The Effect of Hypertonic Osmotic Stress and Its Duration on the Level and Activity of Executioner Caspase Enzymes in Blood Anuclear Cells (Platelets)

Abstract

Background: Caspases are protease enzymes critical for apoptosis. Caspase-3 and caspase-7, known as executioner caspases, are present in anuclear cells such as platelets. Platelet mitochondria can engage in the intrinsic pathway of apoptosis during stress and cellular damage. Hypertonic osmotic stress influences not only cell shape but also induces chemical changes within the cell. This study examines the function of executioner caspases in platelets under hypertonic osmotic stress due to the absence of a caspase-independent apoptosis pathway in these nucleus-free cells. Methods: Platelets were exposed to osmotic stress in a hypertonic environment (1.7% sodium chloride), with an isotonic environment (0.9% sodium chloride) serving as the control. Both environments were maintained at 37°C for durations of 15 minutes and 24 hours. Following this, platelets were lysed using ultrasound waves, and cellular membranes were removed by centrifugation. The supernatants were then used to measure caspase-3 level (ng/mL) using an ELISA method and caspase-3/7 activity (nmol/min/L) using a colorimetric method. Results: The study confirmed the presence of executioner caspases in platelets under non-stressed isotonic conditions. Following 15 minutes and 24 hours of hypertonic osmotic stress, caspase-3 level increased as determined by ELISA. However, caspase-3/7 activity initially decreased. With extended stress duration from 15 minutes to 24 hours, both the level and activity of these enzymes increased significantly (P-value < 0.05). Conclusions: Exposure to a hypertonic osmotic environment containing 1.7% sodium chloride increases caspase-3 level in anuclear cells such as platelets, while initially reducing caspase-3/7 activity. Prolonging the duration of stress to 24 hours enhances both the level and activity of these enzymes.