The role of protein kinase C and the glycoprotein Ibα cytoplasmic tail in anti-glycoprotein Ibα antibody-induced platelet apoptosis and thrombocytopenia

Abstract

Introduction

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet counts. ITP patients with anti-platelet glycoprotein (GP) Ibα (a subunit of GPIb-IX-V complex) autoantibodies, which induce Fc-independent signaling and platelet clearance, are refractory to conventional treatment. Protein kinase C (PKC) is activated by the binding of the ligand von Willebrand factor (VWF) to GPIbα and regulates VWF-GPIbα-induced platelet activation. However, the role of PKC in anti-GPIbα antibody-induced thrombocytopenia remains unknown.

Materials and methods

The anti-GPIbα antibody-induced PKC activation and its underlying mechanisms were first detected by Western blot, and then the effects of PKC inhibitors, PKC knockout, or GPIbα C-terminal removal on anti-GPIbα antibody-induced platelet apoptosis, activation, aggregation, and clearance were investigated by flow cytometry, platelet aggregometry, and platelet posttransfusion, respectively. Meanwhile, platelet retention and co-localization with macrophages in the liver were detected by spinning disc intravital confocal microscopy.

Results

Anti-GPIbα antibody-induced PKC activation depends on GPIbα clustering and phosphoinositide 3-kinase (PI3K) activation and results in Akt phosphorylation. Pharmacologic inhibition or genetic ablation of PKC suppresses anti-GPIbα antibody-induced platelet apoptosis and activation. Moreover, the GPIbα cytoplasmic tail is required for antibody-induced PKC activation, platelet apoptosis, and activation. Inhibition or ablation of PKC and deletion of the GPIbα cytoplasmic tail protect platelets from clearance in vivo.

Conclusions

Our study indicates the important role of PKC and the GPIbα cytoplasmic tail in anti-GPIbα antibody-mediated platelet signaling and clearance and suggests a novel therapeutic target for ITP and other thrombocytopenic diseases.