Whole Blood Procoagulant Platelet Flow Cytometry Protocol for Heparin-Induced Thrombocytopenia (HIT) and Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Testing.

Heparin-induced thrombocytopenia (HIT) is a well-characterized, iatrogenic complication of heparin anticoagulation with significant morbidity. In contrast, vaccine-induced immune thrombotic thrombocytopenia (VITT) is a recently recognized severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria, AstraZeneca) and Ad26.COV2.S (Janssen, Johnson & Johnson) vaccines against COVID-19. The diagnosis of HIT and VITT involve laboratory testing for antiplatelet antibodies by immunoassays followed by confirmation by functional assays to detect platelet-activating antibodies. Functional assays are critical to detect pathological antibodies due to the varying sensitivity and specificity of immunoassays. This chapter presents a protocol for a novel whole blood flow cytometry-based assay to detect procoagulant platelets in healthy donor blood in response to plasma from patients suspected of HIT or VITT. A method to identify suitable healthy donors for HIT and VITT testing is also described.

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.