Thrombopoietin participates in platelet activation in COVID-19 patients

Abstract

Background: The pathogenesis of coronavirus disease 2019 (COVID-19) is characterized by enhanced platelet activation and diffuse hemostatic alterations, which may contribute to immunothrombosis/thromboinflammation and subsequent development of target-organ damage. Thrombopoietin (THPO), a growth factor essential to megakariocyte proliferation, is known to prime platelet activation and leukocyte-platelet interaction. In addition, THPO concentrations increase in several critical diseases, such as acute cardiac ischemia and sepsis, thus representing a potential diagnostic and prognostic biomarker. Furthermore, several data suggest that interleukin (IL)-6 is one of the most important inflammatory mediators involved in these phenomena, which led to explore the potential therapeutic role of IL-6 inhibitors. In this prospective cohort study, we aimed to study THPO and IL-6 concentrations in COVID-19 patients at the time of first clinical evaluation in the Emergency Department (ED), and to investigate their potential use as diagnostic and prognostic biomarkers. In addition, we sought to explore the role of THPO contained in plasma samples obtained from COVID-19 patients in priming in vitro platelet activation and leukocyte-platelet interaction.

Methods: We enrolled 66 patients presenting to the ED with symptoms suggestive of COVID-19, including 47 with confirmed COVID-19 and 19 in whom COVID-19 was excluded (Non-COVID-19 patients). As controls, we also recruited 18 healthy subjects. In vitro, we reproduced the effects of increased circulating THPO on platelet function by adding plasma from COVID-19 patients or controls to platelet-rich plasma or whole blood obtained by healthy donors, and we indirectly studied the effect of THPO on platelet activation by blocking its biological activity.

Findings: THPO levels were higher in COVID-19 patients than in both Non-COVID-19 patients and healthy subjects. Studying THPO as diagnostic marker for the diagnosis of COVID-19 by receiver-operating-characteristic (ROC) statistics, we found an area under the curve (AUC) of 0.73, with an optimal cut-off value of 42.60 pg/mL. IL-6 was higher in COVID-19 patients than in healthy subjects, but did not differ between COVID-19 and Non-COVID-19 patients. THPO concentrations measured at the time of diagnosis in the ED were also higher in COVID-19 patients subsequently developing a severe disease than in those with mild disease. Evaluating THPO as biomarker for severe COVID-19 using ROC analysis, we found an AUC of 0.71, with an optimal cut-off value of 57.11 pg/mL. IL-6 was also higher in severe than in mild COVID-19 patients, with an AUC for severe COVID-19 of 0.83 and an optimal cut-off value of 23 pg/ml. THPO concentrations correlated with those of IL-6 (r=0.2963; p=0.043), and decreased 24 h after the administration of tocilizumab, an IL-6 receptor blocking antibody, showing that the increase of THPO levels depends on IL-6-stimulated hepatic synthesis. In vitro, plasma obtained from COVID-19 patients, but not from healthy subjects, primed platelet aggregation and leukocyte-platelet binding, and these effects were reduced by inhibiting THPO activity.

Interpretation: Increased THPO may be proposed as an early biomarker for the diagnosis of COVID-19 and for the identification of patients at risk of developing critical illness. Elevated THPO may contribute to enhance platelet activation and leukocyte-platelet interaction in COVID-19 patients, thus potentially participating in immunothrombosis/thromboinflammation.

Funding: This work was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) ex 60% to GM and EL.

Keywords: Biomarker; COVID-19; Interleukin-6; Platelet activation; Thromboinflammation; Thrombopoietin.

Figure 1

Comparison of plasma levels of THPO and IL-6 in healthy subjects, Non-COVID-19 patients, and COVID-19 patients and relative receiver-operating characteristics (ROC) curves for the diagnosis of COVID-19. Box-and-whisker plots of THPO (a) and IL-6 (c) plasma concentrations in healthy subjects (n = 18), Non-COVID-19 patients (n = 19), and COVID-19 patients (n = 47). Data were expressed as median (range), and were analyzed by using Kruskal-Wallis one-way analysis of variance on ranks followed by Dunn's multiple comparison test. ROC curves of plasma THPO (b) and IL-6 (d) for the diagnosis of COVID-19. Area under the curve (AUC) values are reported with 95% CI in brackets.

