EP0990157A1

EP0990157A1 - Dysprothrombinaemia

Info

Publication number: EP0990157A1
Application number: EP98934924A
Authority: EP
Inventors: Bernd PÖTZSCH; Simone Hund; Gert MÜLLER-BERGHAUS
Original assignee: Kerckhoff-Klink GmbH
Current assignee: Kerckhoff-Klink GmbH
Priority date: 1997-06-09
Filing date: 1998-06-08
Publication date: 2000-04-05
Also published as: WO1998057178A1; DE19724239A1

Abstract

The present invention relates to a method for detecting thrombophilia in patients and to a test kit containing the substances required for carrying out said method.

Description

Dysprothrombinemia

description

The present invention relates to a method for detecting thrombophilia in patients and to a test kit containing the substances required for carrying out the method.

In a group of patients with clinically suspected congenital thrombosis (thrombophilia), disorders of the hemostasis system can be identified as the molecular cause of thrombophilia in about 50% of patients. The majority of these molecular defects concern the protein C system, which is initiated by binding of the serine protease thrombin to the endothelial cofactor thrombomodulin (TM). The formation of a complex of thrombin and thrombomodulin activates protein C, which then has an anticoagulant effect.

Thrombomodulin in this reaction is not only a cofactor that increases substrate conversion, but also changes the substrate specificity of thrombin in the sense that thrombin loses its procoagulant properties.

The object of the present invention was to provide a possibility of making a prediction in patients as to whether there is an increased risk of thrombosis. It was found in the context of the invention that there are thrombin mutants which have normal procoagulatory (coagulation-promoting) but limited or no anticoagulant activity. Investigations with mutants generated in vitro have found that the molecular regions of the thrombin molecule which are necessary for binding to TM are only partially identical to the regions which mediate the fibrinogen interaction. To detect these postulated mutants, a Test method developed that specifically determines the anticoagulant activity of thrombin.

The object of the invention is therefore achieved by a method for detecting thrombophilia in patients in which the anticoagulant activity of thrombin in blood or blood plasma is determined.

This method is characterized by the knowledge that it is not the total concentration of thrombin that is decisive for the presence of thrombophilia, but rather the pro- and anticoagulatory activities of thrombin are to be considered, since defects in the anticoagulant activity alone cause otherwise unchanged procoagulatory activity Thrombophilia.

In a preferred embodiment of the invention, the total concentration of thrombin and the second the concentration of anticoagulant active thrombin are therefore determined in two separate test batches.

For this purpose, the total concentration of thrombin in the blood plasma is preferably determined after the thrombin formation has been initiated. Although it is also conceivable to include prothrombin in the determination of total thrombin, it is advantageous to initiate the formation of thrombin before the concentration is determined and then to measure the concentration.

For this purpose calcium, phospholipids and tissue factor (TF) are advantageously added to the plasma obtained from patient blood. As phospholipids, those are used which are coagulant. Such phospholipids are known to the person skilled in the art.

In order to rule out an unfavorable influence of other substances present in the plasma, such as, for example, antithrombin or alpha-2 macroglobulin or to keep it so low that it no longer hinders the determination, the plasma is preferably diluted in a ratio of 1: 100 to 1: 500 before the initiation of thrombin formation.

In a particularly preferred embodiment of the invention, the anticoagulant activity is investigated by activating protein C. For this purpose, in addition to determining the total concentration of thrombin in the plasma, the concentration of anticoagulant active thrombin is determined by adding thrombomodulin and protein C to the preferably diluted plasma. The amount of activated protein C formed is subsequently determined and the concentration of anticoagulant active thrombin is concluded therefrom.

In the concentration determinations according to the present invention, that is to say for thrombin and activated protein C, the hydrolysis of specific chromogenic substrates is preferably used as an indicator. Corresponding chromogenic substrates are known to the person skilled in the art and are generally available.

It is again preferred to use substances produced recombinantly, such as tissue factor, thrombomodulin and / or protein C, in the process according to the invention.

Although the process itself does not speak against the use of substances produced in a conventional way, recombinantly produced additives may have the advantage of greater purity, easier availability and thus a lower price, etc.

In another particularly preferred embodiment, the anticoagulant activity of thrombin is determined via the ability to complex with thrombomodulin. An impaired binding activity of thrombin with thrombomodulin can also be a cause of reduced anticoagulant activity. The complex formation- speed is preferably also determined after initiation of the formation of thrombin in the plasma and determination of the total concentration of thrombin.

For this purpose it is preferably initiated again by adding tissue factor and then, after determining total thrombin, thrombomodulin is added. The complex formed can then be detected using methods known per se. Recombinant thrombomodulin can also be used advantageously according to this method.

It is particularly preferred to bring the plasma into contact with thrombomodulin bound to the solid phase. The plasma can then be separated off particularly easily and the complex can be detected particularly preferably by means of a sandwich ELISA.

