A More Uniform Measurement of Factor VIII Inhibitors

1975 ◽  
Author(s):  
C. K. Kasper ◽  
L. Aledort ◽  
D. Aronson ◽  
R. Counts ◽  
J. R. Edson ◽  
...  
Keyword(s):  
Factor Viii ◽  
Incubation Mixture ◽  
Equal Part ◽  
Normal Human Plasma ◽  
Dilution Factor ◽  
Factor Viii Activity ◽  
Blood Diseases ◽  
Normal Human ◽  
Lung Institute ◽  
Methods Of Measurement

A group of hematologists, involved with hemophilia research and care in the U.S.A., met under the sponsorship of the Division of Blood Diseases and Resources of the National Heart and Lung Institute. In order to improve future communication among ourselves, we decided to alter our individual methods of measurement of inhibitors to the extent necessary to permit a uniform, although arbitrary, description of inhibitor units. We agreed to the following standards: (1) The incubation mixture consists of one part citratecl patient plasma, undiluted or diluted, plus an equal part of citrated pooled normal human plasma. (2) A control incubation mixture consists of equal parts of normal pooled plasma and imidazole buffer, as formulated by Dr. Biggs. (3) The mixtures are incubated at 37° C for two hours. (4) Assays specific for Factor VIII are then performed and the Factor VIII activity in the patient mixture is divided by the Factor VIII activity in the control mixture to determine the percent residual Factor VIII activity. (5) A patient plasma giving a residual Factor VIII activity of 50 percent in this test is said to contain one “Bethesda unit” of inhibitor per ml. (6) On a graph, the log percent residual Factor VIII activity is plotted against inhibitor units. If the residual Factor VIII activity of the incubation mixture is between 75 and 25 percent, the inhibitor units are read from the graph. Plasmas containing strong inhibitors are diluted with imidazole buffer before being placed in the incubation mixture. A dilution is sought which will result in a residual Factor VIII activity between 75 and 25 percent. The units of inhibitor read from the graph are then multiplied by the dilution factor to determine the number of Bethesda units of inhibitor per ml of undiluted patient plasma.We invite interested colleagues to join us in the use of this method, and we invite discussion of better methods of describing inhibitor potency.

scholarly journals A Simple Method for the Assay of Factor VIII

Blood ◽  
1960 ◽  
Vol 15 (5) ◽  
pp. 637-645 ◽  
Author(s):  
LUIS JOSÉ BERGNA
Keyword(s):  
Factor Viii ◽  
Bovine Serum ◽  
Factor V ◽  
Normal Human Plasma ◽  
Factor Viii Activity ◽  
Simple Method ◽  
Serum Factors ◽  
Standard Normal ◽  
Normal Human ◽  
Generation Test

Abstract A simple method for the assay of factor VIII activity, based on the thromboplastin generation test of Biggs and Douglas, has been described. Bovine serum is used as a source of factor V and serum factors, and plasma from a hemophiliac is not required. The concentration of factor VIII of the test plasma is compared to that of a standard normal human plasma. The results obtained with the method have been fairly reproducible.

Investigations on the Nature of Hemophilia A

1963 ◽  
Vol 09 (01) ◽  
pp. 030-052 ◽  
Author(s):  
Eberhard Mammen
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Barium Sulfate ◽  
Freeze Drying ◽  
Room Temperature ◽  
Hemophilia A ◽  
Normal Human Plasma ◽  
Normal Human ◽  
And Storage ◽  
Prothrombin Activation

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

Heparin, Protamine and Polybrene

1965 ◽  
Vol 13 (02) ◽  
pp. 550-560 ◽  
Author(s):  
Anthony Britten
Keyword(s):  
Factor Viii ◽  
Incubation Mixture ◽  
Factor V ◽  
Factor Viii Activity ◽  
Rapid Loss

SummaryThe effects of incubating heparin, protamine or Polybrene with plasma were studied. All three drugs cause rapid loss of factor V from decalcified plasma, while Polybrene also accelerates the loss of factor VIII activity. These changes are related to temperature, the period of incubation and the dose of the drug used, and can be partially prevented by inclusion of neutralizing doses of the appropriate antagonist in the incubation mixture.The implications of these findings are discussed.

