The effect of anticoagulation medication on cardiac troponin T degradation: a proof of principle study

Cardiac troponin (cTn) is the prevailing biomarker for diagnosing myocardial infarction (MI). In previous years, different cTn proteoforms, either cardiac troponin I (cTnI) or cardiac troponin T (cTnT) in complex or as free subunits, have been identified in blood (1). Mainly larger cTnT proteoforms of ≥29 kilodalton (kDa) are observed in patients with MI (1), while in patients with end-stage renal disease (ESRD) mainly small 15–18 kDa cTnT fragments are found (2). The type of blood tube used for blood withdrawal then becomes very crucial, with a preference for heparin-based tubes that block the in vitro degradation of cTnT by thrombin (1,3). Importantly, most patients with acute coronary syndromes receive anticoagulation medication at their presentation that indirectly inhibits thrombin, namely a bolus of unfractionated heparin (UFH) for ST-elevated MI (STEMI) patients and a subcutaneous injection of the synthetic pentasaccharide fondaparinux for non-ST-elevated MI (NSTEMI) patients (4). Hence, it could be hypothesized that the larger cTnT proteoforms (≥29 kDa) observed in the circulation of MI patients might be influenced by the presence of anticoagulation medications in their blood. In the present study, we investigated the effect of anticoagulation medication on cTnT degradation over time in vitro using blood samples spiked with UFH and fondaparinux.

In the present study, serum, lithium-heparin (LH) and ethylenediaminetetraacetic acid (EDTA) blood tubes (all BD Vacutainers) were spiked with 2,500 ng/L free intact 40 kDa cTnT prior to venipuncture of a healthy volunteer [high-sensitivity (hs)-cTnT <5 ng/L] (n=2). Additionally, these blood tubes were either unspiked or spiked with anticoagulation medication, either 1,000 U/L UFH (1× UFH; SAHZ S043 100 I.U./mL; comparable to the bolus of 5,000 I.U. administered to STEMI patients at presentation), 20,000 U/L UFH (20× UFH; comparable to the concentration used in heparin-based blood tube as based on personal communication with BD) or 0.5 µg/mL fondaparinux (Aspen Arixtra 5 mg/mL; comparable to a subcutaneous injection that is administered to NSTEMI patients), all based on a general human blood volume of 5 L. Blood tubes were aliquoted and then incubated for 0, 2, 4, 8, 24, and 48 hours at 37 ℃ and subsequently stored at −80 ℃ until further analysis. cTnT in the samples was purified and concentrated using immunoprecipitation and analyzed using Western blotting as previously described (2). The cTnT band intensities were analyzed using Imagelab 6.1 (Bio-Rad Laboratories, Hercules, CA, USA).

Serum spiked with intact 40 kDa cTnT showed immediate and complete degradation to 29 kDa cTnT, in line with previous results (1). Addition of 1× UFH slightly reduced degradation of intact 40 kDa cTnT in serum, resulting in 10–15% of cTnT still being intact cTnT at T0. In contrast, addition of 20× UFH resulted in mostly intact cTnT and after 48 hours only 5% was reduced to 29 kDa cTnT. In the LH blood tube spiked with 1× UFH, the presence of free intact 40 kDa cTnT remained stable at 75–80%, with degradation to the 29 kDa fragment starting at 24 hours. The only difference between heparinized LH-plasma and LH-plasma without any medication, is the delayed degradation to 15–18 kDa fragments up to 48 hours. In the EDTA blood tube, addition of 1× UFH delayed the degradation of intact 40 kDa cTnT from 4 to 8 hours compared to EDTA-plasma without any medication. Fondaparinux did not appear to influence the cTnT degradation in either serum or LH-plasma. However, in EDTA-plasma, fondaparinux resulted in a delay in the degradation of intact 40 kDa cTnT up to 24 hours (Figure 1).

Figure 1 Representative Western blots (n=2) of free intact 40 kDa cTnT spiked in serum (top), LH-plasma (middle), and EDTA-plasma (bottom). Blood tubes, spiked with 40 kDa cTnT, were also spiked with either no medication, 1× UFH, 20× UFH, or fondaparinux (from left to right respectively), and incubated at 37 ℃ up to 48 hours. +, positive control; −, negative control; EDTA, ethylenediaminetetraacetic acid; LH, lithium-heparin; M, protein marker; UFH, unfractionated heparin.

This proof-of-principle study illustrated that interference of heparin on cTnT degradation is limited for a typical clinical dose of UFH, while interference is clearly present for higher concentrations in commonly used blood tubes. Fondaparinux did not appear to have an effect on cTnT degradation except for EDTA-plasma. Both medications target antithrombin, however, UFH inhibits various activated coagulation factors including thrombin (4) while fondaparinux mainly inhibits factor Xa (4). This difference in target can be attributed to the length of the polysaccharide (4) and could possibly explain the difference in degradation pattern we observed.

Overall, new cTnT-based immunoassays are under development that target larger cTnT proteoforms in a different way compared with the present cTnT assay to improve diagnostic specificity for the acute phase of MI (5). A potential effect of interference from anticoagulation medication on cTnT was not yet taken into account. Our in vitro model and preliminary results suggest, though, that this impact might be limited for clinical use-cases because of the relatively low levels of UFH medications and the limited effect of fondaparinux. Future studies should validate the medication impact of all typical anticoagulants, e.g., mentioned in the European Society of Cardiology (ESC) guidelines (6) and influence of thrombin activity in clinical MI patients, either STEMI or NSTEMI patients. The impact for patients who are already on anticoagulation medications before their acute MI, i.e., direct oral anticoagulants (DOACs), also remains to be further investigated.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was a standard submission to the journal. The article has undergone external peer review.

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0013/prf

Funding: This study was supported by the Veni grant (09150161810155) from the Dutch Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO), to A.M.A.M.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2026-1-0013/coif). A.M.A.M. received non-financial support from Abbott Diagnostics and Roche Diagnostics and the Veni grant (09150161810155) from the Dutch Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO). The other authors have no conflicts of interest to declare.

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