TO THE EDITOR:
Ticagrelor, a reversible inhibitor of the adenosine 5′-diphosphate (ADP) receptor P2Y12, is recommended as a first-line P2Y12 receptor antagonist after coronary interventions or acute coronary syndrome.1,2 In contrast to the irreversible inhibitors aspirin, clopidogrel, and prasugrel, ticagrelor binds to the ADP receptor reversibly, and the drug is present at high concentrations in plasma (227-770 ng/mL).3
Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction that is caused by platelet-activating anti–platelet factor 4 (PF4)/heparin antibodies and leads to an increased risk for thrombosis4 . The most widely used system for clinical diagnosis is the 4Ts score (0-8 points).5 The diagnosis is confirmed by laboratory tests. Antigen tests for anti-PF4/heparin antibodies6 have a high sensitivity7 ; however, only a subset of anti-PF4/heparin antibodies that activate platelets is clinically relevant. In some patient populations, such as patients undergoing cardiac surgery, this accounts for <25% of anti-PF4/heparin antibodies detected by antigen tests.8 Clinically relevant anti-PF4/heparin antibodies typically activate washed platelets of healthy donors in the presence of patient serum and heparin in the heparin-induced platelet activation (HIPA) test9 or the serotonin release assay.10,11
Platelet activation by HIT antibodies is caused via the platelet FcγRIIA receptor and depends on cosignaling via the P2Y12 ADP receptor,12 which is blocked by ticagrelor. When we observed a patient with a clinical course typical for HIT, a positive anti-PF4/heparin enzyme immunoassay (EIA) but a negative HIPA test (Figure 1), we systematically assessed whether ticagrelor may lead to a false-negative HIPA test. The in-house anti-PF4/heparin-immunoglobulin G (IgG) EIA and HIPA tests were performed as previously described.9,13,14 Aggregation in ≥3 of 4 donors’ test cells in the presence of low molecular weight heparin (0.2 aFXaU/mL) within 35 minutes was defined as HIT+. Confirmed HIT+ sera were spiked with ticagrelor (final concentrations: 15.6, 31.3, 62.5, 125, 250, 500, and 1000 ng/mL; in vivo range, 227-770 ng/mL; 1% dimethyl sulfoxide was used as carrier control). The IgG fraction from patient sera was prepared using protein G sepharose (optical density [OD] 2.20 in the PF4/heparin EIA). Serum from the index patient (Figure 1) and ticagrelor-spiked known HIPA+ sera were incubated for 30 minutes with 100 μg/mL the ticagrelor antidote PB2452 (kindly provided by PhaseBio, Malvern, PA). A total of 500 μL of HIPA+ sera spiked with ticagrelor was incubated with 20 mg of pulverized activated charcoal tablets (Kohle-Hevert, Hevert Arzneimittel, Berlin, Germany) for 5 minutes, then centrifuged, and the supernatant was used.
In all but 1 tested sera, ticagrelor dose dependently inhibited the HIPA test when concentrations in the serum reached those during clinical steady-state (227-770 ng/mL; Figure 2A). In contrast to the ticagrelor-containing serum, the purified IgG fraction (PF4/heparin EIA OD 2.20) strongly activated platelets in the HIPA test (Figure 2A,B); respiking with ticagrelor again inhibited the HIPA test. Also in the presence of the ticagrelor antidote (PB2452; 100 mg/mL), consisting of monoclonal Fab fragments against ticagrelor that block ticagrelor-platelet interactions,15 the index patient’s serum and ticagrelor (500 ng/mL)–containing patient sera tested positive in the HIPA test (Figure 2C). Finally, when we incubated 3 previously confirmed HIPA+ sera, prespiked with ticagrelor (500 ng/mL) with activated charcoal, the cleared sera again tested positive in the HIPA test (Figure 2D). The Institutional Review Board of the Universitätsmedizin Greifswald, Greifswald, Germany, allowed the use of anonymized patient samples for laboratory studies. The patient described in the case report gave informed consent for publishing the case report.
