A Novel Hemoglobin-Binding Agent Reduces Plasma Free Hemoglobin and Partially Improves Vascular Function In Murine Hemolytic Anemia

Abstract 267

Humans with sickle cell disease (SCD) and hereditary spherocytosis (HS) have been shown to have increased plasma free hemoglobin, vascular dysfunction, and pulmonary hypertension. Increased plasma free hemoglobin (Hb) levels are associated with multiple physiologic findings, including hemoglobinuria, increased blood pressure, platelet activation, and increased mortality. Previously, we showed that SCD and HS mice have impaired systemic vasodilation and mild pulmonary hypertension. Furthermore, there is evidence of increased plasma free hemoglobin and plasma oxidizing potential in mice with SCD and HS, and plasma oxidizing potential correlates with plasma free hemoglobin levels. We hypothesize that increased plasma free Hb impairs systemic vasodilation and increases pulmonary hypertension in SCD and HS mice. To test this hypothesis, we developed a peptide (hE-Hb-B10) that specifically binds human hemoglobin and removes it from the plasma via the heparan sulfate proteoglycan (HSPG)-associated lipoprotein pathway, rather than the haptoglobin-Hb-CD163 receptor pathway. SCD, HS, and control mice were treated with daily intraperitoneal (ip) injections of PBS or peptide hE-Hb-B10 dissolved in PBS (10-20 mg/mouse/day). At the end of three weeks of treatment, anesthetized mice were phlebotomized, as well as utilized for either ex vivo vasodilation studies using the facialis artery (a distal branch of the carotid artery) or assessment of right ventricular systolic pressure (RVsP) as a surrogate measure of pulmonary hypertension. In addition, plasma samples from the mice were analyzed for plasma free hemoglobin (PFH), lactate dehydrogenase (LDH, a marker of hemolysis), and oxidizing potential. PFH, LDH and oxidizing potential were significantly increased in the plasma of PBS-treated SCD and HS mice as compared with levels in control mice. Treatment with hE-Hb-B10 significantly reduced PFH levels in both SCD and HS mice compared to PFH levels in PBS-treated SCD and HS mice. In contrast, hE-Hb-B10 treatment did not reduce oxidizing potential in SCD and HS mice, suggesting that factors other than PFH contribute to the increased oxidizing potential of plasma in SCD and HS mice. hE-Hb-B10 treatment did not alter hematocrit, reticulocyte count, or plasma LDH levels in SCD and HS mice, suggesting that the overall hemolytic rate was unaltered by peptide treatment. Plasma from control mice had low levels of PFH, LDH and oxidizing potential; these levels did not change with hE-Hb-B10 treatment. Finally, facialis artery vasodilation in response to acetylcholine was significantly improved in hE-Hb-B10 treated as compared to PBS-treated SCD and HS mice. However, pulmonary hypertension, as measured by RVsP, in SCD mice was not altered by treatment with hE-Hb-B10, while hE-Hb-B10-treated HS mice showed mild improvement. These data demonstrate that hE-Hb-B10 effectively reduces plasma free hemoglobin in SCD and HS mice. Although reducing PFH improves vascular function in a systemic vascular bed (facialis artery), we found minimal beneficial effects in the pulmonary vascular bed (as measured by RVsP). Our findings confirm that the mechanisms impairing vascular function in SCD and HS are complex, and suggest that targeting only one mechanism (i.e., plasma free Hb) may be insufficient to achieve notable improvements in vascular function in these complex disease states.