Cystic fibrosis transmembrane conductance regulator modulators attenuate platelet activation and aggregation in blood of healthy donors and COVID-19 patients

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators reduce agonist-induced platelet activation and function. CFTR modulators, such as ivacaftor, present a promising therapeutic strategy in thrombocytopathies, including severe COVID-19. https://bit.ly/3HJykdt

FIGURE 1

Pre-treatment with ivacaftor reduces platelet activation, adhesion and aggregation. Platelets from healthy donors (HDs) were pre-treated with vehicle, 10 µM ivacaftor, or 5 µM forskolin prior to stimulation with 5 µM TRAP6 or 10 µM ADP, or without (w/o) stimulation. a) Surface expression of CD62p and CD63 on CD42b⁺ platelets. b) Agonist-induced [Ca²⁺]_i transients traced as fluorescence ratio of Ca²⁺-bound (405 nm) to Ca²⁺-unbound (530 nm) indo-1 AM in CD42b⁺ platelets. c) Quantification of [Ca²⁺]_i transients in (b) as area under the curve (AUC). d) Agonist-induced aggregation of platelets assessed by impedance aggregometry (Roche, Germany) in hirudin blood samples. e) For platelet adhesion, citrated HD blood was stained with anti-CD42b antibody, and perfused through collagen-IV-coated microchannel flow chambers (Ibidi, Germany). Total CD42b⁺ covered area was quantified in five randomly selected regions of interest for each experimental condition (ImageJ, Version 13.0.6). f) Overview of COVID-19 patient and HD cohorts, both without cystic fibrosis (CF), sampled at Charité – Universitätsmedizin Berlin, Germany (Pa-COVID-19 cohort study [15] ethics approvals EA2/066/20 and EA2/075/15), and the Amsterdam University Medical Centers, the Netherlands (ethics approval METc-number 2021.0520) according to the World Health Organization (WHO) clinical progression scale for COVID-19. Patient inclusion required a positive PCR test for SARS-CoV-2 and >18 years of age. Patients treated with anti-platelet drugs were excluded. g) Surface expression of CD62p and CD63 on CD42b⁺ platelets from COVID-19 patients with moderate or severe disease compared to HDs. h) Representative tracings of agonist-induced [Ca²⁺]_i transients in platelets from patients with severe COVID-19 (without CF). i) Quantification of [Ca²⁺]_i transients measured in (h) as AUC. j) Agonist-induced aggregation of platelets from patients with severe COVID-19 (without CF). k) Representative fluorescence microscopy images of CD42b⁺ platelets from patients with severe COVID-19 (without CF) adhering on collagen-IV following perfusion with re-calcified whole blood pre-treated with vehicle, ivacaftor or forskolin, and stimulated with vehicle or ADP. Scale bar: 100 μm‌. l) Quantification of platelet-adhesion assessed in (k) as in (e). Graphical representation: Each circle (a, c, d, e, g, i, j, l) represents platelets from an individual HD/patient; lines represent intra-individual comparisons; bars indicate mean. *: p≤0.05; **: p≤0.005; ***: p≤0.001 by two-way ANOVA with Holm-Šídák's multiple comparisons test (a, c, d, e, g, i, j, l). Data are representative of a) n=12, c) n=8, d) n=7–19, e) n=4–8, g) n=20 (all HDs), n=30 for moderate, and n=22 for severe, i) n=8 each for severe, j) n=6–15 for severe, and l) n=6–12 for severe COVID-19 after ADP- or TRAP6-induced platelet activation following pre-treatment with vehicle, ivacaftor or forskolin. ECMO: extracorporeal membrane oxygenation; NIV: noninvasive ventilation.