#506 Thrombin-activated factor VIII ARG531HIS has increased rate of A2 subunit dissociation responsible for the phenotype of discrepant one stage and two stage activities

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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Molecular genetic background of haemophilia A patients with discrepancy between one-stage and two-stage factor VIII assays

Hämostaseologie ◽

10.1055/s-0037-1619100 ◽

2010 ◽

Vol 30 (S 01) ◽

pp. S153-S155

Author(s):

D. Delev ◽

S. Pahl ◽

J. Driesen ◽

H. Brondke ◽

J. Oldenburg ◽

...

Keyword(s):

Factor Viii ◽

Genetic Background ◽

Molecular Genetic ◽

Haemophilia A ◽

Two Stage ◽

One Stage

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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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An International Collaborative Assay of Factor VIII Clotting Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648661 ◽

1978 ◽

Vol 40 (02) ◽

pp. 260-271 ◽

Cited By ~ 23

Author(s):

T W Barrowcliffe ◽

T B L Kirkwood

Keyword(s):

Factor Viii ◽

World Health ◽

Expert Committee ◽

Two Stage ◽

International Standard ◽

One Stage ◽

Normal Plasma ◽

Freeze Dried ◽

The Mean ◽

International Collaborative

SummaryAn International Collaborative Study was organised to replace the first International Standard for factor VIII. A freeze-dried concentrate, 73/552, and a freeze-dried plasma, 75/510, were assayed against the International Standard, and also compared to fresh normal plasma and local standards.In assays of the concentrate 73/552 against the first I.S. the mean potency was 1.14 i.u./ ampoule and there was no significant difference between one-stage and two-stage methods. When assayed against average fresh normal plasma, the potency was 1.05 “normal plasma units” per ampoule. It was agreed by the participants that the potency of 73/552 be regarded as the mean of these two figures, i.e. 1.10 i. u./ampoule.In assays of the freeze-dried plasma, 75/510, against the first I.S. the mean potency was 0. 68 i. u./ampoule, but the one-stage assays gave significantly higher potencies (mean 0.74 1. u./ampoule) than the two-stage assays (mean 0.59 i. u./ampoule). The same trend was also seen in the fresh normal plasmas, and in the local plasma standards. This finding has important implications for the standardisation of factor VIII.Stability studies on the concentrate 73/552 gave a predicted loss of 0.02% per year at – 20° C. All participants agreed that the material was suitable to serve as an International Standard, and at the 26th meeting of the Expert Committee on Biological Standardisation of the World Health Organization, the material in ampoules coded 73/552 was established as the 2nd International Standard for factor VIII, with a potency of 1.10 i. u./ampoule.

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Automated Techniques for Estimating Factor VIII Levels in Concentrate Preparations

10.1055/s-0039-1689481 ◽

1975 ◽

Author(s):

M. J. Seghatchian ◽

M. Miller-Anderson ◽

D. J. Howarth ◽

Y. Stirling ◽

M. Brozovic

Keyword(s):

Factor Viii ◽

Factor Vii ◽

Two Stage ◽

End Point ◽

One Stage ◽

Different Types ◽

Long Course ◽

Good Agreement

An. automated coagulometer (a modification of Electra 600D) using an optical end-point for clot detection, has been used in both one and twostage methods of assaying factor VIII. Fifity clotting times can be measured in a hour. The methods for which the coagulometer has been used are (1) one-stage assay with kaolin-platelet substitute and haemophilic plasma, (2) one-stage assay with activated cephaloplastin (Dade, Nyegaard) and haemophilic plasma, and (3) two-stage method using combined serum reagent (Diagen). With the first two methods, a shortening of standard and blank clotting times occurred during the hour-long course of the assays (2-5 seconds per hour); this was particularly marked in the presence of kaolin, and due in part to activation of factor VII. With the two-stage method, longer standard and blank clotting times, up to 8 seconds per hour, were often recorded as the assays proceeded. These results emphasize the need to incorporate standards at regular intervals during every run, and to take account of the changes with time that occur.Four different types of concentrate preparations commonly used in the treatment of haemophilia were assayed in two laboratories, each using conventional and automated (Electra 600D) methods. There was good agreement between methods and between laboratories.

