A Simple Method of Studying the Generation of Thrombin in Recalcified Plasma: Application in the Investigation of Haemophilia

Pitney, W. R.; Dacie, J. V.

doi:10.1136/jcp.6.1.9

Cited by 146 publications

(78 citation statements)

References 6 publications

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“…The thrombin generation time (TGT) was originally described by Macfarlane and Biggs [7] for whole blood and by Pitney and Dacy [8] for plasma. Since then, a vast number of variants have been published, all differing in detail from one another.…”

Section: Thrombin Generation Test and Thromboelastographymentioning

confidence: 99%

Diagnosis of factor VIII deficiency

Verbruggen

Meijer²,

Nováková

2008

Haemophilia

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Summary. The correct diagnosis of factor VIII deficiency and the assessment of severity of the disease are essential for a patient-tailored treatment strategy. An optimal diagnostic procedure comprises sensitive and specific screening methods and factor VIII activity assays. Different screening reagents show variable characteristics and receiver operator characteristic curves are presented showing the relation between sensitivity and specificity of eleven activated partial thromboplastin time reagents. The details of the three methods for factor VIII activity assay, onestage and two-stage assay and chromogenic assays, are discussed. The chromogenic assay seems to be more sensitive than the one-stage assay with regard to the detection of severe haemophilia. Discrepant results obtained with one-stage and two-stage assays are reviewed and discussed.

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Section: Thrombin Generation Test and Thromboelastographymentioning

confidence: 99%

Diagnosis of factor VIII deficiency

Verbruggen

Meijer²,

Nováková

2008

Haemophilia

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“…Thrombin Generation Test-This assay is a modification of that carried out by Pitney and Dacie (22). Normal platelet-poor plasma was used as substrate.…”

Section: Screening the Phage Library With Mab Esh8 -The Different M13mentioning

confidence: 99%

Low Molecular Weight Peptides Restore the Procoagulant Activity of Factor VIII in the Presence of the Potent Inhibitor Antibody ESH8

Villard

Piquer

Raut³

et al. 2002

Journal of Biological Chemistry

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Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the 2231 QVDFQKTMKV 2240 sequence of the C 2 domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence 2267 DGHQ 2270 . Upon inspection of the C 2 domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivo in a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs.Coagulation defects attributed to the absence or dysfunction of blood coagulation factor VIII (FVIII) 1 are observed in 0.01-0.02% of the male population (1). This recessive X-linked bleeding disorder is known as hemophilia A. The adequate treatment of this disease relies on infusions of human FVIII concentrates. However, a serious treatment complication is the development of a humoral immune response to FVIII in ϳ25% of patients with hemophilia A. These antibodies (Abs), also called inhibitors, block the FVIII procoagulant activity and complicate seriously the medical care of the patients by precluding further FVIII injections (2). This immune response is not only polyclonal but also heterogeneous in its specificity (3). Nevertheless, epitope mapping studies have shown that inhibitors recognize restricted binding sites, predominantly on the A 2 , C 2 , and A 3 domains on the FVIII molecule (4). The incidence of inhibitor occurrence in hemophilia A patients is difficult to estimate and can vary from 18 to 28% (5). This variation can be the result of a number of parameters, which needs to be considered, including quantitative criteria of inhibitor dosages, the elapsed time from the first apparition, the origin, the viral inactivation procedure applied, the purity of the administered product, and, of course, the patient.A number of different therapeutic approaches have been developed to circumvent complications arising from inhibitors, such as the administration of porcine FVIII, which is less antigenic than its human counterpart (6), the administration of activated human FVII to bypass the intrinsic pathway (7), and the activation of ...

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“…These tests have their natural end point at a time when only 3-5 % of thrombin has been formed. Although it was developed half a century ago [1,2], it is only during the last one and a half decade that the thrombin generation test has attracted increasing attention. It appears that this test may replace coagulation based tests in many cases in the future [3,4], more so as computer aided models of the events leading to coagulation have been developed and been shown to correlate well with biochemical observations [5,6].…”

Section: Introductionmentioning

confidence: 99%

Calibrating Thrombin Generation in Different Samples: Less Effort with a Less Efficient Substrate

Tapp¹,

Grundmann²,

Kusch³

et al. 2009

TOATHERTJ

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Abstract:The technique of thrombin generation has been established as a promising tool for the diagnosis, risk estimation, and perhaps prognosis of coagulation insufficiencies. Without further experimentation an indication can be obtained if a sample donor carries the risk for haemophilia or thrombophilia, either congenital or acquired. A comparison of two different thrombin generation assays is presented, demonstrating that a currently not commercially available test allows an easier and cheaper calibration than its commercial counterpart. This is achieved by employment of a less efficient but highly specific peptide substrate for thrombin and the involvement of low amounts of analyte (plasma samples).

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A Simple Method of Studying the Generation of Thrombin in Recalcified Plasma: Application in the Investigation of Haemophilia

Cited by 146 publications

References 6 publications

Diagnosis of factor VIII deficiency

Diagnosis of factor VIII deficiency

Low Molecular Weight Peptides Restore the Procoagulant Activity of Factor VIII in the Presence of the Potent Inhibitor Antibody ESH8

Calibrating Thrombin Generation in Different Samples: Less Effort with a Less Efficient Substrate

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