Thrombin Potentiation of Factor VIII Procoagulant Activity: Assessment by the Two-Stage Assay

SummaryFactor VIII (FVIII) procoagulant activity is the function of a plasma glycoprotein that is missing or inactive in patients with classic hemophilia. Numerous studies have shown that trace thrombin causes rapid enhancement followed by gradual inactivation of FVIII procoagulant activity. Recent evidence suggests that thrombin activation of the FVIII/von Willebrand factor (vWF) protein is required for inactivation to occur. All of these studies have used the one-stage partial thromboplastin time to assay FVIII activity. Other investigators have used the two-stage assay of FVIII activity and have been unable to demonstrate thrombin-induced enhancement of FVIII activity, although inactivation has consistently occurred. We performed experiments designed to help resolve this disagreement, using the two-stage assay specifically modified to detect thrombin potentiation of FVIII activity. The length of the first-stage incubation time was found to be critical in demonstrating the initial effect of thrombin on FVIII activity. Taking advantage of this finding we were able to show a 4.1 ± 0.5-fold enhancement of FVIII activity upon incubating purified FVin/vWF with 0.04 NIH unit thrombin per ml. The apparent enhancement of FVEQ activity declined with increasing thrombin concentration. Incubation with 0.08, 0.16, and 0.32 NIH unit thrombin per ml resulted in only 3.2 ± 0.5, 2.6 ± 0.5 and 1.5 ± 0.3-fold enhancement, respectively, of FVIII activity. As with results from the one-stage assay, activation was followed by slow inactivation of FVIH/vWF. Using the two-stage assay we also showed 100% inactivation and 100% inhibition of FVIII activity by plasmin and human anti-FVUI IgG, respectively. Plasmin inactivation of FVIII activity showed a dose-response effect. Thrombin was unable to activate plasmin-degraded FVin/vWF. Our results show that thrombin potentiation of FVni activity is easily demonstrable in the two-stage assay. These findings support the contention that activation of FVm activity by thrombin is prerequisite for inactivation and underscore the importance of thrombin activation of FVHI/vWF in the intrinsic clotting system.

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Studies Of Factor VIII Inhibitor Bypassing Activity (FEIBA)

10.1055/s-0038-1651981 ◽

1981 ◽

Cited By ~ 1

Author(s):

T W Barrowcliffe ◽

E Gray ◽

G Kemball-Cook

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Factor Ix ◽

Procoagulant Activity ◽

Two Stage ◽

Human Antibodies ◽

One Stage ◽

Viii And Ix ◽

The One

The activities of an activated Factor IX concentrate (FEIBA, Immuno AG) were studied by two in vitro assays: a one-stage method using VUI-deficient plasma as substrate, and a two-stage assay based on the thrombin generation test. The nature of the active principle was explored by measuring the reduction in activity when FEIBA was incubated with specific antibodies.Incubation of FEIBA with human antibodies to Factor VIII reduced its activity by about 30% in the one-stage assay, and about 50% in the two-stage assay, suggesting that FEIBA contains Factor VIII procoagulant activity. Inactivation of the Factor VIII in FEIBA was somewhat slower than that of normal Factor VIII, indicating partial protection from inhibition. Human antibodies to Factor IX inhibited the one stage activity by about 30%, and incubation with both antibodies also produced a 30% reduction in activity. The remaining procoagulant activity decayed only slowly when incubated with non-inhibitor plasma. In contrast, purified human Factor Xa lost activity rapidly on incubation in normal plasma, as did a purified fraction from a Factor IX concentrate, which had high activity in the one-stage assay.These results suggest that the in vitro activity of FEIBA is due to at least two components. One component appears to be dependent on both Factors VIII and IX and may be a complex of VIII and IXa. The other component acts later than Factors VIII and IX in the coagulation cascade but, unlike purified Factor Xa, is relatively resistant to inactivation by plasma inhibitors such as antithrombin III. FEIBA was also found to contain phospholipid, and it may be that the phospholipid protects both the Factor VIII and activated enzymes from their inhibitors.

