Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis

Abstract

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca 2+ -activity ([Ca 2+ ] i ) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α IIb β 3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca 2+ ] i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated α IIb β 3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca 2+ ] i , P-selectin abundance, α IIb β 3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.H. Cao and A. Al Mamun Bhuyan contributed equally and thus share first authorship.

Acquired platelet antagonism: off‐target antiplatelet effects of malignancy treatment with tyrosine kinase inhibitors

“…On the other hand, thrombin‐induced platelet responses were less strongly inhibited. Further investigation showed that the collagen‐induced phosphorylation of Akt was decreased by afatinib .…”

Heterotrimeric G-protein subunit Gα i2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets

“…Individual values (mean AE SD) of female and male WT and Ga i2 -deficient mice of annexin-V-binding, platelet volume, caspase-3-positive cells, ceramide abundance, P-selectin abundance and activated integrin a IIb b3 are displayed in Table 1A-D. No significant differences were observed between male and female WT or Ga i2 -deficient mice. Phosphatidylserine exposure, a hallmark of platelet apoptosis, was quantified by flow cytometry analysis using annexin-V-binding as described previously (Cao et al 2017). As illustrated in Figure 1A and D, the percentage of PS-positive platelets in untreated resting Ga i2 À/À and WT platelets was not significantly different.…”

“…Phosphatidylserine exposure, a hallmark of platelet apoptosis, was quantified by flow cytometry analysis using annexin‐V‐binding as described previously (Cao et al. ). As illustrated in Figure A and D, the percentage of PS‐positive platelets in untreated resting Gα i2 −/− and WT platelets was not significantly different.…”

“…Next, we explored whether G α i2 ‐mediated platelet apoptosis accompanies cell volume alterations (Cao et al. ). As depicted in Figure E and H, forward scatter, reflecting platelet volume, was not significantly different in resting platelets of either genotype but was significantly reduced following thrombin and CoRP treatment, an effect significantly less pronounced in Gα i2 −/− platelets than in WT platelets (Fig.…”

“…We then sought to elucidate the mechanism(s) regulating Ga i2 -mediated apoptosis. To this end, we analyzed intracellular Ca 2+ levels [Ca 2+ ] i in murine platelets of either genotype using Fluo3 AM fluorescence as described previously (Cao et al 2017). As a result, prior to CoRP treatment, [Ca 2+ ] i in resting platelets was similar in Ga i2 À/À platelets (22.73 AE 0.69, a.u.…”