Proteasome is a multi-catalytic protease complex that enables protein degradation. It exists in several structural forms and tissue-specific variants. The most common is the constitutive proteasome and immunoproteasome, which were recently also found to be present in platelets. It is known that platelets and their characteristic can heavily vary among individuals. Based on this, we hypothesized that there are also differences between individuals in the expression and activity of individual catalytic subunits of the proteasome. Our hypothesis was tested in samples from random donors, in whom we determined the protein levels of constitutive and immunoproteasome catalytic subunits in platelets by western blotting. The results showed inter-individual differences, with a greater variation in the expression of immunoproteasome than constitutive subunits. The same donor samples were then subjected to activity based probe assays to determine the activity of the individual subunits in platelets. Differences were also observed in this parameter, where higher activity of the immunoproteasomal subunits β1i and β5i than of the β1c and β5c subunits. We further investigated the effect of selective inhibitors of the catalytic subunits of the immunoproteasome, using KZR-504 to inhibit the β1i subunit and LMP7-IN-1 to inhibit the β5i subunit. Here, 50 nM concentrations of the two selective inhibitors were shown to result in almost complete inhibition of the corresponding subunits after 3 hours. This concentration of inhibitors was then used in a further experiment, where we were interested to see whether platelet activation by adenosine diphosphate (ADP) had any effect on the activity of the immunoproteasome subunits in the presence of selective inhibitors. We found that under the given conditions, ADP did not affect the catalytic activity of the β1i or β5i subunits. We then tested whether inhibition of the immunoproteasome affects platelet activation by determining the concentration of released chemokines from the platelet granules. We found that platelet activation with ADP leads to an increased release of the chemokine RANTES, however the presence of the inhibitors KZR-504 and LMP7-IN-1 did not cause any statistically relevant changes.
The results show differences in the protein levels and activities of the proteasome catalytic subunits in platelets between individuals. The catalytic subunits can be successfully inhibited after only a few hours of exposure to selective inhibitors. Further studies are needed to investigate the role of the proteasome in the process of platelet activation.
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