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Standardization of Factor VIII – IV. Establishment of the 3rd International Standard for Factor VIII: C Concentrate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665290 ◽

1983 ◽

Vol 50 (03) ◽

pp. 697-702 ◽

Cited By ~ 14

Author(s):

T W Barrowcliffe ◽

A D Curtis ◽

D P Thomas

Keyword(s):

Factor Viii ◽

High Purity ◽

Collaborative Study ◽

World Health ◽

Current Standard ◽

Two Stage ◽

International Standard ◽

One Stage ◽

The One ◽

Health Organization

SummaryAn international collaborative study was carried out to establish a replacement for the current (2nd) international standard for Factor VIII: C, concentrate. Twenty-six laboratories took part, of which 17 performed one-stage assays, three performed two-stage assays and six used both methods. The proposed new standard, an intermediate purity concentrate, was assayed against the current standard, against a high-purity concentrate and against an International Reference Plasma, coded 80/511, previously calibrated against fresh normal plasma.Assays of the proposed new standard against the current standard gave a mean potency of 3.89 iu/ampoule, with good agreement between laboratories and between one-stage and two- stage assays. There was also no difference between assay methods in the comparison of high-purity and intermediate purity concentrates. In the comparison of the proposed standard with the plasma reference preparation, the overall mean potency was 4.03 iu/ampoule, but there were substantial differences between laboratories, and the two-stage method gave significantly higher results than the one stage method. Of the technical variables in the one-stage method, only the activation time with one reagent appeared to have any influence on the results of this comparison of concentrate against plasma.Accelerated degradation studies showed that the proposed standard is very stable. With the agreement of the participants, the material, in ampoules coded 80/556, has been established by the World Health Organization as the 3rd International Standard for Factor VIII :C, Concentrate, with an assigned potency of 3.9 iu/ampoule.

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Identification of Sources of Inter-Laboratory Variation in Factor VIII Assay

10.1055/s-0039-1682402 ◽

1977 ◽

Author(s):

T.B.L. Kirkwood ◽

C.R. Rizza ◽

T.J. Snape ◽

I. Rhymes ◽

D.E.G. Austen

Keyword(s):

Factor Viii ◽

Systematic Difference ◽

Normal System ◽

Two Stage ◽

Initial Dilution ◽

One Stage ◽

Collaborative Studies ◽

Freeze Dried ◽

Sources Of Variation ◽

The One

A repeated finding of national and international collaborative studies of standard Factor VIII preparations has been that systematic differences exist between laboratories in their measurement of the relative activities of the same pairs of Factor VIII preparations.A workshop meeting was held at the Oxford Haemophilia Centre (England) during 23rd-26th November 1976 to investigate which of the possible sources of variation between laboratories were responsible. Participants from 16 British laboratories (9 one-stage, 7 two-stage) performed a total of 273 assays using three freeze-dried preparations of differing purity (a plasma, an intermediate and a high purity concentrate). The results of assays with each participant using their normal system established that, if the participants were a representative cross-section, approximately one-third of one-stage laboratories would show a systematic difference from the overall mean of at least 16%, with a similar figure for the two-stage laboratories of 9%. Various features of the assay systems were then modified in a controlled series of experiments. The results showed conclusively that i) differences between reagents accounted for most of the variation between laboratories and, ii) the two-stage assays were, on average, detecting relatively more activity in the more purified preparations than the one-stage assays. The results also suggested that the use of buffer as opposed to haemophilic plasma for the initial dilution of concentrates did not affect the assay results.

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The Influence of Thrombin on the Clotting Activity of Factor VIII. A Study with Insolubilized Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646733 ◽

1978 ◽

Vol 39 (03) ◽

pp. 600-606 ◽

Cited By ~ 5

Author(s):

Th Vukovich ◽

E Koller ◽

W Doleschel

Keyword(s):

Factor Viii ◽

Test Sample ◽

Test System ◽

Factor Viii Activity ◽

Two Stage ◽

One Stage ◽

Analytical Procedures ◽

The One

SummaryAn investigation of the influence of thrombin on the clotting activity of factor VIII was made. Purified factor VIII and different amounts of thrombin complexed to Sepharose 4 B were mixed and incubated for various periods of time. The factor VIII activities of these incubation mixtures were determined by the one- and two-stage analytical procedures in the presence of the thrombin-sepharose and in its absence following the latter removal from the test sample by filtration. The results so obtained confirm the view that thrombin inactivates factor VIII. Evidences for a thrombin-induced potentiation of the factor VIII activity, seen only in the thrombin-sepharose containing test samples analyzed by the one-stage method, are here interpreted as thrombin-effects peculiar to this factor VIII test system and not as potentiation by thrombin of the factor itself.

