Inhibition of Collagen Related Peptide Induced Platelet Activation and Apoptosis by Ceritinib

Abstract

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca²⁺ channel Orai1, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 – 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 fluorescence, ROS abundance from 2’,7’-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α_IIbβ₃ 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 CRP, ceritinib slightly, but significantly decreased [Ca²⁺]_i without significantly modifying the other measured parameters. CRP significantly increased [Ca²⁺]_i, ROS abundance, P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

Garcinol A Novel Inhibitor of Platelet Activation and Apoptosis

“…Experiments were performed with blood platelets isolated from wild type mice. The mice had free access to water and control chow (Ssniff, Soest, Germany) [40,41,42,43,44,45,46].…”

“…Platelets were prepared as described previously [40,41,42,43,44,45,46]. Platelets were obtained from 10- to 12-week-old mice of either sex.…”

“…For the measurement of the cytosolic Ca 2+ concentration, the platelet preparation was washed once in Tyrode buffer (pH 7.4), stained with 3 µM Fluo-3AM (Biotinium, USA) in the same buffer and incubated at 37 °C for 30 min. Following the indicated experimental treatment, relative fluorescence was measured utilizing a BD FACS Calibur (BD Biosciences, Heidelberg, Germany) [41,42,43,44,45,46,50].…”

“…Washed mouse platelets (1 × 10 6 ) were suspended in modified Tyrode buffer (pH 7.4) containing 1 mM CaCl 2 and antibodies (1:10 dilution) and subsequently subjected to the respective treatments and for the indicated time periods at room temperature (RT). The reaction was stopped by the addition of PBS and the samples were immediately analyzed on a BD FACSCalibur [24,41,42,43,44,45,46].…”

“…Annexin-V-binding reflecting surface exposure of phosphatidylserine was evaluated by flow cytometry utilizing a BD FACSCalibur. In parallel, the forward scatter (FSC) of the platelets was determined by flow cytometry as a measure of platelet size [41,42,43,44,45,46,53].…”