Extracellular methemoglobin primes red blood cell aggregation in malaria: An in vitro mechanistic study

► Methemoglobin exposure causes RBC aggregation. ► The high order aggregates are irreversible and resistant to physical disaggregation. ► Oxidative insult to RBC causes PS externalization to initiate and form aggregates. ► Antioxidant treatment especially inhibits high order RBC aggregate formation. ► Potentials of antioxidant to over-come RBC aggregation in malaria is discussed. Toxic byproducts from infected RBC cause rheological alteration and RBC aggregation. Malaria culture supernatant has the ability to exhibit RBC aggregation. Ammonium sulfate fractionation and immunodepletion of methemoglobin from culture supernatant confirms methemoglobin as a major aggregant. In vitro treatment of RBC with methemoglobin induces irreversible high order RBC aggregates, resistant to shear stress and physical forces. Methemoglobin-mediated ROS generation in the external micro-environment to develop oxidative stress close to RBC membrane seems to be responsible for initiating and forming high order RBC aggregates through phosphatidyl-serine externalization. Removal of oxidative stress through antioxidant treatment abolishes high order RBC aggregate formation. In conclusion, we discovered a novel pathway of methemoglobin-mediated RBC aggregation and its potential role in patho-physiological effects during malaria.