Can somebody tell me (or send me a link to the literature) what are the normal concentrations of active Thrombin in blood (in enzyme Units) ? Also, what are the physiological concentration that Thrombin can reach in disease states ? I would be interested if there are measurements that show how many Units/ml active thrombin can be detected in the blood ...
Thanks !
Hi Ivan
Thrombin is the active component produced following cleavage of prothrombin by factor Xa. Thrombin then is actively involved in the formation of a clot and is not directly measurable as a circulating protein. Rather prothrombin/thrombin time assays are used to measure the time it takes for a clot to form. Prothrombin concentration in plasma is around 100 ug/ml.
Thanks Rosemary ! I wasn't very bright asking what is the physiological concentration of active Thrombin since it is an enzyme involved in blood coagulation and in high concentrations it is probably in a solid clot. I am using thrombin in an in vitro assay in 30U/ml and I was wondering if that is anywhere near the physiological concentrations in which thrombin can be found ....
Hi Ivan, happy to help! I am not a coag expert, but I think 30U/ml of thrombin would be considered a moderate level, at least in an in vitro assay. Some fibrinogen assays use thrombin at so-called 'high' concentration, 100 U/ml. In terms of physiological concentration, I would imagine that the immediate local concentration of thrombin at the site of clot initiation could reach high levels, even if for only a very short duration.
Perhaps another ResearchGate member with coag expertise might like to add further comment.
Hi Ivan,
you can look at plasma thrombin levels in two ways: 1) how much thrombin is present in your sample at the time of blood collection (in vivo); 2) how much thrombin can be generated in your plasma sample upon in vitro stimulation. It is intuitive that they give you completely different information. In the first case, you take a picture of the coagulative status at the moment of blood draw. As thrombin is short-lived in plasma, you cannot measure the active form of the enzyme, but have to look at thrombin markers, e.g. prothrombin fragment 1+2 (marker of prothrombin activation) or thrombin-antithrombin complex (TAT, marker of thrombin activity). By measuring these markers (several ELISA are available), you will have an idea of how much thrombin has been generated in vivo until the blood was drawn and anticoagulated (of course, such determination is only applicable to plasma, as serum will contain artificially high concentrations of such markers). In the second case, you get information on the plasma coagulation potential, because you trigger coagulation and directly measure how much thrombin is generated in the sample under well-defined experimental conditions (you can use tissue factor, FXII activators or calcium only). Calibrated automated thrombinography (CAT) is the gold standard for such measurement (at least for research applications). There is plenty of papers about the levels of coagulation markers or thrombin generation potential in a variety of diseases.
Hope this can be of help.
Fabrizio
I'm sorry, Ivan, I don't know any paper dealing with such a specific issue.
Good luck!
Recalcified citrated plasma clots when there is about 0.1 IU/ml thrombin (measured in RECA). Plasmatic thrombin in vivo binds to antithrombin-1 (fibrin), antithrombin-3, heparin cofactor 2, and alpha-2-macroglobulin. In the normal circulation you can find 5.5 mIU/ml amidolytic thrombin activity, i.e. F2a bound to a2M. The normal range is 5.5+-1.1 mIU/ml (MV+-1SD). Patients with pathologic in vivo thrombin generation have > 6.6 mIU/ml F2a-a2M.
I would say that when coagulation is activated the cocentration of thrombin in plasma is an order of magnitude of 1 - 10 IU/ml.
1 IU/ml of thrombin is about 10 nM. And thrombin generation experiments indicate that thrombin concentration can reach hundreds of nM.
ear Ivan,
Following your last input:
Studies on the eye (similar barrier as BBB) may be of help.
General: Ricker Invest Ophthalmol Vis Sci 2010; 51: 5234
Specific for thrombin: Bastiaans same journal 2014; 55: 4659.
The activity on thrombin peptide substrates is potentially from free thrombin and thrombin complexed to alpha-2-macroglobulin. The test approach could be to measure the hirudin inhibitable portion supposed to be from free thrombin. It is however to be remarked that hirudin also time dependently inhibits the complex that is only accessible for small substrates and inhibitors . Using a more bulky inhibitor such as SBTI is possible, but is not specific for thrombin. Pegylated hirudin or analogues might be the clue.
Regards
Kees Kluft
There is a must read article concerning thrombin production in plasma during clotting:
What is all that thrombin for? K.G. Mann, K; Brummel, S. Butenas.
J Thromb Haemost. 2003 Jul;1(7):1504-14.
In particular, look at figure 3 which is basically the cumulative amount of thrombin present in plasma during clotting. Since 1IU~8nM, fibrin is formed with thrombin concentrations of about 0.1-0.3 IU and the maximum thrombin present during clotting is of about several hundreds of nM, i.e. several 10s of IU.
The normal range of prothrombin concentration is 0.8-2.0 µM.
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