In this case, an anti-thrombin antibody is preferably used as the detection antibody. This antibody can, for example, itself carry a label, possibly also an indirect label, such as conjugation with an enzyme, the presence of which in turn is detected via a chromogenic substrate. On the other hand, however, it is also possible and preferred to have another antibody bound to the existing complex of thrombomodulin, thrombin and anti-thrombin antibody, which is directed against the anti-thrombin antibody. Anti-antibodies of this type are familiar to the person skilled in the art. Such an antibody would then carry a label, whereby all labels known to those skilled in the art are suitable for this.

In a very particularly preferred embodiment of the invention, both the anticoagulant activity of thrombin via the activation of protein C and the ability of thrombin to bind with thrombomodulin is determined immunologically to detect thrombophilia. Such a combination can make a particularly relevant one Statement about the presence of thrombophilia in patients.

Another object of the present invention is a test kit for performing the method according to the invention. Such a test kit includes all the essential substances necessary for carrying out the process or processes. Such test kits can optionally also contain buffer solutions and other substances.

A test kit according to the invention for determining the anticoagulant activity of thrombin in patient blood or plasma contains:

a) Tissue factor, calcium (preferably in the form of a calcium salt) and clotting-active phospholipids to initiate thrombin formation

and

b) Protein C and thrombomodulin

or and

c) solid-phase thrombomodulin;

and, if appropriate, suitable detection substances for thrombin and / or activated protein C and / or a thrombin-thrombomodulin complex formed.

In a preferred embodiment, the kit for the detection of thrombin and / or activated protein C contains a chromogenic substrate. In another preferred embodiment, the kit for detecting a thrombin-thrombomodulin complex formed contains an anti-thrombin antibody. It is possible for a labeled anti-thrombin antibody to be present or, preferably, for another antibody to be present which is directed against the anti-thrombin antibody and which itself bears a detectable label.

Furthermore, it is preferred that the substances tissue factor or / and protein C or / and thrombomodulin are present in recombinant form,

Monoclonal antibodies are particularly preferably used as antibodies.

The following examples and figures are intended to explain the invention further:

FIG. 1 shows a clear correlation between the amount of thrombin formed and the amount of activated protein C formed in healthy people after carrying out the determination according to the invention via activation of protein C.

FIG. 2 shows the correlation between measured activated protein C and expected activated protein C in a patient group with suspected thrombophilia.

example 1

In an amidolytic test procedure, both the thrombin concentration and the concentration of anticoagulant active thrombin are measured in two separate test batches. The plasma used is prediluted 1: 200.

By mixing with a recombinant and commercially available tissue factor in

In the presence of calcium and phospholipids, thrombin formation is initiated. One test batch contains recombinant thrombomodulin (rTM) and recombinant protein C (rPC). The concentrations are chosen so that the amount of activated protein C (APC) formed with the Correlated amount of functionally active thrombin. The APC formed is measured by hydrolysis of the chromogenic substrate S2366 (Chromogenix AB, Mölndal, Sweden, Art. No. 41 222), while in the control batch the amount of thrombin by hydrolysis of the thrombin substrate S2238 (Chromogenix AB, Mölndal, Sweden, Art No. 41 202) is determined. The pipetting schemes of both approaches are shown below:

Total thrombin Thrombin anticoagulant

50 μL plasma 1 .200 50 μL plasma 1: 200 + + 25 μL rTF (Innovin 1: 2) + 25 μL rTF (Innovin 1: 2)

+ 25 μL TBS, 0.1% BSA + 25 μL rTM (1 μM) + 20 μL calcium chloride (25mM) + 20μL calcium chloride (25mM) + 1 0 μL phospholipids (2.5μg / mL) + 10μL phospholipids (2.5μg / mL)

Incubation 2 min at 37 ° C

+ 20 μL TBS, 0.1% BSA + 20 μL rPC (1 00 nM)

Incubation 1 5 min at 37 ° C + 50 μL S2238 (2mM) + 50 μL S2366 / r-hirudin (2 mM / 0.5 μg / mL)

A group of 50 healthy blood donors showed that there is a clear correlation between the amount of thrombin formed and the amount of APC (FIG. 1). Accordingly, the amount of expected APC formed could be extrapolated based on the measured thrombin concentration. With this method, a statistically significant deviation between the amount of expected APC and the amount of APC actually formed was measured in a group of 57 patients with clinically suspected thrombophilia in 9 patients (FIG. 2).

There is a high probability that there is a causal relationship between the low measured anticoagulant activity of the patient Thrombins and the clinically existing tendency to thrombosis. A possible explanatory model for the reduced anticoagulant activity of thrombin is the presence of a mutation in the thrombomodulin binding sequence or the presence of another interference factor.

