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 Study of the Cation-and pH-Dependent Stability of Factors V and VIII in Plasma*

1965 ◽  
Vol 14 (01/02) ◽  
pp. 032-051 ◽  
Author(s):  
HJ Weiss
Keyword(s):  
Factor Viii ◽  
Temperature Coefficient ◽  
Divalent Cations ◽  
Factor V ◽  
Rapid Loss ◽  
Edta Plasma ◽  
Ph Dependent ◽  
Nickel Manganese ◽  
Physical Reaction

SummaryThe in vitro lability of factors V and VIII in plasma has been studied. In agreement with previous reports, an increase in anticoagulant concentration renders both factors more labile (cation-deficient decay), as does an increase in the pH above 7.3 (alkaline-decay). Calcium appears to be the plasma cation which protects factors V and VIII against in vitro loss of activity. The protection obtained by the addition of other divalent cations depended on the type of plasma used. When resin-EDTA plasma was made cation free by dialysis at 4° C and then incubated at 37° C, the rapid loss of factors V and VIII activity could be prevented by prior addition of strontium, manganese and magnesium. In oxalate plasma, nickel, manganese, cadmium and strontium were effective.The alkaline decay of both factors V and VIII is irreversible. Partial reversibility of the cation-deficient decay was demonstrated for factor V, but not for factor VIII. The temperature coefficient for both the cation-deficient and alkaline decay is 2-3, suggesting an enzymatic rather than a physical reaction.There was no evidence to implicate thrombin, plasmin or trypsin since inhibitors of these enzymes failed to modify either type of decay.

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.

scholarly journals An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3067-3072 ◽  
Author(s):  
Cornelis van ‘t Veer ◽  
Neal J. Golden ◽  
Michael Kalafatis ◽  
Paolo Simioni ◽  
Rogier M. Bertina ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor V ◽  
Severe Hemophilia ◽  
Factor Viii Activity ◽  
Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

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.

Characterization of Clotting Factors Associated with Blood Platelets after Gel Filtration

1973 ◽  
Vol 30 (02) ◽  
pp. 289-298
Author(s):  
Oddvar Tangen ◽  
Eva B. Lestrup ◽  
Herbert J. Berman
Keyword(s):  
Factor Viii ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Factor Viii Activity ◽  
Clotting Factors ◽  
Platelet Surface ◽  
Filtration Factor

SummaryAggregation of human and rabbit gel filtered platelets (GFP) has been studied in presence of Ca2+, activated factor X (Xa) and different plasma preparations. It was found that factor Xa by itself is not a platelet aggregating agent. However, the platelets aggregated immediately when platelet poor plasma (PPP) was added to a mixture of GFP, Ca2+ and factor Xa. Aggregation also occurred immediately when factor V-deficient plasma was substituted for PPP, but not when factor II-deficient plasma was used. In the absence of factor Xa, aggregation occurred on addition of factor V- or VIII-deficient plasma, but only after some delay. The platelet aggregation experiments and experiments with centrifugations and resuspensions of the platelets, clotting experiments, and gel filtration of platelet free plasma (PEP) led to the following conclusions : Factors II and X are totally removed from the platelets by gel filtration, factor V is closely associated with the platelet surface, and part of the factor VIII-activity in the plasma is eluted together with the GFP without being associated with the platelets. This factor VIII-activity belonged to an extremely large molecule or molecular complex with a Mw in the order of 2 - 5 · 107.

scholarly journals An Artificial ‘Haemophilic’ Plasma for One-Stage Factor VIII Assay

1977 ◽  
Author(s):  
V. Chantarangkul ◽  
G.I.C. Ingram ◽  
S. Darby
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Factor V ◽  
Contact Activation ◽  
Factor Viii Activity ◽  
One Stage ◽  
Artificial Plasma ◽  
Bovine Factor

An ‘artificial’ haemophilic plasma for one-stage factor-VIII assays is made by incubating human plasma with EDTA, Na2 to destroy factor VIII, and afterwards removing the anticoagulant by dialysis. Bovine factor V is then added to a given level. In the assay, contact activation is controlled by adding contact product.It was confirmed that factor-VIII activity was destroyed and that the EDTA, Na2 was subsequently removed. The fibrinogen in the treated plasma clotted normally with thrombin. Likely variation in factor-V activity would not be critical. The concentration of fibrinogen and other factors was adequate. Variation between batches was small. The artificial plasma yielded assay results closely comparable to haemophilic plasma in samples with factor-VIII activities in the range 0.01-20.0 iu/ml.

