Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1999-03-01
2000-10-31
Chin, Christopher L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 74, 435 4, 435 13, 435 23, 435 24, 435968, 435805, 436 69, 436166, 436169, 436170, 530331, 530802, 2601125, 422 56, 422 57, 422 60, 422 61, C12Q 138, C12Q 156, C07C10352, G01N 3348, G01N 3114
Patent
active
061400629
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of determining the heparin content, in particular in plasma.
The coagulation factors X and II play an important part in the blood coagulation mechanism. The intrinsic and extrinsic systems of the blood coagulation mechanism meet in coagulation factor X. Under the influence of the activated factor X (FXa), factor II (prothrombin) is activated to form IIa (FIIa or thrombin). Thrombin then catalyses the conversion of fibrinogen into fibrin, as a result of which the actual blood coagulation occurs.
An important inhibitor of both FXa and FIIa is antithrombin III (AT), a protein which occurs in blood plasma and which deactivates FXa and FIIa by concomitantly forming a complex in an irreversible manner. AT thus inhibits the blood coagulation. It is known that heparin intensifies the deactivating action of AT by increasing the reaction rate with which AT forms an irreversible complex with FXa or FIIa.
Heparin is a mixture of polysaccharides having molecular weights which vary from 2000 to more than 40,000. It is known that active heparin is composed of two different types of active molecules: heparin molecules having a molecular weight which is higher than 5400 daltons (ACLM) and heparin molecules having a molecular weight of 5400 daltons or less (BCLM). The ACLM variant has both anti-FXa and anti-FIIa activity, while the BCLM variant has only anti-FXa activity. For this reason, the ratio between anti-FIIa activity and anti-FXa activity depends on the distribution of the polysaccharide chains in the heparin mixture.
The object of the present invention is therefore, inter alia, to provide a method with which it is possible to determine both the total heparin content and the separate contents of the ACLM and BCLM variants.
Methods of determining the heparin content of samples are known. Examples are the anti-thrombin test, the anti-FXa test, a test in which the activated partial thromboplastin time (APTT) is measured and various tests in which the decomposition of FXa or FIIa is measured. The results of said tests are often influenced by random variations in the plasma samples. For this reason, a calibration against a standard amount of heparin is necessary for every collection of plasma samples. In these methods, it is assumed that the result of the test with the standard will vary in the same way as with the samples to be tested. However, this is not always the case. An important cause of this is the fact described above that heparin is composed of two different types of active molecules. The ratio between anti-FIIa and anti-FXa activities is dependent on the distribution of the polysaccharide chains in the heparin. The anticoagulation action is dependent on the amount of anti-FIIa activity present. Because the anti-FIIa to anti-FXa activity ratio varies for each sample and for each sample preparation and is also dependent on the type of heparin and the time after which measurement is made, the result of a test which makes no distinction between ACLM and BCLM may result in incorrect interpretations of the test results. Various tests react differently to variations in the ratio between anti-FIIa activity and anti-FXa activity and it is therefore difficult to draw conclusions about the thrombotic behaviour of a patient.
The object of the present invention is therefore also a method of determining the heparin content in a sample in which a distinction can be made between the two types of heparin molecules mentioned above.
Moreover, U.S. Pat. No. 5,308,755 discloses a method of determining the heparin concentration in which a sample having an unknown heparin concentration is mixed with AT and a substrate for factor Xa or thrombin. Factor Xa or thrombin is then added to the sample in such a way that the enzyme is deactivated by the AT and that a portion of the substrate is converted into a product whose concentration can be determined.
According to said document, substrates having a high affinity for the enzyme, such as S2238 and S2222, are used. As a result, it takes a relativ
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Ten Cate H et al. "Automated amidolytic method for determining heparin, a heparinoid, and a low molecular weight heparin fragment based on their anti-factor XA activity" Clin Chem 30 (6). 1984 pp. 860-864.
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Hemker Hendrik Coenraad
Wagenvoord Robert Johan
Chin Christopher L.
Cook Lisa V.
Universiteit Maastricht
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