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Letters to the Editor
nels at constant blood flow of 950/s shear rate for 4 min- utes. Six images were then captured and the surface cov- erage and area of thrombi (ATh) were calculated. Ig (5 mg/mL) (Venital, Kedrion Biopharma), aspirin (100 mmol/L) (Sanofi SPA) or the P2Y12 antagonist cangrelor (1 mmol/L) (The Medicines Company, Parsippany-Troy Hills, NJ, USA) were added in vitro in some experiments.
The suspicion of TTS, based on the co-presence of thrombosis and thrombocytopenia, was supported by the positivity of the ELISA for anti-PF4/polyanions anti- bodies (Figure 1A), which was normalized by heparin at high concentration (100 U/mL). PAT was tested both by LTA and HIMEA after the addition of patients’ sera to normal WPS and normal WB. Different results were obtained in the two patients: only serum from patient 1 induced aggregation of WPS, which was inhibited by heparin at low (0.2 U/mL) and high (100 U/mL) concen- trations (Figure 1B); in contrast, both patients’ sera induced platelet aggregation in normal WB, which was not inhibited by 1 U/mL heparin and was inhibited by 200 U/mL heparin only when induced by patient 2 serum (Figure 1C). The observed discrepant results obtained with WPS and WB might suggest a major role in patient 2 of leukocytes interaction with platelets and anti- PF4/polyanions autoantibodies in the pathogenesis of platelet activation and thrombosis.13 As it has been demonstrated that the in vitro addition of PF4 increases the sensitivity of the PAT test in some patients, experi- ments were repeated in the presence of 10 μg/mL PF4 (Chromatec, Germany): under these conditions, serum from patient 1 induced platelet activation similarly in two separate experiments, while serum from patient 2 induced platelet activation in one experiment, but was still ineffective in two separate experiments (Figure 1B). Following the diagnosis of TTS, anticoagulant treatment, which was initially based on heparin preparations, was switched to alternative anticoagulants: fondaparinux dur- ing hospitalization and edoxaban at discharge for patient 1, argatroban and fondaparinux during hospitalization and apixaban at discharge for patient 2. Both patients were also treated with IVIg, 2 g/Kg body weight over 5 days, which normalized their platelet count (Figure 1D). The time needed to increase the platelet count was simi- lar to that observed in other studies in which the same dose of IVIg was infused over 2 days.3,4,14 No steroids were given to patients. Patient 2 also underwent trans- jugular intrahepatic portosystemic shunt (TIPS), throm- bo-aspiration and loco-regional fibrinolysis in the angiog- raphy room on day 2. The clinical courses were unevent- ful for both patients, who were discharged on days 9 and 16. Platelet counts of both patients were normal up to 7 weeks after completion of IVIg treatment (not shown).
IVIg infusion had additional potentially protective effects: it i) reduced (patient 1) or normalized (patient 2) the serum reactivity detected by the ELISA test (Figure 1A), compatible with inhibition of antibody production;15 ii) reduced or abolished the activation of normal WPS by patients’ sera (Figure 1B); iii) normalized the percentage of circulating platelet/monocyte hetero-aggregates in both patients, a marker of platelet activation and interac- tion with leukocytes, which were increased at baseline (Figure 1E): similar findings were recently reported in other patients;13 iv) blunted the amplifying effect of patients’ sera on in vitro thrombus formation by normal blood (see below).
Considering that markers of platelet hyper-reactivity could be secondary to the patients’ ongoing thrombotic process in vivo and that their improvement after IVIg could also be due to concomitant treatment with antico-
agulants, we elected to evaluate the effects of patients’ sera or plasma on markers of activation and reactivity of platelets from healthy subjects and the inhibitory effects of Ig added in vitro. To this end, the effects of patients’ sera/plasma were compared not only to those of sera/plasma from six to 18 healthy subjects, but also to those of serum/plasma from a 76 years old man (patient 3) who developed thrombocytopenia (69x109/L) and epistaxis 6 days after the first ChAdOx1 nCov-19 injec- tion, but had no thrombotic events and negative ELISA test results for anti-PF4/polyanions antibodies (Figure 1A). Compared to 18 plasma samples from healthy sub- jects, plasma from patients 2 and 1 (albeit less markedly), but not plasma from patient 3, increased thrombus for- mation by normal WB perfused over collagen-coated microchannels at 950/s shear rate (Table 1). Increased thrombus formation was not observed or was less marked with patients’ plasma obtained after IVIg, and was completely prevented by Ig added in vitro (Table 1). Similar effects of patients’ sera/plasma were observed on aggregation of normal WPS, formation of monocyte/platelet hetero-aggregates and binding of annexin V to procoagulant phosphatidylserine on the platelet membrane in normal WB, which were dramati- cally increased, especially by serum from patient 2. These effects were prevented by both the in vivo administration of IVIg and the in vitro addition of Ig, suggesting that Ig mostly inhibit platelet activation through FcgRIIa recep- tors, although a partial contribution by in vivo inhibition of antibody production cannot be ruled out.15 Even though serum/plasma from patient 2 did not activate nor- mal WPS, it was more pro-thrombogenic than serum/plasma from patient 1 in all other tests in which normal WB was used, thus reproducing the discrepant results obtained with WPS and WB in the PAT test (Figure 1B and C). Finally, we also tested the in vitro effects of the antiplatelet drugs aspirin and cangrelor on these parame- ters of platelet reactivity. Both drugs prevented the potentiation of platelet reactivity induced by patients’ sera/plasma, although cangrelor tended to be more effec- tive than aspirin. These results suggest that the throm- boxane A2 and ADP/P2Y12 pathways of platelet activa- tion might play a role in platelet activation in TTS. Whether or not these antiplatelet drugs could benefit TTS patients should only be determined by the results of ad hoc control studies.
In conclusion, we found that IVIg curbed the platelet- activating properties of our patients’ sera and produced a lasting increase in platelet count even in absence of con- comitant corticosteroid treatment.
Mariangela Scavone,1* Bianca Clerici,1* Simone Birocchi,1 Tatiana Mencarini,2 Mariagrazia Calogiuri,1 Claudia Ghali,1 Daniele Prati,3 Silvia Bozzi,2 Paolo Villa,4 Marco Cattaneo1 and Gian Marco Podda1
1Divisione di Medicina Generale II, ASST Santi Paolo e Carlo, Dipartimento di Scienze della Salute, Università degli Studi di Milano; 2Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano; 3Dipartimento di Medicina Trasfusionale e di Ematologia, IRCCS Fondazione Ca' Granda, Ospedale Maggiore Policlinico and 4U.O Medicina d'urgenza Sacco - Medico dell'ASST Fatebenefratelli Sacco, Milano, Italy.
*MS and BC contributed equally as co-first authors Correspondence:
GIAN MARCO PODDA - gmpodda@gmail.com doi:10.3324/haematol.2021.279345
Received: July 1, 2021.
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haematologica | 2021; 106(12)