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561 Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Program: Oral and Poster Abstracts
Type: Oral
Session: 113. Sickle Cell Disease, Sickle Cell Trait and Other Hemoglobinopathies, Excluding Thalassemias: Basic and Translational: Pathophysiology of Sickle Hemoglobinopathies: From Mice to Humans
Hematology Disease Topics & Pathways:
Research, Sickle Cell Disease, Translational Research, Hemoglobinopathies, Diseases
Sunday, December 10, 2023: 5:00 PM

Marissa J.M. Traets1*, Sigrid van der Veen2*, Jennifer Bos1*, Laura van Pelt1*, Aida Kidane, MD3*, Brigitte A. van Oirschot1*, Wouter W. van Solinge1*, Saskia Schols4*, Mandy N. Lauw5, Marjon H. Cnossen3*, Erfan Nur, MD, PhD6,7, Bart J. Biemond8, Eduard J. Van Beers2*, Anita W. Rijneveld, M.D.5, Richard van Wijk1 and Minke A.E. Rab1,5*

1Central Diagnostic Laboratory - Research, University Medical Center Utrecht, Utrecht, Netherlands
2Center for Benign Hematology, Thrombosis and Hemostasis - Van Creveldkliniek, University Medical Center Utrecht, Utrecht, Netherlands
3Department of Pediatric Hematology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
4Department of Hematology, Radboud university medical center, Nijmegen, Netherlands
5Department of Hematology, Erasmus University Medical Center, Rotterdam, Netherlands
6Department of Hematology, Amsterdam University Medical Centers, Amsterdam, Netherlands
7Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands
8Department of Hematology, Amsterdam University Medical Centers, location University of Amsterdam, Amsterdam, Netherlands

Introduction: Sickle cell disease (SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell’s structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology.

Methods: Homozygous HbS (HbSS) and HbS/β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (<3 months) were excluded. Dense RBCs (MCHC >41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson’s correlation).

Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p<0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated.

Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

Disclosures: Kidane: CSL Behring: Research Funding. Schols: Bayer: Research Funding. Cnossen: NWO, NWA, ZonMW, the Dutch Innovatiefonds Zorgverzekeraars, Stichting Haemophilia, Baxter/Baxalta/Shire/Takeda, Pfizer, Bayer Schering Pharma, CSL Behring, Sobi Biogen, Novo Nordisk, Novartis, Roche, and Nordic Pharma: Research Funding; Roche, Bayer and Novartis: Membership on an entity's Board of Directors or advisory committees. Nur: Amgen: Consultancy, Speakers Bureau; VERTEX: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Biemond: BMS: Research Funding; Sanquin: Research Funding; Novo Nordisk: Other: Advisory board; Novartis: Other: Advisory board, Research Funding; Celgene: Other: Advisory board; Global Blood Therapeutics/Pfizer: Other: Advisory board, Research Funding; CSL Behring: Other: Advisory board. Van Beers: Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Pfizer: Consultancy. Rijneveld: BMS: Honoraria; Servier: Honoraria. van Wijk: Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Pfizer, Inc.: Consultancy, Research Funding; RR Mechatronics: Research Funding; Axcella Therapeutics, Inc.: Research Funding. Rab: Agios Pharmaceuticals: Research Funding; Pfizer: Research Funding.

*signifies non-member of ASH