These data show that ticagrelor present in patient’s serum during treatment with ticagrelor inhibits the functional assay for HIT. This inhibition can be ameliorated by 3 methods: isolating the IgG fraction from the patient serum, preincubating the serum with the ticagrelor antidote PB2452, or preincubating the patient serum with 20 mg of charcoal powder.
This finding is of major relevance when patients suspected to have HIT also receive ticagrelor. Although it is not very frequent, HIT can occur in cardiac patients with acute coronary syndrome, as in the index case presented. Cardiac patients with additional major complications are at an especially increased risk for HIT. In the index case, the tissue trauma after resuscitation was probably an additional trigger for the development of HIT. In contrast to medically treated cardiac patients, HIT is highly relevant in patients undergoing cardiac surgery. The incidence of HIT after cardiac surgery is in the range of 1% to 2%,16 and it is ∼0.5% after transcatheter aortic valve replacement.17 Although ticagrelor is usually stopped before surgery, antiplatelet drugs are typically restarted within 3 days after major surgery in patients at high risk for coronary events, whereas HIT typically occurs 5 to 10 days after major surgery. Thus, restart of ticagrelor and the onset of HIT after surgery will usually overlap. Although ticagrelor in the patient’s serum will inhibit the in vitro test, the index patient demonstrates that venous and arterial thrombotic complications can be induced by heparin in acute HIT in vivo, despite concomitant ticagrelor treatment.
An additional clinically important scenario is patients with a (recent) history of HIT who require cardiac surgery. Heparin is still the most preferred anticoagulant during cardiopulmonary bypass surgery. In patients with a history of HIT, who require cardiopulmonary bypass surgery with the heart-lung machine, current guidelines recommend the use of heparin once the functional test becomes negative, even when the antigen test is still positive (and to use nonheparin anticoagulants before and after surgery).7
As a result of the widespread use of ticagrelor, the number of patients in whom HIT is suspected while they are treated with ticagrelor will likely increase. Continuing or restarting heparin based on a false-negative platelet-activation test can be life-threatening. For interpretation of platelet-activation tests, the laboratory needs to know whether the patient is treated with ticagrelor. Removing ticagrelor from the patient’s serum by charcoal powder, using the purified IgG fraction, or inhibiting ticagrelor in the patient’s serum by a specific antidote is a method to obtain reliable results in functional HIT tests.
Although we did not test this in the present study, it is very likely that the other available reversibly binding P2Y12 inhibitor, cangrelor, shows the same effect. However, because of its short half-life, this can be overcome by stopping the drug up to 30 minutes before the blood sample for HIT testing is obtained. We cannot exclude that prasugrel and clopidogrel also show some inhibition at peak plasma concentrations; however, their plasma concentrations are much lower than for ticagrelor. Furthermore, the active metabolites are highly unstable in serum because they contain a thiol group. Because the timespan between taking a blood sample and testing the serum in a functional test is several hours, and the serum is heat inactivated, we consider it highly unlikely that the unstable metabolite of clopidogrel will still be present in the serum at sufficient concentrations.
In summary, free P2Y12 inhibitors in patient plasma can inhibit functional tests for HIT. This is of special importance for the testing of cardiac patients with a history of (recent) HIT who are scheduled for planned re-exposure to heparin during cardiopulmonary bypass surgery. It is very important that clinicians inform the laboratory about the use of ticagrelor in a patient when the clinical suspicion for HIT should be confirmed or ruled out. Laboratory physicians must be aware of the interaction between ticagrelor and functional assays for HIT.
The authors thank Ulrike Strobel and Ricarda Raschke for technical support.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFBTRR240: Z03.
Contribution: J.J.M.E. performed ticagrelor experiments and wrote the manuscript; C.P., T.M., and G.W. managed the patient with HIT under ticagrelor, first recognized that the functional HIT test was likely false negative, and wrote the patient case report; N.K. performed all charcoal experiments; A.G. designed the study, interpreted the results, and wrote the manuscript; and all authors reviewed the final version of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Andreas Greinacher, Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Sauerbruchstraße, 17487 Greifswald, Germany; e-mail: firstname.lastname@example.org.