Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. The exact mechanisms of COVID-19-associated hypercoagulopathy, however, remain elusive. Recently, we observed that platelets (PLTs) from patients with severe COVID-19 infection express high levels of procoagulant markers, which were found to be associated with increased risk for thrombosis. In the current study, we investigated the time course as well as the mechanisms leading to procoagulant PLTs in COVID-19. Our study demonstrates the presence of PLT-reactive IgG antibodies that induce marked changes in PLTs in terms of increased inner-mitochondrial transmembrane potential (Δψ) depolarization, phosphatidylserine (PS) externalization, and P-selectin expression. The IgG-induced procoagulant PLTs and increased thrombus formation were mediated by ligation of PLT Fc-γ RIIA (FcγRIIA). In addition, contents of calcium and cyclic-adenosine-monophosphate (cAMP) in PLTs were identified to play a central role in antibody-induced procoagulant PLT formation. Most importantly, antibody-induced procoagulant events, as well as increased thrombus formation in severe COVID-19, were inhibited by Iloprost, a clinically approved therapeutic agent that increases the intracellular cAMP levels in PLTs. Our data indicate that upregulation of cAMP could be a potential therapeutic target to prevent antibody-mediated coagulopathy in COVID-19 disease.

Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. However, the exact mechanisms of thromboembolic events remain elusive. In this work, we show that immunoglobulin G (IgG) subclass in patients with COVID-19 trigger the formation of procoagulant PLTs in a Fc-gamma-RIIA (FcγRIIA) dependent pathway leading to increased thrombus formation in vitro. Most importantly, these events were significantly inhibited via FcγRIIA blockade as well as by the elevation of PLTs’ intracellular cyclic-adenosine-monophosphate (cAMP) levels by the clinical used agent Iloprost. The novel findings of FcγRIIA mediated prothrombotic conditions in terms of procoagulant PLTs leading to higher thrombus formation as well as the successful inhibition of these events via Iloprost could be promising for the future treatment of the complex coagulopathy observed in COVID-19 disease. Key points - Fc-gamma-receptor IIA mediated PS externalization on the PLT surface triggers increased thrombus formation - Inductors of cAMP inhibit antibody-mediated thrombus formation and may have potential therapeutic advantage in COVID-19

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies reactive to complexes of platelet factor 4 and heparin. Platelets (PLTs) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations to this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient antibodies (Abs) induce relevant changes in PLT phenotype, with the key features being increased P-Selectin expression and procoagulant phosphatidylserine (PS) externalization. Formation of procoagulant PLTs was dependent on engagement of PLT Fc-gamma-RIIA by HIT Abs and resulted in 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 PLTs propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLTs intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of high P-Selectin and PS levels on procoagulant PLTs was dissected. While inhibition of P-Selectin did not affect thrombus formation, the specific blockade of PS with Lactadherin prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLTs are critical mediators of prothrombotic conditions in HIT. Upregulation of cAMP with Iloprost or PS targeting specifc therapeutics could be a promising approach to prevent thromboembolic events in HIT patients.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies reactive to complexes of platelet factor 4 and heparin. Platelets (PLTs) 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 antibodies (Abs) 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-gamma-RIIA by HIT Abs and resulted in 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 PLTs propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLTs 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 PLTs are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.