A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)

1979 ◽  
Author(s):  
E. T. Yin ◽  
W. J. Salsgiver ◽  
O. Tangen
Keyword(s):  
Equilibrium State ◽  
Human Plasma ◽  
Antithrombin Iii ◽  
Circumstantial Evidence ◽  
Incubation Mixture ◽  
Factor X ◽  
Normal Human Plasma ◽  
Plasma Component ◽  
Naturally Occurring ◽  
Normal Human

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F.Xa by F.Xa inhibitor(XaI), (Yin et.al.,Adv.Exper. Med. & Biol., 52 : 239, 1975, Plenum Press, N.Y.).This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-XaI”.In experiments employing purified components, when Anti-XaI was incubated at 37°C with F.Xa, Xal and heparin for two minutes at pH7.5, the amount of F.Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F.Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed.Anti-XaI was found to be neither PF3 nor PF4.These and other data strongly suggest that the “Antithrombin III pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-XaI and XaI.Under certain conditions when the Anti-XaI activity is predominant the rate of F.Xa neutralization bv XaI then becomes slower than the activation of prothrombin to thrombin by F.Xa.

A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)

1979 ◽  
Author(s):  
E.T. Yin ◽  
W.J. Salsgiver ◽  
O. Tangen
Keyword(s):  
Equilibrium State ◽  
Human Plasma ◽  
Antithrombin Iii ◽  
Circumstantial Evidence ◽  
Incubation Mixture ◽  
Factor X ◽  
Normal Human Plasma ◽  
Plasma Component ◽  
Naturally Occurring ◽  
Normal Human

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F. Xa by F. Xa inhibitor (XaI), (Yin et.al., Adv. Exper. Med.& Biol., 52: 239, 1975, Plenum Press, N.Y.).This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-Xal”.In experiments employing purified components, when Anti-Xal was incubated at 37°C with F. Xa. Xal and heparin for two minutes at pH7.5, the amount of F. Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F. Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed. Anti-Xa I was found to be neither PF3 nor PF6.These and other data strongly suggest that the “Antithrombin ill pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-Xal and Xal. Under certain conditions when the Anti-Xal activity is predominant the rate of F.Xa neutralization by Xal then becomes slower than the activation of prothrombin to thrombin by F.Xa.

Human Protein C, Its Inhibitor, And The Combined Deficiency Of Factors V And VIII

1981 ◽  
Author(s):  
Richard A Marlar ◽  
John H Griffin
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Partial Thromboplastin Time ◽  
Protein C ◽  
Human Protein ◽  
Clotting Factor ◽  
Factor V ◽  
Normal Human Plasma ◽  
Normal Plasma ◽  
Normal Human

Human protein C (PC) is a vitamin K-dependent serine protease zymogen present in normal plasma. PC was activated with soluble thrombin, thrombin-Sepharose, trypsin-Sepharose or Russell’s viper venom. When activated protein C (APC) at 10 μg/ml was added to normal plasma for 3 min prior to assays, clotting was greatly inhibited. In prothrombin time, activated partial thromboplastin time, and partial thromboplastin time assays, the clotting times were prolonged from 24 to 68 sec, 57 to 616 sec, and 247 to > 1200 sec, respectively. Both calcium and phospholipid were needed for this potent anticoagulant activity. The thrombin time was unaffected. To test which coagulation factors in plasma were inactivated by APC, APC was added to normal human plasma for 3 min and residual clotting factor activities were assayed using appropriate deficient substrate plasmas. Only Factor V and Factor VIII activities were decreased by APC. PC and DFP-inhibited APC were not anticoagulant and did not decrease Factors V and VIII. APC hydrolyzed synthetic peptide substrates. The amidolytic activity of APC was inhibited by normal human plasma (50% of APC activity was lost in 10 min). APC inhibitory activity was not detectable in plasmas from 4 unrelated patients with combined Factor V/VIII deficiency whereas it was present in normal amounts in plasmas from patients with simple Factor V deficiency or Factor VIII deficiency. Thus, it appears that APC may regulate blood coagulation by enzymatically inactivating two critical cofactors, Factor Va and Villa. Moreover, APC itself may be regulated by a previously undescribed plasma protein inhibitor. It is suggested that the molecular basis for combined Factor V/VIII deficiency disease that exhibits simple autosomal recessive inheritance is a deficiency of APC inhibitor.