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In Vivo Recovery of Factor VIII: A Comparison of One-Stage and Two-Stage Assay Methods

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657018 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1230-1239 ◽

Cited By ~ 46

Author(s):

I M Nilsson ◽

T B L Kirkwood ◽

T W Barrowcliffe

Keyword(s):

Factor Viii ◽

Half Life ◽

In Vitro Assays ◽

Two Stage ◽

One Stage ◽

Significant Difference ◽

Assay Methods ◽

The One ◽

Fraction I

SummaryThe recovery and half-life of VIII: C in the plasma of severely haemophilic patients was measured by one-stage and two-stage assays after injection of two Factor VIII concentrates (Hemofil, Hyland and Fraction I-O, Kabi). Plasma volumes were measured with an Evans� Blue technique, and both concentrates and post-infusion samples were measured against the same plasma standard.There was a highly significant difference in recoveries estimated by the two assay methods. The one-stage assays gave the most consistent results, in that the average recovery was 100%, whereas the two-stage assays gave only about 80% of the value expected from in vitro assays. There was no difference in recoveries between the two concentrates.The two-stage assays gave a slightly shorter half-life than the one-stage assays, and the half-life of Hemofil was also shorter than that of Fraction I-O.

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Disparity Between One- and Two-Stage Factor VIII Assays in Measuring VIIIC in Heparinized Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660132 ◽

1985 ◽

Vol 54 (04) ◽

pp. 780-783 ◽

Cited By ~ 4

Author(s):

G Rock ◽

D S Palmer

Keyword(s):

Factor Viii ◽

Procoagulant Activity ◽

Two Stage ◽

Adsorption Step ◽

One Stage ◽

Heparin Plasma ◽

The Difference

SummaryThe level of Factor VIII procoagulant activity (VIIIC) was found to be more than 30% higher in heparinized plasma than in citrated plasma from the same donor when determined by a one-stage assay (1.34 U/ml versus 1.0 U/ml). However, after Al(OH)3 adsorption and two-stage assay, the VIIIC levels were not significantly different for the two types of plasma (0.90 ± 0.25 U/ ml versus 0.82 ± 0.23 U/ml). If a one-stage assay was employed after adsorption, a similar low result was obtained (0.92 ± 0.21 U/ ml versus 0.91 ± 0.12 U/ml). This indicated that the adsorption step itself was responsible for the difference in results. Parallel analyses of other VIII markers demonstrated that more VIIIC antigen (VIIICAg) was removed by adsorption of heparinized rather than citrated plasma (0.33 U/ml versus 0.12 U/ml) although there was no difference in the level of VUI-related antigen (VIIIRAg). The results show that adsorption of heparinized plasma prior to either one- or two-stage assay leads to a greater loss of both VIIIC and VIIICAg activity than observed with citrated plasma and indicate that, when applied to heparin plasma the two-stage assay for VIIIC underestimates the actual VIIIC content.

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Decreased Potency Of Factor VIII Concentrates

10.1055/s-0038-1652637 ◽

1981 ◽

Cited By ~ 3

Author(s):

C K Kasper

Keyword(s):

Factor Viii ◽

In Vitro Assays ◽

Assay Method ◽

Two Stage ◽

One Stage ◽

Plasma Factor ◽

Factor Viii Concentrates ◽

The One

Plasma factor VIII recoveries after infusions of factor VIII concentrates into patients with classic hemophilia have been measured in this laboratory for 14 years. Recently, we observed a decline in the in vivo recovery of factor VIII per factor VIII unit infused. In 1980, plasma factor VIII levels were measured by a one-stage APTT-based assay before and 10 min after 150 infusions of 46 lots of 3 brands of factor VIII concentrate produced in the U.S.A. Our pooled normal plasma reference was calibrated against WHO International Standard 2 and results expressed in International factor VIII units. Observed in vivo factor VIII recovery was compared to the value expected from calculations based on the unitage stated on the label. The ratio of observed/expected recovery averaged 56% per lot for brand A, 60% per lot for brand B, and 103% per lot for brand C. In vitro assays were performed on 22 lots on 36 occasions, and the ratio of observed/labelled units average 46% per lot for brand A, 53% for brand B and 75% for brand C. The two-stage factor VIII assay method of Pool and Robinson was also used to assay plasma samples from 18 infusions, and results averaged 135% of the one-stage values for infusions of brand A, 160% for brand B, and 109% for brand C. (Brand A is assayed by the manufacturer by a two-stage method, brands B and C by one-stage methods.)Decreased clinical efficacy was observed when postinfusion plasma factor VIII levels were lower than customary. The decline in potency of brands A and B has necessitated more frequent assay of patients and use of larger amounts of concentrate, with greatly-increased expense. Investigation of the effect of different assay methods and different factor VIII standards and references on the apparent factor VIII content of concentrates has begun.

