Conclusions
In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.
Methods
Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.
Results
Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces. Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.