In vitro determination of apoptotic effect of heparin on lymphoblasts by using flow cytometric DNA analysis and measurements of caspase-9 activation and cytochrome C level.

Heparin induces apoptosis on peripheral neutrophils, mononuclear cells of the healthy controls, and on lymphoblasts of the patients with acute lymphoblastic leukemia, in vitro. We studied the caspase-9 activity and cytochrome C level as the indicators of the apoptotic effect of heparin on lymphoblasts by the intrinsic pathway of apoptosis. Twenty samples of the patients with acute lymphoblastic leukemia were included in the study. Cytochrome C level and caspase-9 activity were concomitantly determined with the percentage of apoptotic lymphoblasts when incubated in 0, 10, and 20 U/mL heparin concentrations at 0, 1, and 2 hours. The percentages of apoptosis of lymphoblasts at the first hour were higher than those at 0 and 2 hours in 10 and 20 U/mL heparin concentrations, separately (P<0.05). The mean percentage of apoptosis of lymphoblasts in 20 U/mL heparin levels was significantly higher than those in 0 and 10 U/mL heparin levels at 1 and 2 hours (P<0.05). The highest apoptotic effect of heparin on lymphoblasts was determined at the first hour in 20 U/mL heparin concentration. The mean caspase-9 activitity at the first hour was significantly higher than the values at 0 and 2 hours in 10 and 20 U/mL heparin levels, separately (P<0.05). The mean caspase-9 activity in 20 U/mL heparin concentration was significantly higher than values in 0 and 10 U/mL heparin concentrations at 1 and 2 hours (P<0.05). The highest caspase-9 activity was determined in 20 U/mL heparin levels at the first hour. The mean cytochrome C level at the first hour was significantly higher than those at 0 and 2 hours in 10 and 20 U/mL heparin concentrations, separately (P<0.05). The highest cytochrome C level was determined in 20 U/mL heparin concentration at the first hour. We claimed that heparin induces the apoptosis of lymphoblasts by the activation of the intrinsic pathway.