SARS-CoV-2 Spike Protein and Neutralizing Anti-Spike Protein Antibodies Modulate Blood Platelet Function.

Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.

Antibody-mediated procoagulant platelet formation in COVID-19 is AKT dependent.

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.

Endothelial Injury and Glycocalyx Degradation in Critically Ill Coronavirus Disease 2019 Patients: Implications for Microvascular Platelet Aggregation.

OBJECTIVES: Coronavirus disease 2019 is caused by the novel severe acute respiratory syndrome coronavirus 2 virus. Patients admitted to the ICU suffer from microvascular thrombosis, which may contribute to mortality. Our aim was to profile plasma thrombotic factors and endothelial injury markers in critically ill coronavirus disease 2019 ICU patients to help understand their thrombotic mechanisms. DESIGN: Daily blood coagulation and thrombotic factor profiling with immunoassays and in vitro experiments on human pulmonary microvascular endothelial cells. SETTING: Tertiary care ICU and academic laboratory. SUBJECTS: All patients admitted to the ICU suspected of being infected with severe acute respiratory syndrome coronavirus 2, using standardized hospital screening methodologies, had daily blood samples collected until testing was confirmed coronavirus disease 2019 negative on either ICU day 3 or ICU day 7 if the patient was coronavirus disease 2019 positive. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Age- and sex-matched healthy control subjects and ICU patients that were either coronavirus disease 2019 positive or coronavirus disease 2019 negative were enrolled. Cohorts were well balanced with the exception that coronavirus disease 2019 positive patients were more likely than coronavirus disease 2019 negative patients to suffer bilateral pneumonia. Mortality rate for coronavirus disease 2019 positive ICU patients was 40%. Compared with healthy control subjects, coronavirus disease 2019 positive patients had higher plasma von Willebrand factor (p < 0.001) and glycocalyx-degradation products (chondroitin sulfate and syndecan-1; p < 0.01). When compared with coronavirus disease 2019 negative patients, coronavirus disease 2019 positive patients had persistently higher soluble P-selectin, hyaluronic acid, and syndecan-1 (p < 0.05), particularly on ICU day 3 and thereafter. Thrombosis profiling on ICU days 1-3 predicted coronavirus disease 2019 status with 85% accuracy and patient mortality with 86% accuracy. Surface hyaluronic acid removal from human pulmonary microvascular endothelial cells with hyaluronidase treatment resulted in depressed nitric oxide, an instigating mechanism for platelet adhesion to the microvascular endothelium. CONCLUSIONS: Thrombosis profiling identified endothelial activation and glycocalyx degradation in coronavirus disease 2019 positive patients. Our data suggest that medications to protect and/or restore the endothelial glycocalyx, as well as platelet inhibitors, should be considered for further study.