scholarly journals The Apparent Separation of Factor VIII Related Antigen and Coagulant Activity from von Willebrand Factor Activity by an Immunoadsorbent

1977 ◽  
Author(s):  
H. A. Cooper ◽  
D. Lee ◽  
M. A. Lamb ◽  
R. H. Wagner
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Human Factor ◽  
Solid Phase ◽  
Residual Activity ◽  
Normal Human Plasma ◽  
Human Factor Viii ◽  
Coagulant Activity ◽  
Plasma Fractions ◽  
Normal Human

An antibody was raised in rabbits to the small active fragment of human factor VIII. The antigen was obtained by Ca2+ dissociation of a human factor VIII preparation made from a multidonor pool of plasma. After two adsorptions with 0.1 volume of normal human plasma, the antibody neutralized the F. VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions nor did it neutralize vWF activity as measured by ristocetin aggregation of fixed washed platelets. A solid phase immunochemical reagent was prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were similarly prepared with IgG fractions from a normal non-immunized rabbit. Using a batch technique the beads were studied for their ability to remove F. VIII coagulant, F. VIII Ag, and vWF activity from normal human plasma. Assay of the supernatant plasma after 2 hrs, 22°, from 10 replicate experiments gave the following results for residual activity, as per cent of non-immune bead control:F. VIII (37.5±4), F. VIII-Ag (30.8±9.7), and vWF (72.1±16). The experiment was repeated with 6 replicate samples with higher ratio of beads to plasma with essentially similar results. This unexpected separation of F. VIII-Ag from vWF activity prompted further investigation into how these activities are related to the molecular structure of F. VIII and vWF.

Measurement of factor VIII activity of B-domain deleted recombinant factor VIII

2001 ◽  
Vol 38 (2, Suppl 4) ◽  
pp. 13-23 ◽  
Author(s):  
M. Mikaelsson ◽  
U. Oswaldsson ◽  
M. A. Jankowski
Keyword(s):  
Factor Viii ◽  
Recombinant Factor Viii ◽  
Factor Viii Activity

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

1975 ◽  
Vol 33 (03) ◽  
pp. 547-552 ◽  
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.

Plasma Components which Interfere with Ristocetin-induced Platelet Aggregation

1975 ◽  
Vol 33 (03) ◽  
pp. 540-546 ◽  
Author(s):  
Robert F Baugh ◽  
James E Brown ◽  
Cecil Hougie
Keyword(s):  
Platelet Aggregation ◽  
Human Plasma ◽  
Plasma Proteins ◽  
Binding Protein ◽  
Plasma Heating ◽  
Protein S ◽  
Fold Increase ◽  
Normal Human Plasma ◽  
Preliminary Examination ◽  
Normal Human

SummaryNormal human plasma contains a component or components which interfere with ristocetin-induced platelet aggregation. Preliminary examination suggests a protein (or proteins) which binds ristocetin and competes more effectively for ristocetin than do the proteins involved in ristocetin-induced platelet aggregation. The presence of this protein in normal human plasma also prevents ristocetin-induced precipitation of plasma proteins at levels of ristocetin necessary to produce platelet aggregation (0.5–2.0 mg/ml). Serum contains an apparent two-fold increase of this component when compared with plasma. Heating serum at 56° for one hour results in an additional 2 to 4 fold increase. The presence of a ristocetin-binding protein in normal human plasma requires that this protein be saturated with ristocetin before ristocetin-induced platelet aggregation will occur. Variations in the ristocetin-binding protein(s) will cause apparent discrepancies in ristocetin-induced platelet aggregation in normal human plasmas.

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