The role of proteasome activity for activating and inhibitory signalling in human platelets.

Platelets express key proteins of the proteasome system, but its functional role in the regulation of platelet integrity, however, is not fully understood yet. Therefore, this study evaluated activating and inhibitory platelet signalling pathways using the potent and selective proteasome inhibitor bortezomib. In washed platelets, the effect of bortezomib on viability and on aggregation was assessed. In addition, fibrinogen binding and CD62P expression were determined. The influence on activating and inhibitory signalling was detected by phosphorylation levels of essential messenger molecules. Platelet viability was maintained after incubation with 0.01 µM to 1 µM bortezomib, but tampered with 100 µM bortezomib. Agonist-induced aggregation was only reduced under 100 µM bortezomib and with weak induction by 10 µM adenosine diphosphate. Similarly, phosphorylated kinase levels of the activating signalling pathways were not affected by 0.01 µM to 1 µM bortezomib. In contrast, proteasome inhibition resulted in the reduction of inhibitor-induced vasodilator-stimulated phosphoprotein phosphorylation, accompanied with the partial decrease of induced inhibition of fibrinogen binding and CD62P expression. In conclusion, platelet activation and aggregation are not dependent on proteasome activity. Instead, inhibitory signalling is partially attenuated under proteasome inhibition. Supramaximal inhibitory concentrations of bortezomib (above 1 µM) lead to heterogeneous effects on activating or inhibitory systems, probably caused by decreasing platelet viability.