Effect of Bexarotene on Platelet Activation and Apoptosis

Abstract

Background/Aims: The retinoid X receptor (RXRs) stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl] benzoic acid) is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP). The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 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, and relative platelet volume from forward scatter. Results: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca²⁺]_i, but did not significantly modify P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca²⁺]_i, P-selectin abundance, active α_IIbβ₃ integrin, annexin-V-binding, and caspase activity. The effects of thrombin on [Ca²⁺]_i, annexin-V-binding, cell volume, and caspase activity as well as the effects of CRP on [Ca²⁺]_i, P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, cell volume, and caspase activity were significantly augmented in the presence of bexarotene. Conclusions: Bexarotene sensitizes blood platelets for thrombin and/or CRP induced 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].…”