Abstract During pathophysiological conditions characterized by extensive hemolysis (e.g. acquired and genetic hemolytic diseases), free heme and cell-free hemoglobin (Hb) are released into the blood stream and elicit a variety of adverse effects, namely: vasoconstriction, hypertension, and end organ damage. Thus treatment of hemolytic conditions would benefit from scavengers of free heme and cell-free Hb such as hemopexin (Hpx) and haptoglobin (Hp), respectively. A possible functional alternative to Hpx is apohemoglobin (apoHb). ApoHb is derived by removing heme from Hb, and its vacant heme-binding pockets have a high affinity for heme. Hence, apoHb could serve as a novel in vivo heme scavenger instead of Hpx. However, major potential issues with the use of apoHb as an in vivo heme scavenger are its low thermal stability at physiological temperature, and short circulatory half-life (similar to Hb, 5 min). Fortuitously, previous studies have shown that, similar to Hb, apoHb can bind to Hp forming a highly stable complex. The apoHb-Hp complex retains its ability to bind heme, and is more stable at physiological temperature compared to free apoHb. In addition to being able to bind free heme, the apoHb-Hp complex can scavenge free Hb by exchanging Hp bound apoHb αβ dimers for Hb αβ dimers. Therefore, we hypothesize that the apoHb-Hp complex will have the dual ability to bind and detoxify free heme and Hb that are produced during states of hemolysis. The resulting Hb-Hp complex is much less toxic than free heme or cell-free Hb and is readily cleared from circulation via CD163 mediated monocyte/macrophage uptake. To test this hypothesis, we propose the following specific aims. Specific Aim 1: Biophysical and biochemical characterization of the apoHb-Hp complex and its ability to bind heme and Hb in vitro. Specific Aim 2: In vivo determination of heme and Hb transfer and binding to apoHb-Hp. Specific Aim 3: Systematic pre-clinical evaluation of apoHb-Hp to prevent and/or halt the progression of intravascular hemolysis (Hb/heme)-induced pulmonary cardiovascular disease. Specific Aim 4: Microvascular evaluation of apoHb-Hp to prevent vaso-occlusion and reduce vascular inflammation.