PS1519 DECREASED ACTIVITY AND STABILITY OF PYRUVATE KINASE IN SICKLE CELL DISEASE AND THALASSEMIA: A POTENTIAL TARGET FOR THERAPY
Authors: Rab M. , van Oirschot B. , van Straaten S. , Biemond B.J. , Bos J. , Kosinski P. , Kung C. , van Beers E. , van Wijk R. 
Published: June 2019

Background:Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of sickle cell disease (SCD) and thalassemia. In SCD, auto‐oxidation of red blood cell (RBC) hemoglobin produces high levels of ROS that exceed anti‐oxidant capacity. In β–thalassemia, auto‐oxidation of unpaired α‐globins produces high levels of ROS. Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's only source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in hemoglobinopathies impairs proper enzyme function and thereby compromizes RBC energy metabolism. This may contribute to disease pathophysiology.Aims:To investigate if secondary deficiency of PK is common in SCD and thalassemia, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG‐348.Methods:Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK thermal stability (residual activity of PK) in order to assess relative PK activity and enzyme stability. RBC lysates were incubated with AG‐348 (3.33 uM) for 2 hours after which PK thermal stability, PK activity and ATP response was measured. Red blood cells of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior (Rab et al, Am J Hematol, 2019).Results:Twenty‐eight patients and 18 healthy controls (HC) were included. The patient cohort consisted of patients with HbSS (n = 16), HbS/β‐thalassemia (n = 1), β‐thalassemia major (n = 3, regularly transfused), and HbSC (n = 3). Patients showed reticulocytosis and a concomitant increase in HK activity. However, PK activity was relatively decreased significantly compared to HK in SCD and β‐thalassemia patients, but not in HbSC patients (Figure 1A). PK thermal stability also was significantly decreased in SCD patients compared to healthy controls. In HbSC and β‐thalassemia patients PK‐thermal stability was comparable to HC (Figure 1B). When purified RBCs were incubated with 3.33uM of the allosteric PK‐activator AG‐348 an increase in PK activity was seen in all patients and HCs, with a median of 122% in SCD (range 120–138%, n = 3), 127% in β‐thalassemia and 142% in HC (range 130–166%, n = 5, Figure 1D). Accordingly, ATP‐levels increased in all patients and HCs, with a median of 126% in SCD (range 121–129%, n = 3), 146% in 1 patient with β‐thalassemia, and 122% in HCs (range 100–148%). In SCD patients PK‐thermal stability inversely correlated significantly with oxygenscan parameters, in particular the point‐of‐sickling (PoS). This indicates that RBCs tend to sickle at a higher pO2 when PK is more unstable. Interestingly, the 3 patients with most stable PK are HbSC patients (2) and a HbS/dβ‐thalassemia patient with HbF levels of 35% (Figure 1C).Summary/Conclusion:PK enzyme activity and stability is compromised in patients with SCD and β‐thalassemia. In SCD, reduced PK thermal stability is associated with higher PoS, which is associated with more severe disease. Current studies are in progress to further substantiate the underlying mechanism(s), and to investigate whether AG‐348 may ameliorate pathophysiological features such as hemolysis and sickling tendency.image