Pyruvate Kinase Function Correlates With Red Blood Cell Properties and Clinical Manifestations in Sickle Cell Disease

Abstract

Pyruvate kinase (PK) is a key enzyme involved in the final step of glycolysis, essential to produce adenosine triphosphate (ATP). Relatively decreased red blood cell (RBC) PK activity (reflected by a lower PK/hexokinase [HK] ratio) and PK thermostability (PK activity after exposure to heat) were recently identified as pathophysiological features of sickle cell disease (SCD). In this study, we investigated whether impaired PK function is associated with sickle RBC properties and SCD-related clinical manifestations. This study included 97 non-transfused patients with SCD (88 HbSS, 9 HbS/β0 thalassemia). PK thermostability was correlated with RBC parameters such as reticulocyte count (r = -0.402, p < 0.0001) and hemoglobin F (r = 0.394, p < 0.0001), and indicators of impaired functional properties of sickle RBCs, such as the point of sickling (r = -0.417, p < 0.0001), oxygen affinity (r = 0.408, p < 0.001) and RBC adhesion to laminin (r = -0.322, p = 0.024). Additionally, a low PK/HK ratio correlated with decreased PK thermostability (r = 0.308, p = 0.002), decreased RBC deformability (r = 0.268, p = 0.009), and elevated 2,3-diphosphoglycerate levels (r = -0.244, p = 0.016). Multivariate Poisson regression analysis demonstrated that reduced PK thermostability and PK/HK ratio were associated with a higher incidence of SCD-related clinical complications. For every 10-unit decrease in PK thermostability and 1-unit decrease in PK/HK ratio, the incidence of SCD-related clinical complications increased by 11% (p = 0.012) and 10% (p = 0.019), respectively. Altogether, these findings indicate that impaired PK function is related to compromised sickle RBC properties and SCD-related clinical manifestations. This supports the relevance and underlines the potential of PK activation therapy.

Keywords: pyruvate kinase; red cells metabolism; sickle cell disease.

FIGURE 1

Linear regression with Spearman correlation coefficient of PK thermostability measurements and laboratory parameters. PK thermostability, expressed as the residual PK activity after 60 min of incubation at 53°C, is significantly correlated with reticulocyte count, hemoglobin, dense RBCs, and hemoglobin F (HbF). Additionally, patients with low PK thermostability showed increased RBC adhesion to laminin, reduced oxygen affinity (reflected by a higher p50), a tendency to sickle at higher oxygen tensions upon deoxygenation (reflected by a higher point of sickling) and decreased deformability (reflected by lower EI_max and EI_min values).

FIGURE 2

Enzymatic measurements and oxygen gradient ektacytometry curves of RBCs after density separation. Bars represent median values with interquartile range. Only significant comparisons were shown. All other comparisons between fractions were nonsignificant. Enzyme activity measurements in Fraction 1 were not feasible due to an insufficient volume. (A) Percoll gradient density separation of five different patients with HbSS. Fraction 1 contains RBCs with the lowest density, and Fraction 4 contains RBCs with the highest density. The distribution of fractions differs between patients. (B) The mean corpuscular hemoglobin concentration (MCHC) increase reflects the increase in density. (C) Reticulocyte levels were the highest in the low dense fraction; however, these results were not statistically significant. (D, E) Enzymatic pyruvate kinase (PK) and hexokinase (HK) activity decreases with increasing density. (F) PK/HK ratio is significantly higher in Fraction 4 compared to the PK/HK ratio of the total RBCs. (G) PK thermostability is not significantly different compared to the total of all fractions. (H, I) RBCs with the highest density have the lowest deformability and sickle earlier upon deoxygenation. [Color figure can be viewed at wileyonlinelibrary.com]