Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways.

Elmira R Mordakhanova,Gulnaz E Synbulatova,John W Weisel,Lubica Rauova,Tatiana A Nevzorova,Rustem I Litvinov

doi:10.3390/ijms21072556

Abstract

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

Highlights

Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse reaction to heparin, a broadly used anticoagulant [1]
These immune complexes bind to the FcγRIIA receptors of platelets, resulting in platelet activation associated with the exposure of procoagulant phosphatidylserine (PS) on the platelet membrane and on platelet-derived microparticles [3] as well as the expression of P-selectin [4]
No significant difference was detected when comparing the expression of active αIIbβ3, PS and microvesiculation between platelets treated with platelet factor 4 (PF4) mixed with RTO, or PF4, KKO or RTO alone, similar to the untreated platelets presented in Figure 1B,D; Figure 1F,H (PS); and Figure 1J,L

Summary

Introduction

Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse reaction to heparin, a broadly used anticoagulant [1]. KKO, unlike RTO, causes PF4 to oligomerize in solution, forming ultra-large complexes that cluster on cell surfaces, which probably activate the cells and predispose to Ab-induced thrombosis [13]. This suggests that KKO is an appropriate model Ab for studies of HIT pathology, and comparisons between KKO and RTO might help to define the difference between the pathogenic and non-pathogenic human anti-PF4 Abs that underlie their dissimilar clinical impact. Using flow cytometry and Western blot analysis, we showed that, after activation, platelets undergo a complex death pathway that combines molecular signs of apoptosis and activation of calpain

Subpopulations of Live Activated and Dying Platelets

Assay of Calpain Activity

Statistical Analysis

Conclusions