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Thrombin-Mediated Degradation of Human Cardiac Troponin T.

Cardiac isoforms of troponin T (cTnT) (5) and troponin I (cTnT) are the most important biomarkers of acute myocardial infarction (AMI) (1). Following a heart attack, troponins are released into the bloodstream from damaged myocardial cells and can be measured in blood samples by immunoassays. Characterization of the stability of troponins in the circulation and in the collected blood samples is important for their precise measurement. Both cTnI (2, 3) and cTnT (4) have been shown to be prone to proteolytic degradation. A stable region of cTnI located in the central part of the molecule has been characterized (2), but the degradation of cTnT is less well understood.

Several studies devoted to the analysis of cTnT from serum samples of AMI patients revealed a set of proteolytic fragments with apparent molecular masses of 29, 19, 18, and 16 kDa, with the 29-kDa fragment being the predominant form (4-9). A similar pattern of degradation was observed when cTnT [in a form of native ternary troponin complex (ITC)] was incubated in normal human serum (NHS) (9, 10). Analysis of the 29-kDa fragment by mass spectrometry (MS) showed that it composes central and C-terminal portions of the cTnT molecule and is a product of cleavage of cTnT between amino acidresidues (aar) 54-69 (9); 16-19-kDaproteolytic fragments were determined as products of cTnT degradation between aar 54-84 at the N-terminus, and aar 191-230 at the C-terminus (9).

In cell culture experiments with cardiomyocytes, it was shown that cTnT is cleaved between the R68/S69 amino acid residues, and it was suggested that the cytosolic enzyme [mu]-calpain (calpain-1) is responsible for this degradation (11, 12). It was assumed that, in serum, the cleavage of cTnT between residues R68/S69 also takes place under the action of [mu]-calpain (9). However, [mu]-calpain is an intracellular protease (13) and its activity in blood would be difficult to explain. At the same time, no degradation of cTnT was observed during the incubation of ITC in normal human heparin plasma (NHP) (10). These differences were explained by the protease inactivation in plasma samples (9, 10, 14). Taking into consideration that serum differs from plasma in regard to the activation of coagulation cascade, we have suggested that the degradation of cTnT in serum could be explained by the action of activated coagulation enzymes rather than by the action of [mu]-calpain. Herein we study the proteolytic action of the most abundant coagulation enzyme--thrombin--on the degradation of cTnT.

Materials and Methods

Recombinant human cardiac troponin T (recTnT), ITC, and monoclonal antibodies (mAbs) that are specific to different epitopes of the cTnT molecule came from HyTest Ltd. If not stated otherwise, all other chemicals came from Sigma-Aldrich.

BLOOD SAMPLES

All patients selected for this study (n = 17) had an ST-elevation myocardial infarction followed by coronary angiography and stenting. All percutaneous interventions were performed within 4 h after the onset of chest pain. Serum and heparin plasma samples were collected by a standard technique (see the Data Supplement that accompanies the online version of this article at http:// www.clinchem.org/content/vol63/issue6) over a period of 6-20 h after the onset of chest pain (2-16 h after stenting). During the period when samples were taken, all patients received clopidogrel bisulfate (Plavix or Plagril, 300 mg, 2 times--at admission and 12 h after the initial dose), and intravenous heparin (5000 U in the ambulance and 10 000 U during the percutaneous intervention). Pooled cTnT-negative NHS and NHP samples were prepared from the blood of 5 apparently healthy volunteers.

Taking into consideration that all patients received heparin to prevent blood clotting, all serum samples were tested for the presence of fibrinogen by the in-house 2-step sandwich IFA assay (see online Data Supplement). Only serum samples that did not contain fibrinogen (thus were not subjected to the action of the injected heparin) were included into study.

All volunteers were informed in accordance with the current revision of the Declaration of Helsinki (15).

MEASUREMENTS OF cTnT CONCENTRATION

The concentration of cTnT in all samples (in a form of heparinized whole blood) was measured by the TropT Quantitative assay on the Cobas h232 system (Roche), right after the samples were taken. The samples in which concentration of cTnT exceeded 2 ng/mL (upper limit for TropT Quantitative assay) were re-measured (in a form of heparin plasma) by the in-house assay that used mAb TnT9 as a capture antibody, and mAb TnT1A11 conjugated with stable [Eu.sup.3+] chelate, for detection. Native human ITC complex was used as a calibrator. The limit of detection (LoD) of the assay was 1.2 ng/mL, limit of blank (LoB) = 1.0 ng/mL, with a CV of 11% at an antigen concentration of 0.2 ng/mL; it did not cross-react with unrelated proteins. For the detailed information please see the online Data Supplement.

