Method for determining the concentration of thrombin inhibitors

1. A method for determining the concentration of a thrombin inhibitor in a non-turbid body liquid or a non-turbid extract from a body liquid, comprising

a) obtaining a body liquid from a living body and optionally separating turbid matter from said body liquid;

b) obtaining a solution or a mixture, by adding to the non-turbid body liquid of (a),

a coagulation-inhibiting substance which does not interfere in the transformation of educt prothrombin into an active meizothrombin or meizothrombin-des fragment 1 (Mtdesfg1) product;

a chromogenic or fluorogenic substrate which is not dissociable by said active meizothrombin or said Mtdesfgl product; and

a substance which dissociates prothrombin into meizothrombin or Mtdesfgl optionally together with prothrombin;

c) subjecting the solution or mixture of (b) to a wavelength-selective light absorption or light emission measurement as a function of the time; and

d) comparing the per time unit absorption or emission measurement of (c) to a standard; and

e) determining the concentration of said thrombin inhibitor from d).

2. A method for determining the concentration of a thrombin inhibitor in a subject, comprising

(a) obtaining a body sample from said subject and optionally separating turbid matter from said body sample to generate a non-turbid liquid;

(b) adding, to the non-turbid liquid of (a),

a coagulation-inhibiting substance which does not interfere in the transformation of educt prothrombin into an active meizothrombin or meizothrombin-des fragment 1 (Mtdesfg1) product;

a chromogenic or fluorogenic substrate which is dissociable by said active meizothrombin or said Mtdesfg1 product; and

a substance which dissociates prothrombin into meizothrombin or Mtdesfg1, optionally together with prothrombin;

(c) determining the level of said chromogenic or fluorogenic substrate in said solution or mixture of (b);

(d) comparing said levels in (c) to one or more standards; and

(e) determining the concentration of said thrombin inhibitor.

3. The method according to claim 2 , wherein the standard comprises pooled human citrate plasma that is treated with known concentrations of hirudin.

4. The method according to claim 2 , comprising employing a standard curve comprising a plot of optical density (OD) vs. hirudin concentration.

5. The method according to claim 2 , wherein the non-turbid liquid is synthetically produced.

6. The method according to claim 2 , wherein the coagulation inhibiting substance which does not interfere in the transformation of prothrombin to active meizothrombin or Mtdesfg1 is a calcium-complex forming agent, heparin, a heparinoid, anti-thrombin III, protein C, a fibrin polymerization inhibiting substance, or a mixture thereof.

7. The method according to claim 2 , wherein the substance that dissociates prothrombin into meizothrombin or Mtdesfg1 is a snake venom or a fraction thereof.

8. The method according to claim 2 , wherein the substance which dissociates prothrombin into meizothrombin or Mtdesfg1 is ecarin.

9. The method according to claim 2 , wherein the chromogenic substrate dissociable by active meizothrombin or Mtdesfg1 releases p-nitroanilin upon dissociation, and the levels thereof is determined by making measurements of optical density (OD) at 405 nm.

10. The method according to claim 2 , wherein step (c) comprises a first determining step comprising measuring the absorption or emission after 0-100 s, and a second determining step comprising measuring the absorption or emission after another 10-1,000 s, said measurements being made following addition of the substance which dissociates prothrombin into meizothrombin or Mtdesfg1.

11. The method according to claim 2 , wherein the thrombin inhibitor is hirudin, a hirulog or a synthetic thrombin inhibitor.

12. The method according to claim 2 , wherein step (b) comprises adding to said solution or mixture,

a substance which dissociates prothrombin into meizothrombin or Mtdesfg1, and

prothrombin or Mtdesfg1.