Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death

Tatiana A Nevzorova,John W Weisel,John W Weisel,Giang Le Minh,Amina G Daminova,Amina G Daminova,Anastasia A Ponomareva,Anastasia A Ponomareva,Lubica Rauova,Lubica Rauova,Elmira R Mordakhanova,Izabella A Andrianova,Rustem I Litvinov,Rustem I Litvinov

doi:10.1038/s41420-019-0188-0

Abstract

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.

Highlights

Heparin-induced thrombocytopenia (HIT) is a common autoimmune disorder that develops in ∼1–5% of patients treated with heparin
The immune complexes composed of platelet factor 4 (PF4) and a nonpathogenic antibody RTO did not change the fraction of Annexin V-positive platelets
The immune platelet activation in HIT is mediated through the interaction of the Fc region of IgG with the FcγRIIA receptor on the platelet surface[10,16,17,18,19,20]

Summary

Introduction

Heparin-induced thrombocytopenia (HIT) is a common autoimmune disorder that develops in ∼1–5% of patients treated with heparin. Official journal of the Cell Death Differentiation Association. Nevzorova et al Cell Death Discovery (2019)5:106. After exposure of patients to heparin, it binds to PF4 and promotes PF4 aggregation, so that they form ultra-large PF4/heparin complexes with antigenic properties. Some patients develop antibodies against PF4/heparin complexes that cause HIT. The reasons for the immune response to the formation of the PF4/heparin complex as an autoantigen remain largely unclear. Anti-PF4/heparin antibodies were detected in 3.1–4.4% of healthy subjects, but occur in ~27–61% of patients after cardiac surgery and in 8–17% of medical and surgical patients treated with heparin. It is estimated that only 5–30% of patients with antibodies against heparin develop HIT4

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