IMMUNOPRECIPITATION OF cTnT AND ITS FRAGMENTS

For affinity chromatography, anti-cTnT mAbs were immobilized on Sepharose CL-4B (GE Healthcare; 5 mg of mAbs per 1 mL of Sepharose), using a conventional BrCN method (16). Three affinity matrices were prepared: the Sorbent 1, with mAbs specific to the N-terminal portion of cTnT, contained mAbs TnT35 (specific to aar 2-21), TnT195, (15-34) and TnT103 (41-47). Sorbent 2 contained mAbs specific to the central and C-terminal portions, namely mAbs TnT110 (aar 119-138), TnT1A11 (145-164), TnT104 (184-203), TnT155 (262-281), TnT199 (275-288). All of the antibodies were present in affinity matrices in equimolar quantities. Finally, the Sorbent 3 was prepared as a mixture of both of the above-mentioned matrices, and therefore was specific to all parts of the cTnT molecule.

To extract cTnT and its fragments, 500 [micro]L of sample with concentration of cTnT >2 ng/mL or 3000 [micro]L of sample with concentration of cTnT <2 ng/mL was mixed with 50 [micro]L or 100 [micro]L of the affinity matrix, respectively, and was incubated for 90 min at 4 [degrees]C under gentle shaking. The mixture of the sample and matrix was added into the Pierce Paper Filter Spin Cups (Thermo Fisher Scientific) and centrifuged for 2 minutes at 1000g. Flow-through was discarded, while the resin was washed 5 times with 300 [micro]L of washing buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L Ca[Cl.sub.2], pH 7.5). Proteins were eluted by the boiling of the affinity matrix in 100 [micro]L of the sample buffer for electrophoresis. To prevent the elution of mAbs immobilized on the matrix, the sample buffer did not contain [beta]-mercaptoethanol. After boiling, the samples were centrifuged for 2 minutes at 1000g, and [beta]-mercaptoethanol was added to the eluates to the concentration of 2% (v/v). Samples were then boiled again for 5 minutes, and frozen at -20 [degrees]C until use. The recovery of cTnT following the immunoprecipitation procedure exceeded 90% (data not shown).

WESTERN BLOTTING AND ENHANCED CHEMILUMINESCENCE

Proteins were separated via 16% Tris-Tricine SDS-PAGE according to Schagger and von Jagow (17), blotted onto Amersham Hybond--enhanced chemiluminescence (ECL) nitrocellulose membranes (GE Healthcare), immunostained by anti-cTnT mAbs conjugated with horseradish peroxidase (HRP), and visu alized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) on the ChemiDoc Touch Imaging System (Bio-Rad) (for details see the online Data Supplement).

Image quantification was performed using ImageLab 5.2.1 software (Bio-Rad). The ratios of individual bands were expressed as percentage of the total signal measured on each lane.

IN VITRO CLEAVAGE OF cTnT BY THROMBIN

RecTnT or ITC ternary complex was either spiked into a 20 mmol/L Tris-HCl, 150 mmol/L KCl, 5 mmol/L Ca[Cl.sub.2], 75 g/L BSA, 0.15 g/L Na[N.sub.3] pH 7.5 buffer containing 3 NIH units/mL of native human thrombin, or into NHS, or into heparin NHP samples, and incubated for 3 h at 37 [degrees]C. The final concentration of cTnT was 50 ng/mL. In control samples, thrombin was inhibited by preincubation with either 2.5-300 USP (United States Pharmaceutical) units/mL of heparin (sodium salt from porcine intestinal mucosa) or 2.5-100 IU/mL of recombinant hirudin (from Saccharomyces cerevisiae) before troponin was added.

PROTEOLYSIS BY Glu-C V8 ENDOPROTEINASE

Proteolysis of full-sized cTnT and its 29-kDa fragment was performed by means of in-gel digestion by Glu-C V8 endoproteinase. For details please see the online Data Supplement.

MS ANALYSIS

MS and MS/MS analysis of the intact recTnT, and its fragments obtained by thrombin-mediated cleavage, was performed by an UltrafleXtreme[TM] MALDI-TOF/TOF mass spectrometer (Bruker) that was equipped with a Smartbeam-II laser (Nd:YAG, 355 nm). For details please see the online Data Supplement.

