Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients

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

The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia

Life threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against SARS-CoV-2. This phenomenon is now termed as vaccine-induced immune thrombotic thrombocytopenia (VITT). Pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT), and associated with platelet-activating antibodies against platelet factor 4 (PF4). Therefore, current guidelines suggest non-heparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux and argatroban with VITT-Ab/PF4 complexes using ex vivo model for thrombus formation as well as in vitro assays to analyze antibody binding and platelet activation. We found that IgGs from VITT patients induce increased adherent platelets/thrombus formation in comparison to IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid, argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation, however it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation, but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce antibody-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made