Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1982-06-09
1985-01-29
Kepplinger, Esther M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 13, 435 23, C12Q 156, C12Q 138
Patent
active
044966539
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with a process for the determination of a new thrombin inhibitor, which is called antithrombin BM, and with a reagent for carrying out the process.
The proteolytic enzyme thrombin plays an important part in the blood coagulation system. It is known that, in the coagulation system, a specific inactivator for thrombin is present which is able to inhibit thrombin in the presence of heparin and is called antithrombin III (AT III). Therefore, the use of heparin as an anticoagulent plays an important part in therapy and research. Furthermore, AT III, which is also called heparin cofactor, is an important component of test systems for the coagulation properties of plasma. However, AT III also inhibits thrombin in the absence of heparin in a time-dependent reaction and, apart from thrombin, also inhibits, for example, trypsin, plasmin and Factor Xa, and thus is relatively non-specific.
Surprisingly, we have now found a new specific thrombin inhibitor which differs from AT III in many respects. It is described in more detail in Federal Republic of Germany Patent Specification (Patent Application) No. 30 38 163.
The new thrombin inhibitor inhibits thrombin in the presence of dextran sulphate at least twice as strongly as in the presence of heparin, but plasmin and trypsin are practically not inhibited at all and thus it differs immunologically from AT III.
Since the new thrombin inhibitor binds to heparin less strongly than AT III, it is called AT-BM (antithrombin binding moderately to heparin).
Whereas, for optimum thrombin inhibition, AT III only requires a heparin concentration of the order of 0.02 USP/ml., in the case of AT-BM, the heparin concentration necessary for optimum thrombin inhibition is more than 1.0 USP/ml.
Whereas human AT III also inhibits, inter alia, Factor Xa, plasmin and trypsin and does not react substantially stronger with human thrombin as with bovine thrombin, the new human AT-BM is almost completely thrombin-specific and inhibits human thrombin much more strongly than bovine thrombin. Furthermore, the thrombin inhibition by AT III is much more strongly promoted by heparin than by other activators, such as dextran sulphate. On the other hand, in the case of AT-BM, a 2 to 3 times stronger thrombin inhibition is achieved with dextran sulphate than in the presence of heparin.
Whereas AT III inhibits thrombin in a time-dependent reaction, even in the complete absence of cofactors, such as heparin, under these conditions AT-BM shows no inhibiting action whatsoever.
On the basis of the above properties, the new thrombin inhibitor AT-BM according to the present invention can be used not only similarly to AT III in therapeutic and diagnostic processes and thus is an interesting research agent but can also be determined analytically in the presence of AT III.
Therefore, according to the present invention, the determination of AT-BM takes place by utilising the specificity differences towards AT III in the case of various cofactors.
Thus, according to the present invention, there is provided a process for the determination of antithrombin BM, wherein an antithrombin-BM cofactor, thrombin and a determinable thrombin substrate are added to a sample solution and the splitting of the thrombin substrate is determined as a measure of the antithrombin-BM activity, whereby
(a) the specificity differences of antithrombin-BM towards antithrombin III in the presence of various amounts of the cofactors heparin, xylan or .lambda.-carrageenan are utilised and the difference determined of the activities obtained in the case of high and low cofactor concentrations or
(b) the antithrombin-BM activity is measured directly with dextran sulphate, mucopolysaccharide polysulphuric acid esters, pentosan polysulphates or short-chained heparin derivatives produced by the chemical or enzymatic splitting of heparin as cofactor.
The specificity differences of AT-BM in comparison with AT III in the presence of different amounts of heparin are given in the following Table 1:
TABLE 1 __________
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Lill Helmut
Schrenk Jurgen
Wunderwald Peter
Boehringer Mannheim GmbH
Kepplinger Esther M.
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