ABSTRACT
BACKGROUND: Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2. Recently, we observed that platelets from patients with severe COVID-19 infection express procoagulant phenotype. The molecular mechanisms that induce the generation of procoagulant platelets in COVID-19 patients are not completely understood. OBJECTIVES: In this study, we investigated the role of AKT (also known as Protein Kinase B), which is the major downstream effector of PI3K (phosphoinositid-3-kinase) (PI3K/AKT) signaling pathway in platelets from patients with COVID-19. PATIENTS AND METHODS: Platelets, Sera and IgG from COVID-19 patients who were admitted to the intensive care unit (ICU) were analyzed by flow cytometry as well as western blot and adhesion assays. RESULTS: Platelets from COVID-19 patients showed significantly higher levels of phosphorylated AKT, which was correlated with CD62p expression and phosphatidylserine (PS) externalization. In addition, healthy platelets incubated with sera or IgGs from ICU COVID-19 patients induced phosphorylation of PI3K and AKT and were dependent on Fc-gamma-RIIA (FcγRIIA). In contrast, ICU COVID-19 sera mediated generation of procoagulant platelets was not dependent on GPIIb/IIIa. Interestingly, the inhibition of phosphorylation of both proteins AKT and PI3K prevented the generation of procoagulant platelets. CONCLUSIONS: Our study shows that pAKT/AKT signaling pathway is associated with the formation of procoagulant platelets in severe COVID-19 patients without integrin GPIIb/IIIa engagement. The inhibition of PI3K/AKT phosphorylation might represent a promising strategy to reduce the risk for thrombosis in patients with severe COVID-19.
Subject(s)
COVID-19 , Proto-Oncogene Proteins c-akt , Blood Platelets , Humans , Phosphatidylinositol 3-Kinases , Platelet Activation , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex , SARS-CoV-2ABSTRACT
The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.