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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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Comparative Investigations on Factor VIII Assays

10.1055/s-0039-1689477 ◽

1975 ◽

Author(s):

R. Pflugshaupt ◽

S. Moser ◽

K. Züger ◽

R. Bütler

Keyword(s):

Factor Viii ◽

High Activity ◽

Hemophilia A ◽

Statistical Evaluation ◽

Two Stage ◽

One Stage ◽

Normal Plasma ◽

Calibration Curves ◽

Factor Viii Concentrates

Six one stage methods and one two stage method were tested for precision and reproducibility. With each method twenty calibration curves of normal plasma and two lots of Factor VIII concentrates were established. Statistical evaluation revealed only minor differences. Neither one of the methods was optimal for both the physiological-pathological region and the region of high activity preparations.Three selected methods were tested in vivo for accuracy: nine patients with hemophilia A were treated with equal amounts of Factor VIII concentrates or kryoprecipitates respectively. The methods showed different activities for preparations as well as for patient’s plasma. The discrepancy between measured and expected recovery differed for each method.

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Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa In Vivo

Blood ◽

10.1182/blood.v93.1.176 ◽

1999 ◽

Vol 93 (1) ◽

pp. 176-183 ◽

Cited By ~ 80

Author(s):

S.W. Pipe ◽

A.N. Eickhorst ◽

S.H. McKinley ◽

E.L. Saenko ◽

R.J. Kaufman

Keyword(s):

Factor Viii ◽

Missense Mutation ◽

Time Course ◽

Hemophilia A ◽

Specific Activity ◽

Metabolic Labeling ◽

Two Stage ◽

One Stage ◽

Thrombin Cleavage

Abstract Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional factor VIII (FVIII) protein and are termed cross-reacting material (CRM)-positive. FVIII is a heterodimer (domain structure A1-A2-B/A3-C1-C2) that requires thrombin cleavage to elicit procoagulant activity. Thrombin-activated FVIII is a heterotrimer with the A2 subunit (amino acid residues 373 to 740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIII. Recently, a phenotype of CRM-positive hemophilia A patients has been characterized whose plasma displays a discrepancy between their FVIII activities, where the one-stage clotting assay displays greater activity than the two-stage clotting assay. One example is a missense mutation whereARG531 has been substituted by HIS531. An FVIII cDNA construct was prepared containing theARG531HIS mutation and the protein was expressed in COS-1 monkey cells by transient DNA transfection. Metabolic labeling with [35S]-methionine demonstrated that ARG531HIS was synthesized at an equal rate compared with FVIII wild-type (WT) but had slightly reduced antigen in the conditioned medium, suggesting a modest secretion defect. A time course of structural cleavage of ARG531HISdemonstrated identical thrombin cleavage sites and rates of proteolysis as FVIII WT. Similar to the patient phenotypes,ARG531HIS had discrepant activity as measured by a one-stage activated partial thromboplastin time (aPTT) clotting assay (36% ± 9.6% of FVIII WT) and a variation of the two-stage assay using a chromogenic substrate (COAMATIC; 19% ± 6.9% of FVIII WT). Partially purified FVIII WT and ARG531HISproteins were subjected to functional activation by incubation with thrombin. ARG531HIS demonstrated significantly reduced peak activity and was completely inactivated after 30 seconds, whereas FVIII WT retained activity until 2.5 minutes after activation. Because the ARG531HIS missense mutation predicts a charge change to the A2 subunit, we hypothesized that theARG531HIS A2 subunit could be subject to more rapid dissociation from the heterotrimer. The rate of A2 dissociation, using an optical biosensor, was determined to be fourfold faster forARG531HIS compared with FVIII WT. Because the two-stage assay involves a preincubation phase before assay measurement, an increased rate of A2 dissociation would result in an increased rate of inactivation and reduced specific activity.

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In Vivo Recovery and Survival of Monoclonal-Antibody-Purified Factor VIII Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646492 ◽

1991 ◽

Vol 66 (06) ◽

pp. 730-733 ◽

Cited By ~ 12

Author(s):

Carol K Kasper ◽

Hugh C Kim ◽

Edward D Gomperts ◽

Kenneth J Smith ◽

Phyllis M Salzman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Factor Viii ◽

In Vitro Assays ◽

Two Stage ◽

Double Blind ◽

One Stage ◽

Factor Viii Concentrates ◽

In Vivo Recovery

SummaryIn response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

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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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