ИСТИНА

Studying the stability of cardiac troponin T (cTnT) in samples of patients with acute myocardial infarction (AMI) we noticed that cTnT is much more stable in plasma samples than it is in serum samples. We suggested that this could be caused by a cleavage of cTnT by coagulation enzymes, known to be activated in serum. In this work we studied the thrombin-mediated cTnT degradation. The degradation of cTnT was studied using gel electrophoresis, immunoblotting and mass spectrometry analysis (MS). The incubation of recombinant cTnT or native ternary troponin complex in buffer solution containing thrombin resulted in a rapid cleavage of cTnT at the N-terminus and a formation of a 25-kDa product. No cTnT degradation was observed if thrombin was pretreated with hirudin, which inhibits its activity. Similar degradation of cTnT was observed in serum (but not in plasma) samples, collected from AMI patients, as well as in in vitro experiments, when both, recombinant cTnT or ternary troponin complex, were incubated in serum. This cleavage was abolished by preincubation of serum samples with either heparin or hirudin. In addition, no degradation was observed when cTnT was spiked into citrate, EDTA or heparin plasma samples. When the products of thrombin-mediated proteolysis were analyzed by MS, two fragments of 2-68 aar and 69-288 aar were identified. This indicates that thrombin cleaves cTnT between R68 and S69. The results of this study show that cTnT is cleaved in serum by thrombin. This should be considered when studying cTnT degradation or developing new immunodiagnostic systems.