Congenital Combined Deficiency of Factor V and Factor VIII

1969 ◽  
Vol 22 (02) ◽  
pp. 316-325 ◽  
Author(s):  
H Saito ◽  
M Shioya ◽  
K Koie ◽  
T Kamiya ◽  
O Katsumi
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Plasma Levels ◽  
Hemophilia A ◽  
Dental Extraction ◽  
Factor V ◽  
Factor Viii Activity ◽  
History Of ◽  
Fresh Plasma ◽  
Combined Deficiency

SummaryA case of congenital combined deficiency of factor V and factor VIII was reported. The patient, a 9 year old boy, gave a history of epistaxis, hematomas, and of hemorrhages following dental extraction since the age of 2 ; plasma levels of factor V and factor VIII were found to be 16% and 8% of normal, respectively. No one in his family had a deficiency of either factor. The effects of transfusion of normal fresh plasma and whole blood from a patient with hemophilia A were studied. While the former were similar to the pattern as seen in classical hemophilia, the latter consisted of an immediate increase of factor V activity and a delayed increase of factor VIII activity, despite the fact that factor VIII activity was almost absent from the donor’s blood.

Non-Decalcified Barium Sulfate-Adsorbed Plasma A Potentially Useful Reagent for Studying Blood Clotting, Platelets or Complement

1982 ◽  
Vol 47 (02) ◽  
pp. 182-184 ◽  
Author(s):  
Marjorie B Zucker ◽  
John Owen
Keyword(s):  
Factor Viii ◽  
Vitamin K ◽  
Divalent Cation ◽  
Barium Sulfate ◽  
Blood Clotting ◽  
Factor V ◽  
Fibrinopeptide A ◽  
Factor Viii Activity ◽  
Clotting Factors ◽  
Plasma Factor

SummaryPlasma was prepared by rapid centrifugation of native blood followed by adsorption of vitamin K-dependent clotting factors with BaSO4. Compared to fresh citrated plasma, BaSO4-treated plasma contained about 80% of factor V and factor VIII and the same concentration of fibrinopeptide A. Assays were also carried out after overnight incubation of citrated plasma and BaSO4-adsorbed plasma with and without added citrate and compared to assays of fresh citrated plasma. Factor V decreased to about 25% in both citrated samples, factor VIII decreased to 45% in both samples of BaSO4-treated plasma, and fibrinopeptide A did not change. Thus loss of factor V activity depended on reduction in divalent cation concentration whereas loss of factor VIII activity may have resulted from effects of early traces of thrombin.

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

Fibrinogen and its Derivatives: Cofactors in the Intrinsic Generation of Thrombin

1976 ◽  
Vol 35 (02) ◽  
pp. 305-313 ◽  
Author(s):  
D.C Triantaphyllopoulos ◽  
L.T Ryan
Keyword(s):  
Factor Viii ◽  
Vitamin K ◽  
Bovine Serum ◽  
Thrombin Generation ◽  
Factor V ◽  
Simultaneous Addition ◽  
Lower Effect

SummaryThe simultaneous addition of suboptimal concentrations of factor VIII and intact or plas-min-lysed fibrinogen into mixtures of the vitamin K dependent factors, phospholipids, adsorbed bovine serum (supplier of factor V) and calcium, increased the amount of thrombin which was generated three to twenty times over the sum of the amounts which were generated when factor VIII, or fibrinogen, or its derivatives were added separately into the thrombin generating mixture. When factor VIII was added together with both fibrinogen and its derivatives, the amount of thrombin generated was even greater, about 130% larger than the amount which was generated in the presence of equal concentrations of only intact fibrinogen plus factor VIII. Addition of albumin instead of fibrinogen or its derivatives has a similar but significantly lower effect on thrombin generation. It appears, therefore, that both intact fibrinogen and its plasminolytic derivatives, singly or in combination, and to a lesser extent albumin, act as cofactors in the reaction which is regulated by factor VIII.

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