COVID-19 induces a hyperactive phenotype in circulating platelets

Abstract

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Fig 1. Routine clinical laboratory parameters are associated with disease severity in COVID-19.

Platelet counts, MPV, Neutrophil counts, and PNRs of patients with severe (n = 34) or nonsevere (n = 20) COVID-19 and hospitalised controls (n = 20). (A) On the day of admission, patients who subsequently developed severe COVID-19 had significantly higher MPV (p = 0.015) and neutrophil counts (p = 0.013) and significantly lower PNR (p = 0.0067) compared to those who did not subsequently develop severe COVID-19. (B) On day 7 of hospitalisation, nonsevere COVID-19 patients had a significantly higher platelet count compared to severe COVID-19 patients on the day of transfer to intensive care (p = 0.014). Boxplots represent the data median (line inside the box) and the IQR (outline of the box) together with data maximum and data minimum (whiskers) and individual observations (see individual data in S1 Data). Statistical analysis was performed using a two-tailed t test, and p-values were adjusted for multiple comparisons using a Holm–Bonferroni post hoc test. COVID-19, Coronavirus Disease 2019; IQR, interquartile range; MPV, mean platelet volume; PNR, platelet-to-neutrophil ratio.

Fig 2. Agonist-induced ADP release was dramatically higher in COVID-19 patients compared with non-COVID-19 hospitalised patients.

Platelet dense granule release was measured in duplicate in severe (n = 5) and nonsevere (n = 4) COVID-19 patients compared to hospitalised (n = 3) and healthy controls (n = 6). Platelets were stimulated with 0.1 U/ml thrombin (A), 0.5 U/ml thrombin (B), 1 μM U46619 (C), and 6.6 μM U46619 (D) and ATP release (surrogate for ADP) was measured using a Chronolume luciferase assay. ADP release is expressed as pmol/10⁶ platelets. Boxplots represent the data median (line inside the box) and the IQR (outline of the box) together with data maximum and data minimum (whiskers) and individual observations (see individual data in S1 Data). Statistical analysis was performed using a two-tailed t test and p-values were adjusted for multiple comparisons using a Holm–Bonferroni post hoc test. COVID-19, Coronavirus Disease 2019; IQR, interquartile range.

Fig 3. Circulating levels of PF4, sP-selectin levels, and TPO were significantly elevated in COVID-19.

(A) PF4, (B) sP-selectin (sCD62P), and (C) TPO plasma levels from patients with severe (n = 6) and nonsevere (n = 6) COVID-19 compared to hospitalised (n = 6) and healthy (n = 6) controls were measured in triplicate by ELISA. Boxplots represent the data median (line inside the box) and the IQR (outline of the box) together with data maximum and data minimum (whiskers) and individual observations (see individual data in S1 Data). Statistical analysis was performed using a two-tailed t test, and p-values were adjusted for multiple comparisons using a Holm–Bonferroni post hoc test. COVID-19, Coronavirus Disease 2019; ELISA, enzyme-linked immunosorbent assay; IQR, interquartile range; PF4, platelet-factor 4; sP-selectin, soluble P-selectin; TPO, thrombopoietin.