Distinct Biomarkers Reflect Pathophysiological Differences of Sickle Cell Disease Sub-Phenotypes: Viscosity-Vaso-Occlusion Vs. Hemolysis-Endothelial Dysfunction

Background: Patients with Sickle Cell Disease (SCD) have variable phenotypes, with different severity of pain and other symptoms, including lung injury, stroke, leg ulcers, renal injury with proteinuria, osteonecrosis, and systemic and pulmonary hypertension. These phenotypes result from erythrocyte injury caused by HbS and its deoxygenation-induced polymerization. Recently, two distinct sub-phenotypes have been defined: a) Patients with the Viscosity-Vaso-Occlusion sub-phenotype (VVO) suffer mainly from vaso-occlusive pain crises with a relatively high hemoglobin concentration and b) patients classified as the Hemolysis-Endothelial Dysfunction sub-phenotype (HED) suffer from stroke and pulmonary hypertension with an elevated concentration of LDH (Kato et al. J Clin Invest 2017;127:750).

Aim: In this context, we aimed to explore the correlation of key biomarkers with the two sub-phenotypes of the disease, namely: Placental growth factor (PlGF) a member of the vascular endothelial growth factor superfamily, which plays an important role in both inflammation and neo-angiogenesis; von Willebrand Factor antigen (vWF:antigen), a multimeric plasma glycoprotein secreted by the endothelium; Growth Differentiation Factor-15 (GDF-15), a member of the Transforming Growth Factor-beta superfamily, which expression is strongly upregulated in response to oxidative stress, inflammation and tissue injury, as well as in conditions related to ineffective erythropoiesis. We tested these biomarkers in patients with compound heterozygous SCD and beta-thalassemia (HbS/βthal).

Patients and Methods: Ninety adult Caucasian patients with HbS/βthal were included in the study, while 20 apparently healthy individuals, of similar age and gender, served as controls. Patients with HbS/βthal were divided in two groups according to their LDH levels: High-LDH (LDH>270U/L) (HED-phenotype) group (42 patients) and the Normal-LDH (LDH<270U/L) (VVO-phenotype) group (48 patients). Along with hematologic and blood chemistry parameters determination, measurements of circulating levels of PlGF, vWF:antigen, GDF-15, hs-CRP, Cystatin C, hs-TnT and D-Dimers were performed in both groups of patients and controls using RUO and IVD immunoenzymatic techniques.

Results: We found that patients with the HED-phenotype compared to the VVO-phenotype of the disease had lower Hb levels (p<0.001), higher Reticulocyte Production Index and higher serum bilirubin (p<0.001 and p=0.004, respectively), while there were no differences regarding Hb F levels between the two groups. PlGF levels were significantly elevated only in patients with the HED-phenotype (22.6±7.1pg/mL) compared to the controls (15.2±2.4pg/mL) (p<0.001) and patients with the VVO-phenotype (18.2±7.6pg/mL) (p=0.005). vWF:antigen concentrations were markedly elevated in both groups of patients compared to controls (186.4±81.7 and 157.8±73.4 vs 85.3±22.1IU/dL, p<0.001), with the increase of vWF:antigen levels to be more pronounced in patients with the HED-phenotype (p=0.008). Similarly, GDF-15 levels were also markedly elevated in both groups of patients compared to controls (2346.2±1295.6 and 1693.5±1398.3 vs 665.4±221.9pg/mL; p<0.001), with the increase of GDF-15 levels to be more pronounced in patients with the HED-phenotype (p=0.006). Regarding other parameters tested we found significant higher levels of D-Dimers in patients with the HED-phenotype (p<0.001) compared to patients with the VVO-phenotype, while no differences were found in parameters of inflammation and renal function.

Conclusions: These findings demonstrate for the first time in the literature the correlation and involvement of PlGF, vWF:antigen and GDF-15 proteins in the pathophysiological mechanisms of the Hemolysis-Endothelial Dysfunction sub-phenotype of SCD in patients with the HbS/βthal genotype. Although, there is a degree of overlapping between the two sub-phenotypes of SCD, the differences in the specific biomarkers were significant. Thus, these markers along with the clinical profile could better identify the two subtypes of SCD patients and drive an innovative approach with the use of direct personalized therapies for each specific sub-phenotype by targeting the predominant mechanism in this multifactorial disorder.