Possible Role Of Circulating Adenine Nucleotides and Platelet Activation In The Pathogenesis Of Sickle Cell Disease: Observations In a Murine Model

Vaso-occlusive crisis (VOC) is common in patients with sickle cell disease (SCD). Although hemolysis of erythrocytes has been associated with VOC, the underlying mechanism remains unclear. In the present study, we conducted pathophysiological analyses in Berkeley SCD mice to examine the possible role of adenine nucleotides and platelet activation in SCD. Berkeley SCD mice are genetically modified mice whose murine α-globin and β-globin genes have been deleted, and replaced with human α-, γ-, and δ-globins and sickle β-globin. Control mice were heterozygous littermates expressing normal β-globin.

Histological analyses revealed congestion of blood vessels in the liver vasculature and renal medulla of 7-week-old SCD mice associated with aggregated sickle-shaped erythrocytes. Further analysis revealed severe hemolytic anemia in these 7-week-old SCD mice. Plasma adenine nucleotide measurements using LC-MS/MS showed that the concentrations of ADP in 7-week-old SCD mice were significantly higher (∼2.7-fold increase) compared with age-matched control mice. These findings suggest that the intravascular hemolysis of sickled erythrocytes and/or congestion in various organs results in the elevation of circulating concentrations of ADP in this SCD model.

Platelet function was assessed by flow-cytometric analysis of platelet-surface activated GPIIbIIIa and P-selectin. These biomarkers of platelet activation were determined in vehicle-treated control mice, vehicle-treated SCD mice, and prasugrel (1 mg/kg/day)-treated SCD mice. Vehicle or prasugrel were orally administered to 7-week-old mice for 1 week and 8 weeks. The expression of activated GPIIbIIIa on platelets was assessed with or without 20 μM ADP stimulation. The expression of platelet P-selectin was assessed with or without 300 μM PAR4 TRAP. These platelet function analyses showed that circulating platelets in SCD mice were activated compared to control mice; the basal expression of activated GPIIbIIIa on platelets in 8- and 15-week-old SCD mice and P-selectin on platelets in 15-week-old SCD mice were increased significantly compared with those in control mice. In vitro stimulation with ADP and PAR4 TRAP increased the expression of activated GPIIbIIIa and P-selectin on platelets in SCD mice. Of note, agonist-stimulated activation was enhanced with age. In 8-week-old mice, the extent of agonist-induced activation of GPIIbIIIa and P-selectin in SCD mice was equivalent to that in control mice; however, in 15-week-old mice, the extent of agonist-induced activation of platelet GPIIbIIIa and P-selectin in SCD mice was significantly higher compared with that in age matched control mice. Prasugrel treatment for 1 week and 8 weeks did not result in significant inhibition of basal expression of activated GPIIbIIIa or P-selectin on circulating platelets in SCD mice, suggesting P2Y₁₂ independent activation of circulating platelets. In contrast, prasugrel treatment for 1 week and 8 weeks did result in highly significant inhibition of ADP-induced GPIIbIIIa activation on SCD mouse platelets. PAR4 TRAP-induced P-selectin expression on SCD mouse platelets was also inhibited by prasugrel.

In conclusion, in a murine model of SCD, the present study found significant elevation of circulating ADP possibly resulting from the in vivo hemolysis of sickled erythrocytes. However, P2Y₁₂ inhibition by prasugrel did not appear to reduce the activation of circulating platelets in this SCD model. In contrast, ex vivo stimulation of SCD mouse platelets by the ex vivo addition of ADP and PAR4 TRAP was effectively inhibited by prasugrel administration. The ex vivo addition of ADP may mimic increased exposure to ADP in vivo during VOC thus suggesting a role for prasugrel in the treatment of SCD.