Thrombin cleaves cardiac troponin T between R68 and S69 during serum sample preparation, resulting in a 29-kDa fragment, whereas cTnT remains full-sized (35 kDa) in heparin plasma.
The 29-kDa fragment of cardiac troponin T commonly observed in AMI serum samples is an in vitro artifact caused by thrombin cleavage during serum preparation.
BACKGROUND: Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation. METHODS: cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis. RESULTS: The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69. CONCLUSIONS: The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.
Katrukha et al. (Fri,) conducted a other in Acute myocardial infarction. Serum sample preparation (thrombin exposure) vs. Heparin plasma or hirudin pretreatment was evaluated on cTnT degradation/cleavage. Thrombin cleaves cardiac troponin T between R68 and S69 during serum sample preparation, resulting in a 29-kDa fragment, whereas cTnT remains full-sized (35 kDa) in heparin plasma.