Voxelotor and Red Blood Cell Pyruvate Kinase Activator Affect Clot Strength in Sickle Cell Disease

Introduction:

Sickle cell disease (SCD) is a common genetic red blood cell (RBC) disorder that results from a single substitution of valine for glutamic acid in the β-globin gene, resulting in sickle hemoglobin (HbSS). Under hypoxia, HbS polymerizes and forms fibers within the RBCs, thus making the cells rigid, deformed, and sickle-shaped [1]. Deformed RBCs influence the compactness and dynamics of blood clots [2]. In addition, people with SCD are susceptible to thrombophilia [2,3,4]. Recently, there has been a heightened interest in ameliorating SCD using RBCs pyruvate kinase (PKR) activators, which increase ATP and decrease 2,3 DPG, resulting in increased oxygen affinity and improved RBCs deformability [5]. Voxelotor is also an oxygen affinity-modifying drug, recently FDA-approved. Voxelotor delays deoxy-HbSS formation, preventing in vitro HbSS polymerization and RBC sickling, thus improving RBC deformability [6]. The in vitro impact of PKR activators and Voxelotor on blood clotting in clinical samples from people with SCD has not been examined. Here we use a microfluidic dielectric sensor, termed ClotChip^® [7,8], to investigate the effects of Voxelotor and a PKR activator on the clotting time and clot strength of samples from people with SCD.

Methods:

Venous blood samples were collected in sodium-citrate from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. ClotChip^® microsensors were fabricated as previously described [7]. Blood samples were centrifuged at 200 g for 10 min. Plasma and buffy coats were removed and stored at 4 ^°C for 6 hours. Isolated, washed RBCs were then re-suspended in PBS at 20% hematocrit and then mixed with 67 mg/mL of Voxelotor (GBT440; Selleckchem) to a final concentration of 600 µM (in DMSO) or a PKR activator (PKR activator 3; MedChem Express) at 10 mM (in DMSO) and incubated at 37 ^°C for 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS containing 0.5% v/v DMSO and incubated at 37 ^°C for 6 hours. After incubation, HbSS+Vox, HbSS+PKR, and control samples were centrifuged at 500 g for 10 min to remove PBS. Each sample’s plasma was used to reconstitute RBCs at 20% hematocrit. CaCl₂ was added to the samples to induce coagulation, and the samples were injected into the ClotChip^® microfluidic sensors. An Agilent 4294A impedance analyzer was used to obtain the ClotChip^® readout curve defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz (see Figure 1A). Based on our previous studies [7,8], the time to reach a permittivity peak (T_peak) parameter was taken to indicate the clotting time, whereas the maximum change in permittivity after the peak (ΔƐ_r,max) parameter was taken to indicate the clot strength. Data are reported as mean ± standard deviation (SD).

Results:

We analyzed the ClotChip^® T_peak and ΔƐ_r,max readout parameters for HbSS (n=11), HbSS+Vox (n=11), HbSS+PKR (n=11), and HbAA control (n=8) samples (Figure 1B). The clot strength was ~50% higher in HbAA than HbSS (HbAA vs. HbSS: p= 0.001, Mann-Whitney test). However, when the SCD samples were treated with Voxelotor, the clot strength was observed to increase by ~10% (HbSS vs. HbSS+Vox, p=0.01, Paired t-test). Treatment with a PKR activator increased the clot strength by ~30% (HbSS vs. HbSS+PKR, p=0.003, Figure 1B). A significant difference was observed in the clotting time between HbAA vs. HbSS (p=0.001) but treatment with Voxelotor or a PKR activator did not change the clotting time for HbSS (HbSS vs. HbSS+Vox, p=0.43; HbSS vs. HbSS+ PKR, p=0.29) (Figure 1B). (p values were determined using a non-parametric Mann-Whitney test and paired t-test).

Discussion :

We found that treatment of HbS-containing RBCs with a PKR activator or voxelotor improves clot strength but not clotting time. PKR activators improved the strength of the clot significantly more than Voxelotor. Patients with SCD had a faster clotting time than healthy participants, which agrees with earlier researchers on hypercoagulability in SCD [4]. These data suggest that HbS polymerization and RBC health affect clot characteristics but not clot kinetics in SCD. Further investigations may be required to relate RBC deformability, which is well-known to impact SCD pathophysiology, to blood clotting and venous thromboembolism.