Results

CTNT DEGRADATION IN PLASMA AND SERUM SAMPLES OF AMI PATIENTS

To compare cTnT fragmentation in plasma and serum samples, we studied cTnT immunopurified from the samples of 17 AMI patients (concentration of cTnT was 0.27-79.0 ng/mL) that underwent coronary artery stenting. cTnT and its fragments were extracted on an affinity matrix with mAbs, specific to the different epitopes of the cTnT molecule (Sorbent 3). Extracted proteins were separated by SDS-PAGE and cTnT and its fragments were detected by ECL with the mAb TnT313, which is specific to the central portion of cTnT [aar 119-138; this epitope is similar to the epitope of one of the antibodies used in modern immunodiagnostic systems (18)].

A comparison of the fragmentation profiles of cTnT in the serum and heparin plasma samples of all studied AMI patients revealed a pronounced difference between them. cTnT in heparin plasma samples was mainly present as a full-sized molecule (apparent molecular mass approximately 35 kDa) with a minor presence of proteolytic fragments, whereas in serum samples cTnT was mainly present as a 29-kDa fragment with almost no full-sized molecules (see Fig. 1). This observation suggested that the fragmentation of cTnT was dependent on the sample type used for analysis. We proposed that the appearance of 29-kDafragment, and apparently 16-19-kDa fragments, might occur because of the activation of proteases during the preparation of serum samples.

THROMBIN-MEDIATED cTnT PROTEOLYSIS

To verify the hypothesis that cTnT is cleaved by coagulation enzymes during the collection and processing of AMI serum samples, we studied the degradation of cTnT by the most abundant protease activated in the coagulation cascade--serine protease thrombin. Incubation of both recTnT or cTnT in the form of ITC ternary complex with purified thrombin resulted in a complete cleavage of cTnT witnessed by the formation of a 29-kDa fragment that was similar to that found in serum samples of AMI patients (see Fig. 2). If thrombin was preincubated with its specific inhibitor hirudin before the addition of troponin, no degradation was observed.

The incubation of recTnT in NHS also resulted in the formation of the same 29-kDa fragment. In contrast, no degradation was observed during the incubation of recTnT in heparin plasma or in NHS, which was spiked with hirudin before the addition of cTnT (the addition of 10 IU/mL of hirudin was enough for the complete inhibition of cTnT degradation). The addition of heparin into NHS (2.5-300 USP units/mL) resulted in a dose-dependent inhibition of recTnT cleavage, with up to 97% of protein being uncleaved in the NHS sample containing 300 USP units/mL of heparin (see online Supplemental Fig. 1). These results support the hypothesis that thrombin-mediated cTnT cleavage is responsible for the appearance of the 29-kDa fragment in serum.

LOCALIZATION OF THE THROMBIN CLEAVAGE SITE ON cTnT BY WESTERN BLOTTING

To localize the cleavage site(s) of cTnT by thrombin, the products of recTnT degradation were immunostained with mAbs specific to aar 55-64 (TnT103), aar 73-80 (TnT175), aar 85-95 (TnT8), aar 119-138 (TnT313), and aar 275-288 (TnT199) of cTnT (Fig. 3). It appeared that TnT103 only stained the full-sized molecule and was unable to stain the 29-kDa fragment of cTnT, while TnT175 was able to stain both bands. This indicates that the site of cleavage is located between the epitopes of these 2 mAbs. Unfortunately, none of the mAbs recognizing the N-terminal portion of cTnT were able to immunostain the small N-terminal fragment. The mAbs TnT8, TnT313, and TnT199 stained the same 29-kDa fragment as the mAb TnT175, which means that there are no additional cleavage sites between the epitopes of mAbs TnT175 and TnT199.

During the analysis of the sequence of cTnT between aar 60 and 75 only one possible thrombin cleavage site was found at R68/S69.

LOCALIZATION OF THE THROMBIN CLEAVAGE SITE BY MS

To precisely localize the site of thrombin cleavage we performed MS to analyze the fragments formed during the proteolysis of recTnT by thrombin in a buffer solution.

First, the central and C-terminal portions of cTnT were extracted by using Sorbent 2 and then N-terminal fragments were extracted from the flow-through using Sorbent 1.

Analysis of the eluate from Sorbent 1 by MS showed 2 peaks at 7704 and 3852 Da (Fig. 4A). The mass of the first peak was in good agreement with the mass of the aar 2-68 fragment of cTnT (expected molecular mass: 7703.7 Da), and the second peak corresponded to the doubly charged ion of the same peptide.

