In washed platelets, unstimulated or stimulated with different agonists and pre-incubated with various bortezomib concentrations, the proteasome activity was determined by a fluorometric assay. The levels of poly-ubiquitinated proteins were assessed by an immunoassay kit. Platelet aggregation was measured by light transmission aggregometry in platelet-rich-plasma.
Platelet agonists stimulate both, the proteasome activity and the accumulation of poly-ubiquitinated proteins in platelets. Bortezomib inhibits the basal and the agonist induced proteasome activity and increased the content of poly-ubiquitinated proteins in a concentration dependent manner. Bortezomib concentrations in the nM-range causing complete blockade of platelet proteasome activity do not affect agonist induced platelet aggregation, indicating that the level of platelet proteasome activity is not directly linked with the induction of platelet aggregation. Bortezomib in the µM-range may tamper platelet aggregation, possibly due to unspecific and toxic effects.