Figure 2

Comparison of THPO and IL-6 plasma levels in mild and severe COVID-19 patients and relative receiver-operating characteristics (ROC) curves for the development of severe COVID-19. Box-and-whisker plots of THPO (a) and IL-6 (c) plasma concentrations in mild (n = 36) and severe (n = 11) COVID-19 patients. Data were expressed as median (range), and were analyzed by using Mann-Whitney rank sum test. ROC curves of plasma THPO (b) and IL-6 (d) for the development of severe COVID-19. Area under the curve (AUC) values are reported with 95%CI in brackets.

Figure 3

Decision curves for THPO and IL-6 for the diagnosis of COVID-19 (a) and for the risk of severe COVID-19 development (b).

Figure 4

Effect of tocilizumab (TCZ) administration on THPO and IL-6 plasma levels in six COVID-19 patients. Scatter plots of THPO (a) and IL-6 (b) plasma levels measured pre- and post-TCZ administration in COVID-19 patients. Data were analyzed by using Wilcoxon matched-pairs signed rank test.

Figure 5

Effect of plasma from healthy subjects and COVID-19 patients on in vitro platelet aggregation in platelet-rich plasma (PRP). Bar graph showing the in vitro priming activity induced by plasma from healthy subjects and COVID-19 patients on platelet aggregation in PRP induced by epinephrine (EPI) (a), adenosine-diphosphate (ADP) (b), and thrombin (THR) (c). Bar graph showing the in vitro effect of plasma from healthy subjects and COVID-19 patients on monocyte-platelet aggregation induced in whole blood by epinephrine (EPI) (d), adenosine-diphosphate (ADP) (e), and thrombin (THR) (f). Bar graph showing the in vitro effect of plasma from healthy subjects and COVID-19 patients on granulocyte-platelet aggregation induced in whole blood by epinephrine (EPI) (g), adenosine-diphosphate (ADP) (h), and thrombin (THR) (i). A minimum of five experiments for each experimental conditions was performed. Data represent means ± SE, and were analyzed by using Student's t-test.

Figure 6

Inhibitory effect of rhTHPOR and Ab αTHPOR on in vitro platelet aggregation in platelet-rich plasma (PRP). Bar graph showing the effects exerted by plasma of COVID-19 patients pre‐incubated or not with rhTHPOR on platelet aggregation induced in PRP by EPI (a), ADP (b), and THR (c). Bar graph showing the effects exerted by plasma of COVID-19 patients, after pre-incubation of PRP or not with Ab αTHPOR, on platelet aggregation induced in PRP by EPI (d), ADP (e), and THR (f). A minimum of five experiments for each experimental conditions was performed. Data were analyzed by using paired Student's t-test.

Figure 7

Inhibitory effect of rhTHPOR and Ab αTHPOR on in vitro monocyte-platelet aggregation in whole blood. Bar graph showing the effects exerted by plasma of COVID-19 patients pre‐incubated or not with rhTHPOR on monocyte-platelet aggregation induced in whole blood by EPI (a), ADP (b), and THR (c). Bar graph showing the effects exerted by plasma of COVID-19 patients, after pre-incubation of whole blood or not with Ab αTHPOR, on monocyte-platelet aggregation induced in whole blood by EPI (d), ADP (e), and THR (f). A minimum of five experiments for each experimental conditions was performed. Data were analyzed by using paired Student's t-test.

Figure 8

Inhibitory effect of rhTHPOR and Ab αTHPOR on in vitro granulocyte-platelet aggregation in whole blood. Bar graph showing the effects exerted by plasma of COVID-19 patients pre‐incubated or not with rhTHPOR on granulocyte-platelet aggregation induced in whole blood by EPI (a), ADP (b), and THR (c). Bar graph showing the effects exerted by plasma of COVID-19 patients, after pre-incubation of whole blood or not with Ab αTHPOR, on granulocyte-platelet aggregation induced in whole blood by EPI (d), ADP (e), and THR (f). A minimum of five experiments for each experimental conditions was performed. Data were analyzed by using paired Student's t-test.

Figure 9

Effect of plasma obtained from six COVID-19 patients before and after tocilizumab (TCZ) administration on in vitro platelet aggregation in platelet-rich plasma (PRP). Scatter plots showing the in vitro priming activity induced by plasma obtained from COVID-19 patients before and after TCZ administration on platelet aggregation in PRP induced by epinephrine (EPI) (a), adenosine-diphosphate (ADP) (b), and thrombin (THR) (c). Data were analyzed by using Wilcoxon matched-pairs signed rank test.