1. Thrombin-Mediated Degradation of Human Cardiac Troponin T
I.A. Katrukha, A.E. Kogan, A.V. Vylegzhanina, M.V. Serebryakova, E.V. Koshkina, A.V. Bereznikova, A.G. Katrukha
June 2017
© Copyright 2017 by the American Association for Clinical Chemistry

2. Introduction
Cardiac troponin T (cTnT) is an established biomarker of acute myocardial infarction (AMI).
It is present in serum as a set of proteolytic fragments.
It is believed that the cTnT degradation is mediated by μ-calpain.
But is μ-calpain active in blood?
In this study we suggest that it is not μ-calpain but thrombin that cleaves cTnT in serum samples.
Editorial. Bodor GS. Cardiac Troponins: Molecules of Many Surprises. Clinical Chemistry 2017; 63:6.

3. Materials and Methods
Serum and heparin plasma samples were simultaneously collected from patients with STEMI (n=17) over a period of 6–20 h after the onset of chest pain.
Immunoprecipitation of cTnT and its fragments was performed on three affinity matrices, with N-terminus, C-terminus and full length cTnT specific monoclonal antibodies (mAbs).
Western blot studies were performed by anti-cTnT mAbs conjugated with horseradish peroxidase.

4. cTnT in serum differs from cTnT in heparin plasma
Serum and heparin plasma samples collected simultaneously from the same AMI patients contained different forms of cTnT.
All heparin plasma samples contained full-sized cTnT (~35 kDa) while in serum samples cTnT was present as a 29-kDa fragment.

5. cTnT in serum differs from cTnT in heparin plasma
1 - RecTnT standard
2 - NHS
3 - heparin NHP
4, 6, 8, 10 - serum (S) samples of AMI patients
5, 7, 9, 11 - heparin plasma (P) samples of AMI patients
Figure 1. cTnT fragmentation in serum and heparin plasma samples of AMI patients.
cTnT and its fragments were immunoprecipitated from the serum and heparin plasma samples of 4 representative AMI patients and immunostained in WB by mAb TnT313, specific to epitope 119 –138.

6. 29-kDa cTnT fragment is a result of thrombin-mediated cleavage
We have suggested that in serum cTnT is cleaved by thrombin – protease activated in the process of serum preparation, which is absent in heparin plasma.
cTnT or ternary troponin complex (ITC) were incubated in buffer with purified thrombin or in normal human serum (NHS). In both cases the 29-kDa fragment was detected.
No cTnT degradation was observed when thrombin or NHS were pretreated with hirudin – a specific thrombin inhibitor.
No cTnT degradation was observed during incubation of troponin in normal heparin plasma.

7. Thrombin cleaves сTnT in vitro
1 – recTnT incubated in a buffer solution
2 - recTnT + thrombin
3 - recTnT + hirudin-pretreated thrombin
4 - ITC in a buffer solution
5 - ITC + thrombin
6 - ITC + hirudin-pretreated thrombin
7 - cTnT from AMI serum sample
8 - recTnT incubated in NHS
9 - recTnT in NHS pretreated with heparin
10 - recTnT in NHS pretreated with hirudin
11 - recTnT incubated in heparin NHP
Figure 2. Thrombin-mediated degradation of troponin T.
RecTnT or ITC was incubated in a buffer with or without thrombin, in NHS or in NHP, for 3 h at 37°C. To inhibit thrombin activity, preincubation of thrombin with hirudin and NHS with either heparin or hirudin was performed. cTnT and its fragments were immunoprecipitated and stained in WB by mAb TnT313.

8. cTnT is cleaved not only by thrombin?
A small amount of fragments can be seen in the heparin plasma samples of AMI patients (Fig. 1). This might be explained by the cleavage of cTnT either by thrombin that is activated in the process of thrombus formation or because of systemic blood coagulation activation, or by action of some intracellular proteases (for example, µ-calpain) in the necrotic tissue.
Some other protease(s) cause the further degradation of the 29-kDa fragment to form 16–19-kDa fragments.

9. Thrombin cleaves сTnT only between epitopes 55-64 and 73-80
1, 3, 5, 7, 9 - recTnT incubated for 3 h at 37 °C in a buffer solution
2, 4, 6, 8, 10 - recTnT incubated for 3 h at 37 °C in a buffer solution with thrombin
Figure 3. Localization of the thrombin cleavage site.
RecTnT and its thrombin-mediated proteolysis fragments were immunoprecipitated, the eluates were analyzed by WB and immunostaining with different anti-TnT mAbs. The epitope of each antibody is indicated in parentheses above the lanes.

10. Thrombin cleaves сTnT between Arg68 and Ser69
Figure 4. Mass spectrum analysis of cTnT fragments.
RecTnT was incubated with thrombin and resulting fragments were captured either with N-terminus specific anti-TnT mAbs (A) or with antibodies specific both to C-terminus and central part of TnT (B).

11. Conclusions
The 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cTnT cleavage by thrombin between R68 and S69 during serum sample preparation.
Thrombin-mediated cleavage of cTnT should be considered during selection of antibodies for new generations of cTnT assays as well as for selection of sample types used for analysis.
Does the cleavage by thrombin affect the current immunoassays?