patients receiving factor VIII-SD developed such antibodies. All patients acquiring an inhibitor had previously been clinically tolerant to transfused factor VIII with 200 to more than 1,000 days of exposure to factor VIII prior to May 1990. Patients with inhibitors were transfused daily with 30 U factor VIII-SD per kg body weight, which was associated with a gradual decline of the inhibitor level. In all patients the antibodies were relatively slow-acting and predominantly directed towards the light chain of factor VIII.This study demonstrates a higher than expected incidence of factor VIII inhibitors associated with the use of a specific factor VIII concentrate in multitransfused haemophilia A patients. It indicates the usefulness of evaluating newly introduced concentrates in prospective, randomized trials.

scholarly journals A Clinical and Experimental Study of Acquired Inhibitors to Factor VIII

Blood ◽  
1965 ◽  
Vol 26 (6) ◽  
pp. 805-818 ◽  
Author(s):  
HAROLD R. ROBERTS ◽  
MARGARET B. SCALES ◽  
JOHN T. MADISON ◽  
WILLIAM P. WEBSTER ◽  
GEORGE D. PENICK
Keyword(s):  
Experimental Study ◽  
Factor Viii ◽  
Mode Of Action ◽  
Exchange Transfusion ◽  
Hemophilia A ◽  
Retroperitoneal Hematoma ◽  
Factor Viii Inhibitor ◽  
Viii Inhibitor ◽  
Potent Factor

Abstract Factor VIII inhibitors which developed in four patients with hemophilia A are described. These inhibitors are apparently specific for Factor VIII and are capable of inducing a transient hemophilic state when injected into dogs. The genesis, properties, and mode of action of these inhibitors can be explained on an immunologic basis and it seems most likely that they represent an antibody to Factor VIII. One hemophilia A patient, with retroperitoneal hematoma and a potent Factor VIII inhibitor, was successfully treated by an exchange transfusion followed by administration of purified porcine Factor VIII.

Human Factor VIII Inhibitor Alloantibodies with a C2 Epitope Inhibit Factor Xa-catalyzed Factor VIII Activation: A new Anti-factor VIII Inhibitory Mechanism

2002 ◽  
Vol 87 (03) ◽  
pp. 459-465 ◽  
Author(s):  
Keiji Nogami ◽  
Katsumi Nishiya ◽  
Yoshihiko Sakurai ◽  
Ichiro Tanaka ◽  
John Giddings ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Inhibitory Mechanism ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Human Factor Viii ◽  
Sds Page ◽  
Viii Inhibitor ◽  
C1 Domains

SummaryFactor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-C1 domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in onestage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)’2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

scholarly journals Postpartum Acquired Hemophilia: A Rare Cause of Postpartum Hemorrhage

2013 ◽  
Vol 2013 ◽  
pp. 1-2 ◽  
Author(s):  
Srikanth Seethala ◽  
Sumit Gaur ◽  
Elizabeth Enderton ◽  
Javier Corral
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Vii ◽  
Normal Vaginal Delivery ◽  
Factor Viii Inhibitor ◽  
Activity Levels ◽  
Factor Viii Activity ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Viii Inhibitor

A 36-year-old female started having postpartum vaginal bleeding after normal vaginal delivery. She underwent hysterectomy for persistent bleeding and was referred to our institution. An elevation of PTT and normal PT made us suspect postpartum acquired hemophilia (PAH), and it was confirmed by low factor VIII activity levels and an elevated factor VIII inhibitor. Hemostasis was achieved with recombinant factor VII concentrates and desmopressin, and factor eradication was achieved with cytoxan, methylprednisolone, and plasmapheresis.

scholarly journals Feiba Immuno: A Preparation with Factor Eight Inhibitor Bypassing Activity

1977 ◽  
Author(s):  
F. Elsinger
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Active Principle ◽  
Factor Viii Inhibitor ◽  
Factor V ◽  
In Vitro Experiments ◽  
Factor X ◽  
Viii Inhibitor

FEIBA IMMUNO is a preparation in which a new activity is generated capable of bypassing factor VIII. The preparation which is used to treat patients with inhibitors (especially inhibitors to factor VIII) is standardized in FEIBA units, i.e. in terms of its in vitro capacity to shorten the activated PTT of a factor VIII inhibitor plasma.It could be concluded from different in vitro experiments that none of the classic’ activated coagulation factors is responsible for the factor VIII bypassing reaction; FEIB-activity seems to be correlated to a new complex of coagulation factors.To get an answer to the question which coagulation factors are essential for FEIB-activity, we tried to generate this activity from different deficient plasmas; from these experiments the following conclusions could be drawn:, the presence of at least factors VII, IX, and X is essential for the generation of the molecular species responsible for factor VIII as well as factor X bypassing activity, but factor V is not bypassed. This activity is not factor Xa itself. Factors VIII and V are not necessary for the generation of this active principle, but factor V is finally needed for its bypassing action.

Hemophilic Dog Model For Evaluating Hemostatic Efetcacy Of Plasma Protein Fractions

1981 ◽  
Author(s):  
H S Kingdon ◽  
T M Hassell
Keyword(s):  
Factor Viii ◽  
Plasma Protein ◽  
Hemophilia A ◽  
Protein Fractions ◽  
Factor Viii Inhibitor ◽  
Repeat Dose ◽  
The Usa ◽  
Sharp Dissection ◽  
Viii Inhibitor ◽  
Uncontrolled Bleeding

About 15% of patients with hemophilia A develop inhibitors to Factor VIII. Because in many cases the inhibitor renders the patient refractory to treatment with Factor VIII, plasma protein fractions designed to bypass the inhibitor have been developed. In the USA these are referred to genetically as Anti Inhibitor Ccrplex Concentrates (AICCs). Development of AICCs has been hampered by lack of a suitable irodelin which to judge hemostatic efficacy. Similarly, lack pf a model has impeded research on the mechanism of action Of AICCs. Therefore, we chose to evaluate AICCs in dogs with hemophilia A, reasoning that a material capable of bypassing & Factor VIII inhibitor should be effective in Factor VIII deficient recipients with or without inhibitors. Under local anesthesia a standardized gingival biopsy was performed losing a flexible plastic template and a modified scalpel handle holding two #11 Bard-Parker blades. The parallel time is ions were 5 mm long, 2 nm apart, and 1.5 mm deep. The tissue block thus defined was removed by sharp dissection. In normal dogs, bleeding from this wound ceased in 5 ± 2min, the wound was filled with concave clot, and bleeding did not recur. In contrast, hemophilic dogs formed an abnormal (very large) clot, and rebled for several days if untreated. The hematocrit usually dropped by 2-10 percentage points in 24 hr of uncontrolled bleeding. An experimental AICC fraction under development by Cutter Laboratories was evaluated in 5 dogs, and shown to be hemostatically effective. The dose required to achieve hemostasis was 25-75 units/kg; in some dogs a second dose was required 6 hr after the first dose to maintain hemostasis. A single dog with a low titer inhibitor to Factor VIII was successfully treated with 39 u/kg, followed by a repeat dose of 39 u/kg 6 hr after the first dose. We conclude that this AICC preparation brings about nemostasis in Factor VIII deficient individuals with or without inhibitors to Factor VIII.

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