Example 2

The developed method represents a combined functional immunological assay. Recombinant thrombomodulin (rTM) is immobilized on a microtiter plate. After adding diluted plasma, thrombin formation is initiated by adding tissue factor (rTF) and then the amount of bound thrombin with a polyclonal anti-thrombin antibody (Alexis Corporation, San Diego, CA, USA, Order No. 60760-1 ) certainly. The assay procedure in detail:

Coating the microtiter plates:

100 μg rTM per well

Formation of thrombin:

50 μl plasma (1: 500/1: 1,000) + 25 μl rTF (Innovin 1: 2) + 1 5 μl calcium chloride (25mM) + 10 μl phospholipids (2.5μg / m

Incubation 1 5 minutes at room temperature

Determination of thrombin: rabbit anti-thrombin (10 0ng / ml) pig anti-rabbit IgG, peroxidase conjugated. The binding of thrombin can be measured specifically in a concentration range between 0.04 and 2.5 U / ml thrombin. The coefficients of the inter- and intra-assay variance were <1 2 and <10% in all concentration ranges. In the patient population mentioned above (example 1), a reduced TM binding was determined in all conspicuous patients (6 outside the x-2s range and 3 decreased borderline).

These data support the hypothesis that a reduced anticoagulant activity of thrombin can be caused by a restricted binding of thrombin to thrombomodulin.

Claims

claims

1. A method for the detection of thrombophilia in patients, ie that the anticoagulant activity of thrombin in the blood or blood plasma is determined.

2. The method of claim 1, d a d u rc h g e k e n n z e i c h n et that the total concentration of thrombin and the second the concentration of anticoagulant active thrombin is determined in two separate test batches.

3. The method of claim 2, d a d u rc h g e k e n n ze i c h n et that the total concentration of thrombin in the blood plasma is determined after the thrombin formation has been initiated.

4. The method of claim 3, d ad u rc h g e ke n n z e i c h n et that initiation of thrombin formation by adding calcium, phospholipids and tissue factor to the blood plasma.

5. The method according to claim 3 or 4, d a d u r c h g e ke n n z e i c h n et that the blood plasma is diluted before the initiation of thrombin formation in a ratio of 1: 100 to 1: 500.

6. The method according to claim 1 or 2, dadu rc hgekennzeichn et, that thrombomodulin and protein C are added to the blood plasma to determine the concentration of anticoagulant active thrombin and the amount of activated protein C formed is subsequently determined.

7. The method according to any one of the preceding claims, d a d u rc h g e k e n n ze i c h n et that the concentration of thrombin and / or activated protein C is determined by hydrolysis of chromogenic substrates.

8. The method according to any one of the preceding claims, d a d u rc h g e k e n n z e i c h n et that recombinant tissue factor, recombinant thrombomodulin and / or recombinant protein C is used.

9. The method according to any one of claims 1 to 5, so that the binding ability of the thrombin to thrombomodulin present in patient blood or plasma or formed there after initiation is determined.

10. The method according to claim 9, so that the thrombin formation is initiated in plasma by adding tissue factor, then thrombomodulin is added and the binding to thrombomodulin is determined by a sandwich ELISA.

11. The method of claim 9 or 10, d a d u rc h g e ke n n z e i c h n et that recombinant thrombomodulin is used.

12. The method according to any one of claims 9 to 11, This means that the plasma is brought into contact with thrombomodulin bound to solid phase and, after removal of the plasma, thrombin bound is detected by means of an anti-thrombin antibody.

13. The method according to claim 12, d a d u rc h g e k e n n ze i c h n et that for the detection another, labeled and directed against the anti-thrombin antibody is used.

14. The method of claim 1, d ad u rc h g e ke n n zei c h n et that one determines the anticoagulant activity by means of a method according to claims 2 to 9 and / or according to claims 9 to 13.

15. Kit for determining the anticoagulant activity of thrombin in patient blood or plasma, so that it a) Tissue factor, calcium and phospholipids to initiate thrombin formation and b) protein C and thrombomodulin or / and c) solid-phase-bound thrombomodulin; as well as in each case suitable detection substances for thrombin and / or activated protein C and / or a thrombin-thrombomodulin complex formed.

16. Kit according to claim 15, dadu rc hge ke nnzeichn et, that it contains a chromogenic substrate for the detection of thrombin.

17. Kit according to claim 16, d ad u rc h g e ken nze i c h n et that it contains a chromogenic substrate for the detection of activated protein C.

18. Kit according to claim 16, so that it contains an anti-thrombin antibody for the detection of the thrombin-thrombomoduiin complex formed.

19. Kit according to claim 18, d ad u rc h g e ke n n ze i c h n et that the anti-thrombin antibody is labeled.

20. Kit according to claim 18, d a d u r c h g e k e n n ze i c h n et that additionally contains a labeled antibody directed against the anti-thrombin antibody.