The MS analysis of the eluate from Sorbent 2 containing the central/C-terminal fragment(s) of recTnT revealed the presence of 3 peaks at 26797, 13384, and 8926 Da (Fig. 4B). The mass of the first peak (26797 Da) was in good agreement with the mass of aar 69 -288 fragment of cTnT (expected molecular mass: 26773.3 Da) and with 2 other peaks corresponding to the doubly charged and triply charged ions of the same peptide.

To confirm the position of the cleavage site, the thrombin-derived fragments were separated by means of SDS-PAGE. The 29-kDa band was excised from the gel, proteolyzed, and the obtained peptides were analyzed by MS/MS. As in the case of Western blotting (WB), we were unable to detect any band that might correspond to the N-terminal fragment. Therefore, we only analyzed the peptides that were obtained from the full-sized recTnT (used as a reference) and the 29-kDa fragment. For the proteolysis, we used Glu-C endoproteinase from Staphylococcus aureus V8, which cleaves proteins C-terminally from Glu residues. This protease was used instead of trypsin as the in silico analysis had predicted that trypsin cleaves cTnT at R68/S69, which is a presumed site of thrombin cleavage. MS/MS of the peptides obtained by the proteolysis of the whole recTnT molecule revealed, apart from other masses, a characteristic mass of 2334.1 Da which corresponded to the aar 62-83 peptide SKPKPRSFMPNLVPPKIPDGE (expected molecular mass: 2333.3 Da) which contained the un-cleaved site R68/S69 (see online Supplemental Fig. 2 and Supplemental Table 1). MS/MS of peptides derived from the 29-kDa fragment revealed, apart from the others, the masses of 1639.8 and 1655.8 Da, which corresponded to the peptide SFMPNLVPPKIPDGE (aar 69-83, expected molecular mass: 1639.7 Da) as well as its form with oxidized methionine (expected molecular mass: 1655.8 Da) that appear after the cleavage between R68/S69 (see online Supplemental Fig. 3 and Supplemental Table 2). These results confirm that the 29-kDa band corresponds to the aar 69 -288 fragment of cTnT and locate the site of thrombin cleavage between the amino acid residues R68/S69 of cTnT.

Discussion

When comparing cTnT in serum and heparin plasma samples collected simultaneously from the same AMI patient we observed a substantial difference in the composition of the fragments. cTnT extracted from heparin plasma samples was mostly present as the full-sized molecule, while cTnT extracted from serum samples was fully degraded, forming the 29-kDa (major) and 16-19-kDa (minor) fragments (Fig. 1). This difference suggests that in blood of AMI patients cTnT is mostly present as the full-sized molecule, whereas fragmentation observed in the serum occurs after taking the blood sample.

The major difference between plasma and serum preparation is the stimulation of coagulation in the latter leading to the activation of a number of proteases (19, 20). One of the most abundant proteases, which are activated during coagulation, is thrombin.

Thrombin is a 37-kDa serine protease that appears in blood during the activation of the coagulation system following prothrombin cleavage by factor Xa (19, 20). Thrombin is a rather indiscriminate protease and is capable of cleaving a large number of proteins. Although the main target of thrombin is fibrinogen, it is also known to be able to cleave other coagulation proteins, including factor V, factor VIII, factor XI, factor XIII, protein C, platelet receptors, and others (21). For successful cleavage, thrombin requires an Arg at P1 position, Pro/Thr/Ala/Val/Ile at P2, Ser/Ala/Gly/Thr at P1' and a nonacidic aar at P2' (22).

Our experiments showed that thrombin cleaves cTnT, forming a 29-kDa fragment similar to one that observed in AMI serum samples. Thrombin substrate specificity made it possible for us to propose that the thrombin cleavage site on cTnT lies between aar R68/ S69. This assumption was confirmed by the MS and MS/MS analysis of the cTnT fragments that bordered the N-terminal peptide by aar 2-68, and the C-terminal peptide by aar 69-288. In addition, the results of both WB and MS make it possible to suggest that R68/S69 is the only site of thrombin cleavage on cTnT.