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Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/ Two-stage Activity Discrepancy

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613302 ◽

2002 ◽

Vol 88 (11) ◽

pp. 781-787 ◽

Cited By ~ 30

Author(s):

William Hakeos ◽

Hongzhi Miao ◽

Nongnuch Sirachainan ◽

Geoffrey Kemball-Cook ◽

Evgueni Saenko ◽

...

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Homology Model ◽

Hydrophobic Core ◽

Two Stage ◽

Threefold Axis ◽

Subunit Dissociation ◽

One Stage ◽

Fviii Activity ◽

A Domains

SummaryThrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. A homology model (Blood 89:2413, 1997) of the triplicated A domains of factor VIII (FVIII) predicts a pseudo-threefold axis at the tightly packed hydrophobic core with several interdomain interactions. These lie at the interface of A1-A2, A2-A3 and A1-A3. We have previously demonstrated that hemophilia A mutations (R531H, A284E, S289L) within the predicted A1-A2 and A1-A3 interface disrupt potential intersubunit hydrogen bonds and have the molecular phenotype of increased rate of inactivation of FVIIIa due to increased rate of A2 subunit dissociation. Patients with these mutations exhibit a clinical phenotype where the FVIII activity by one-stage(1-st) assay is at least two-fold higher than by two-stage(2-st) assay. We have now also explored mutations within the predicted A2-A3 interface (N694I, R698W and R698L) that also have the phenotype of 1-st/2-st activity discrepancy. These mutations exhibit the same molecular mechanism of increased instability of FVIIIa as those mutations described along the A1-A2 and A1-A3 interfaces. This suggests that the entire tightly packed hydrophobic core within the predicted pseudo-threefold axis contributes to stabilization of FVIIIa.

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Influence Of High Molecular Weight Factor VIII On The Expression Of Factor VIII Procoagulant Activity

10.1055/s-0038-1652746 ◽

1981 ◽

Author(s):

W Muntean ◽

W E Hathaway ◽

R R Montgomery

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

High Molecular Weight ◽

Procoagulant Activity ◽

Two Stage ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease ◽

The One ◽

Relationship Of ◽

The Relationship

The relationship of the high molecular weight (HMW) moiety and low molecular weight (LMW) moiety of factor VIII in expressing procoagulant activity (VIII C) was studied. LMW VIII C was prepared by immunoadsorbent chromatography; HMW VIII was prepared by chromatographing hemophilic cryo- precipitate in 4% agarose. The LMW VIII C obtained by immunoadsorbent chromatography gave higher VIII C values when tested in the one stage partial thromboplastin time (PTT) system using von Willebrand’s disease plasma as substrate than using hemophilic plasma as substrate. This finding was shown to be due to the VIII related antigen (VIIIR:Ag) in the substrate plasmas. When the VIIIR:Ag was removed from the hemophilic substrate plasma by immuno-adsorption, the VIII C values obtained for the LMW VIII C were higher. Also, adding purified HMW VIII to the von Willebrand’s disease substrate plasma resulted in lower VIII C values for the LMW VIII C in the PTT system.When the LMW VIII C was tested in the two stage assay, all VIII C was adsorbed to A1(0H)3. The adsorption of the LMW VIII C was prevented by mixing it with hemophilic plasma. From normal undiluted plasma only 5-21% of VIII C and no VIII related antigen were adsorbed to A1(OH)3, but after activation of the factor VIII of normal plasma by small amounts of thrombin, most of the VIII C was adsorbed. No VIII related antigen was adsorbed after activation.Nevertheless, when unadsorbed LMW VIII C was assayed by the two stage method both with and without HMW VIII or VIIIR:Ag, the results were the same.Our studies suggest that VIIIR:Ag prevents to some extent the activation of LMW VIII C. LMW VIII C that is not bound or protected by VIIIRiAg is adsorbed from plasma by A1(0H)3. These findings may help explain the differences for VIII C found in some patients and certain clinical circumstances with the one and two stage assays.

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