The Proteasome Inhibitor Bortezomib Induces Apoptosis and Activation in Gel-Filtered Human Platelets

Harriet Ghansah,Ildikó Beke Debreceni,János Kappelmayer,Béla Nagy,Zsolt Fejes

doi:10.3390/ijms22168955

Harriet Ghansah, Ildikó Beke Debreceni + Show 3 more

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https://doi.org/10.3390/ijms22168955

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Abstract

Bortezomib (BTZ) has demonstrated its efficacy in several hematological disorders and has been associated with thrombocytopenia. There is controversy about the effect of BTZ on human platelets, so we set out to determine its effect on various types of platelet samples. Human platelets were investigated in platelet-rich plasma (PRP) and as gel-filtered platelets (GFPs). Mitochondrial inner membrane potential depolarization and phosphatidylserine (PS) and P-selectin expression levels were studied by flow cytometry, while thrombin generation was measured by a fluorescent method. In PRP, BTZ caused negligible PS expression after 60 min of treatment. However, in GFPs, PS expression was dose- and time-dependently increased in the BTZ-treated groups, as was P-selectin. The percentage of depolarized cells was also higher after BTZ pretreatment at both time points. Peak thrombin and velocity index increased significantly even with the lowest BTZ concentration (p = 0.0019; p = 0.0032) whereas time to peak and start tail parameters decreased (p = 0.0007; p = 0.0034). The difference between PRP and GFP results can be attributed to the presence of plasma proteins in PRP, as the PS-stimulating effect of BTZ could be attenuated by supplementing GFPs with purified human albumin. Overall, BTZ induces a procoagulant platelet phenotype in an experimental setting devoid of plasma proteins.

Highlights

Proteasome, present in the nucleus and cytoplasm of eukaryotic cells, is responsible for the degradation of damaged, misfolded or unfolded proteins [1], regulating a variety of cellular pathways, including apoptosis, cell growth and proliferation, transcription, DNA repair, immune responses and signaling processes [2,3]
These gel-filtered human platelets (GFPs) were treated with increasing BTZ concentrations similar to those used in the platelet-rich plasma (PRP)
For PS expression, pretreatment with BTZ was 60 min, since we found that BTZ did not cause significant PS expression in PRP at this time, unlike gel-filtered platelets (GFPs)

Summary

Introduction

Proteasome, present in the nucleus and cytoplasm of eukaryotic cells, is responsible for the degradation of damaged, misfolded or unfolded proteins [1], regulating a variety of cellular pathways, including apoptosis, cell growth and proliferation, transcription, DNA repair, immune responses and signaling processes [2,3]. The most studied proteasome complex is the 26S proteasome consisting of a 20S catalytic core and one or two 19S regulatory subunits on either end of the 20S core. The alpha rings regulate the entry of the substrate into the 20S core and the interaction with the regulatory subunit. The beta rings constitute the proteolytic core where β1, β2 and β5 subunits have caspase-like, trypsin-like and chymotrypsin-like activities, respectively [5,8]. Proteasome inhibitors cause the accumulation of otherwise degradable proteins within the cell that eventually induces cell death [9,10]. The main target of proteasome inhibitors is the chymotrypsin-like β5 subunit of the proteasome [11]. Three proteasome inhibitors are currently in clinical use. Others in clinical trials include marizomib (NPI-0052) [14,15], oprozomib (ONX-0912) [14,15] and delanzomib (CEP-18770) [14]

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