To prove that cTnT cleavage in serum samples takes place because of the specific thrombin-mediated proteolysis, we analyzed the effect of endogenous thrombin on cTnT. Blood plasma contains 100-180 mg/L of prothrombin yielding concentrations during coagulation as high as 500 nmol/L (23) or approximately 6 NIH U/mL of thrombin. According to other data, 1 mL of blood plasma can generate up to 1500 nmol/L or approximately 17.5 NIH U/mL of thrombin (24). The incubation of both recTnT and the ITC ternary complex in NHS resulted in the degradation of troponin and the formation of a 29-kDa fragment. To show that it is thrombin that cleaves cTnT in serum, we inhibited thrombin in the serum samples before the spiking of cTnT. In blood, thrombin is inhibited by such serpins (serine protease inhibitors) as antithrombin III, heparin cofactor II or protein C inhibitor (21, 25, 26), with antithrombin playing the predominant role. The inhibiting activity of all 3 proteins is markedly increased in the presence of polysaccharide heparin (27). The ability of heparin to block thrombin activity is used to prevent intravascular coagulation in AMI patients and to prepare heparin plasma. However, antithrombin III and the protein C inhibitor are able to inhibit proteases other than thrombin (26). To specifically inhibit thrombin we used hirudin (28). This 64-aar peptide, which is produced in the salivary glands of the medical leech Hirudo medicinalis, possesses an extremely high inhibiting capacity ([K.sub.i] = [10.sup.-14]) and specificity towards thrombin (29). In our experiments, the addition of hirudin to both the buffer solution containing purified human thrombin and to the NHS sample completely suppressed the degradation of cTnT, which suggests it is thrombin that is responsible for the cTnT cleavage in serum (Fig. 2). In addition, we saw no degradation of recTnT incubated in heparin NHP, although we were unable to achieve a complete inhibition of recTnT degradation even by adding 300 USP units of heparin per mL of NHS. Following the incubation at 37 [degrees]C, approximately 3% of recTnT added to the NHS pretreated with heparin was present by the 29-kDa fragment. We presume that this minor degradation could be explained by the action of either meizothrombin or [beta]-thrombin, both of which possess protease activity (30, 31). They are readily inhibited by hirudin (32, 33) but are much less sensitive to the inhibition by heparin/antithrombin complex (30, 33, 34).

Many authors who have described the degradation of cTnT in serum attribute this effect to [mu]-calpain. However, there is no reliable evidence that [mu]-calpain from human myocardium is active in serum. On the other hand, a small amount of the fragments can be seen in the heparin plasma samples of AMI patients. This might be explained by the cleavage of cTnT by thrombin that is activated in the process of intravascular clot formation or because of systemic blood coagulation activation that might happen during acute coronary syndromes (35), or by some intracellular proteases (for example, [mu]-calpain) in contact with cTnT in the necrotic tissue.

It is also worth noting that apart from thrombin, some other protease(s) cause the further degradation of the 29-kDa fragment to form 16-19-kDa fragments in serum--as observed by us and others (8, 9)--by cleavage of cTnT between aar 201-240 (9).

Presently, only assays that utilize 2 mAbs specific to the aar 125-147 fragment of cTnT are available for clinical use (18). These assays should not be affected by thrombin-mediated cTnT proteolysis. But, since in recent years cTnT has been shown to be a robust and sensitive AMI marker, we can expect the appearance of new assays (more sensitive and precise than existing generations) that might utilize mAbs specific to other epitopes. Thus, the knowledge of sites of cTnT degradation is very important both: (a) for the selection of the antibodies that are not affected by thrombin-mediated cTnT proteolysis; and (b) for the selection of the proper matrix to be used for cTnT measurements.

Conclusion

We suggest that the degradation of cTnT observed in serum samples that results in the formation of the 29-kDa fragment mainly occurs due to the activation of thrombin during the preparation of serum samples. cTnT is cleaved by thrombin at R68/S69. The thrombin-mediated cTnT degradation that takes place during the process of serum sample preparation should be considered during the investigation of cTnT degradation and in the development of new immunochemical methods of cTnT measurement.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: A.E. Kogan, HyTest Ltd; A. Bereznikova, HyTest Ltd; A.G. Katrukha, HyTest Ltd.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: MALDI MS was available by means of the Moscow State University development program, PNG 5.13.

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, and final approval of manuscript.

Acknowledgments: The authors thank Nadezda Kuzina and Elena Kochkareva for their kind assistance in the realization of this work, and Karina Seferian for her assistance in the preparation of the manuscript.

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Ivan A. Katrukha, [1,2] * Alexander E. Kogan, [1,2] Alexandra V. Vylegzhanina, [1] Marina V. Serebryakova, [3] Ekaterina V. Koshkina, [4] Anastasia V. Bereznikova, [1,2] and Alexey G. Katrukha [1,2]

[1] HyTest Ltd., Turku, Finland; [2] Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia; [3] Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; [4] 67 City Hospital, Moscow, Russia.

