Aspirin Induces Platelet Apoptosis

Abstract 5144

Aspirin is widely used in the treatment of a number of clinical conditions, such as fever, pain, strokes, and heart attacks. Although aspirin is being thought of a relatively “safety” medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. The effectiveness of aspirin has been attributed to its ability to inhibit prostaglandin production by inhibiting the cyclooxygenase (COX) enzyme. Interestingly, it has been reported recently that aspirin induces apoptosis in many cell types. Platelet apoptosis induced by either physical or chemical compounds or platelet storage occurs widely. We have reported that the glycoprotein Ibα-von Willebrand factor interaction, hyperthermia, and calmodulin antagonists induce platelet apoptosis, suggesting that platelet apoptosis might play important roles in controlling the number and function of circulated platelet under physiological conditions, or in the development of platelet-related hemorrhagic diseases. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. Washed platelets (3×10⁸/ml) were incubated with various concentrations of aspirin (2. 5 mM, 5 mM, 10 mM, 20 mM) at 37 °C for 2 hours. The effect of aspirin on platelet mitochondrial membrane potential (ΔΨm) depolarization and phosphatidylserine (PS) exposure were analyzed by cell-permeable lipophilic cationic dye tetramethylrhodamine ethyl ester (TMRE) and annexin V binding, respectively. The data show that ΔΨms depolarization and PS exposures were dose-dependently induced by aspirin in platelets. In order to further confirm that aspirin incurs platelet apoptosis, caspase-3 activation was measured in platelets incubated with aspirin by detecting 32 kD pro-caspase-3 and 17 kD cleaved caspase-3 fragment, and the result shows that aspirin induced caspase-3 activation. Furthermore, platelet shrinkage appeared in platelets incubated with aspirin (20 mM). Caspase inhibitor z-VAD-fmk inhibited apoptotic platelet shrinkage, and blocked ΔΨm depolarization, but had no effect on PS exposure, suggesting that caspase-3 acts upstream of ΔΨm depolarization, and plays a key role in aspirin-induced platelet apoptosis. Whereas, protein kinase C (PKC), protein kinase B (PKB), p38 mitogen-activated protein kinases (MAPK), protein 53 (p53), reactive oxygen species (ROS), and mitogen-activated protein kinase kinase (MAPKK) were not involved in aspirin-induced platelet apoptosis. In addition, platelets incubated with another COX inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure in platelets. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation, and independent of the COX signaling.