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DOI: 10.1160/TH08-08-0523
The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution
Publication History
Received:
14 August 2008
Accepted after minor revision:
09 November 2008
Publication Date:
23 November 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200901/lookinside/thumbnails/10-1160-th08-08-0523-1.jpg)
Summary
Assessing the clotting function inevitably brings about dilution of plasma. With the existing techniques of thrombin generation (TG) measurement, dilution ranges from 2:3 to 1:8. However, the possibility that dilution alters procoagulant and anticoagulant pathways differently has not been examined. We investigated the effects of dilution on the thrombin generation process and found that the anticoagulant pathways are far more affected by dilution than the procoagulant pathways. That is, when prothrombin and antithrombin concentrations are kept constant, dilution of plasma does not significantly affect tissue factor (TF)-driven thrombin generation. We demonstrate that dilution of plasma slows down the inhibitory activity of tissue factor pathway inhibitor (TFPI) to a greater extent when compared with the down regulation by diluting procoagulant factors. Dilution of plasma has also a negative effect on the participation of the anti-haemophiliac factors VIII and IX in TG driven by contact activation or low TF concentration. We also investigated the effect of dilution on the participation of the anticoagulant system that consists of thrombomodulin, protein C and protein S (APC system). We found that plasma dilution causes a loss of sensitivity towards TM and APC. Furthermore, at high dilutions (> 1:12) a second wave of prothrombinase-activity was observed that could be attributed to the suppression of protein S-dependent inhibition. In conclusion, the mechanism of TG is profoundly disturbed by plasma dilution. As a consequence, the less a plasma sample is diluted, the better a TG experiment represents the physiological process.
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References
- 1 Lo K, Diamond SL. Blood coagulation kinetics: high throughput method for real-time reaction monitoring. Thromb Haemost 2004; 92: 874-882.
- 2 Hemker HC. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32: 249-253.
- 3 Tchaikovski SN. et al. Development of a calibrated automated thrombography based TG test in mouse plasma. J Thromb Haemost 2007; 05: 2079-2086.
- 4 Dusel CH. et al. Identification of prothrombin as a major thrombogenic agent in prothrombin complex concentrates. Blood Coagul Fibrinolysis 2004; 15: 405-411.
- 5 Grundman C. et al. Prothrombin overload causes thromboembolic complications in prothrombin complex concentrates: in vitro and in vivo evidence. Thromb Haemost 2005; 94: 1338-1339.
- 6 Varadi K. et al. Monitoring the bioavailability of FEIBA with a TG assay. J Thromb Haemost 2003; 01: 2374-2380.
- 7 De Smedt E. et al. The technique of measuring TG with fluorogenic substrates: 1. Necessity of adequate calibration. Thromb Haemost 2008; 100: 343-349.
- 8 Bajaj SP. et al. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem 1981; 11: 397-412.
- 9 Thaler E, Schmer G. A simple two-step isolation procedure for human and bovine antithrombin II/III (heparin cofactor): a comparison of two methods. Br J Haematol 1975; 31: 233-243.
- 10 Hendrix H. et al. Activation of human prothrombin by stoichiometric levels of staphylocoagulase. J Biol Chem 1983; 258: 3637-3644.
- 11 Hemker HC. et al. A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17.
- 12 Beguin S. et al. The consumption of antithrombin III during coagulation, its consequences for the calculation of prothrombinase activity and the standardisation of heparin activity. Thromb Haemost 1992; 68: 136-142.
- 13 Wagenvoord R. et al. The limits of simulation of the clotting system. J Thromb Haemost 2006; 04: 1331-1338.
- 14 Heeb MJ. et al. Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-2877.
- 15 Heeb MJ. et al. Protein S binds to and inhibits factor Xa. Proc Natl Acad Sci USA 1994; 91: 2728-2732.
- 16 Hackeng TM. et al. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
- 17 van Hylckama Vlieg A. et al. Elevated endogenous thrombin potential is associated with an increased risk of a first deep venous thrombosis but not with the risk of recurrence. Br J Haematol 2007; 138: 769-774.
- 18 Besser M. et al. High rate of unprovoked recurrent venous thrombosis is associated with a high thrombin generating potential in a prospective cohort study. J. Thromb. Haemost 2008; 06: 1720-1725.