* Address correspondence to this author at: HyTest, Intelligate 1,6th Floor, Joukahaisen katu 6,20520Turku, Finland. Fax +358-2-512-0909; e-mail ivan.katrukha@hytest.fi.

Received September 9, 2016; accepted January 26, 2017.

Previously published online at DOI: 10.1373/clinchem.2016.266635

[C] 2017 American Association for Clinical Chemistry

[5] Nonstandard abbreviations: cTnT, human cardiac troponin T; cTnI, human cardiac troponin I; AMI, acute myocardial infarction; ITC, human cardiac ternary cTnI-cTnT-TnC complex; NHS, normal human serum; MS, mass spectrometry analysis; aar, amino acid residues; NHP, normal human plasma; recTnT, recombinant human cardiac troponin T; mAb, monoclonal antibody; LoD, limit of detection; LoB, limit of blank; WB, Western blotting; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; USP, United States Pharmaceutical.

Caption: Fig. 1. cTnT fragmentation in serum and heparin plasma samples of AMI patients. cTnT and its fragments immunoprecipitated from the serum and heparin plasma samples of 4 representative AMI patients, analyzed by WB. cTnT is stained by mAb TnT313, specific to the aar 119-138.1-RecTnTstandard; 2-NHS; 3-heparin NHP; 4,5-serum (S) and heparin plasma (P) samples of AMI patient 1 (Pati), 15 h after the onset of chest pain, [c.sub.(cTnT)] = 0.39 ng/mL [please note that in order obtain a clearer picture larger initial volumes of the samples (3 mL) were used for immunoprecipitation, as well as longer exposure of the membrane]; 6, 7-serum (S) and heparin plasma (P) samples of AMI patient 2 (Pat2), 6 h, [c.sub.(cTnT)] = 45.0 ng/mL; 8, 9-serum (S) and heparin plasma (P) samples of AMI patient 3 (Pat3), 13 h, [c.sub.(cTnT)] = 35.0 ng/mL; 10,11-serum (S) and heparin plasma (P) samples of AMI patient 4 (Pat4), 12 h, [c.sub.(cTnT)] = 47.7 ng/mL.

Caption: Fig. 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 [degrees]C. To inhibit thrombin activity, preincubation of thrombin with hirudin and NHS with either heparin or hirudin was performed. cTnT and its fragments were extracted from samples by means of immunoprecipitation and stained in WB by mAb TnT313. 1, recTnT incubated in a buffer solution; 2, recTnT incubated ina buffersolution with thrombin (3 NIH units/mL); 3, recTnT incubated in a buffer solution with hirudin-pretreated thrombin; 4, ITC ternary complex incubated in a buffer solution; 5, ITC ternary complex incubated in a buffer solution with thrombin (3 NIH units/mL); 6, ITC ternary complex incubated in a buffer solution with hirudin-pretreated thrombin; 7, cTnT from AMI serum sample; 8, recTnT incubated in NHS; 9, recTnT incubated in NHS pretreated with heparin (300 USP units/ mL); 10, recTnT incubated in NHS pretreated with hirudin (10 IU/mL); 11, recTnT incubated in heparin NHP.

Caption: Fig.3. Localization of the thrombin cleavagesite. RecTnT and its thrombin-mediated proteolysis fragments were immunoprecipitated and eluates were analyzed by means of WB and subsequent immunostaining with mAbTnT103, (tracks 1,2); mAb TnT175 (tracks 3, 4); mAb TnT8, (tracks 5, 6); mAb TnT313 (tracks 7,8) and mAb TnT199 (tracks 9-12). Epitopes of each antibody are indicated in brackets above the tracks. Tracks 1, 3, 5, 7, and 9: recTnT incubated for 3 h at 37 [degrees]C in a buffer solution; tracks 2, 4, 6, 8, and 10: recTnT incubated for 3 h at 37 [degrees]C in a buffer solution with thrombin (3 NIH units/mL).

Caption: Fig. 4. MS of cTnT fragments obtained by thrombin cleavage in a buffer solution. (A), MS of the eluate pH 2.0 from the affinity matrix, utilizing mAbs specific to the N-terminal portion of cTnT. (B), MS of the eluate pH 2.0 from the affinity matrix, utilizing mAbs specific to the central and C-terminal portions of cTnT.
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Title Annotation:Proteomics and Protein Markers
Author:Katrukha, Ivan A.; Kogan, Alexander E.; Vylegzhanina, Alexandra V.; Serebryakova, Marina V.; Koshkin
Publication:Clinical Chemistry
Date:Jun 1, 2017
Words:5397
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