Abstract Sun1003: A recombinant protein oxygen carrier with reduced nitric oxide scavenging as a blood substitute for resuscitation

Abstract

Background: Hemorrhagic shock remains a leading cause of preventable death. No artificial oxygen carrier based on hemoglobin or hemoproteins has been approved in the US as an alternative to donated blood. A major challenge for the hemoprotein oxygen carrier is the vasoconstrictive effects due to the nitric oxide (NO) scavenging. We have engineered a modified Regulator of Carbon Monoxide Metabolism (RcoM) heme-containing protein which shows no vasoconstrictive effects. We aim to validate the oxygen binding, NO dioxygenation rate, stability, and resuscitative effects of RcoM. Hypothesis: We hypothesize that RcoM is well tolerated and can behave as an artificial oxygen carrier due to ideal oxygen binding rate, low autoxidation rate, slow NO dioxygenation rate, and high thermal/chemical stability. Approach: We assessed biochemical features using UV-visible spectroscopy methods to determine the transitions between RcoM oxidation state and ligand binding and dissociation. We performed measures of oxygen binding and autoxidation. We carried out competition experiments in which equimolar amounts of RcoM and Hb were exposed to NO to measure NO deoxygenation rate. To assess oxygen carrier capabilities of Rcom, we used a murine model of traumatic, hemorrhagic shock utilizing anesthetized mice with a 2cm abdominal incision and left femoral artery and vein catheterization. Hemorrhage was induced by a 30-minute controlled bleeding to a target blood pressure of 25 to 40 mmHg, shock was maintained for 30 minutes, then infusion by lactated Ringer (LR) solution, 2mM stroma-free Hb, or 2mM RcoM at volume equal to removed blood and monitored for 30 minutes. Mean arterial pressure post resuscitation were compared to assess the tolerance and hemodynamics effects. Results: RcoM displays appropriate oxygen binding capacity and has an NO deoxygenation rate ten-fold lower than Hb. In the murine hemorrhagic model, there was no difference for the baseline MAP or in blood removed during the bleeding phase. Post-resuscitation MAP was significantly higher for RcoM (67.0 ± 6.3 mmHg) and Hb (60.4 ± 16.0 mmHg) compared to LR (25.9 ± 21.4 mmHg, p = 0.0029). There was no difference between Rcom and Hb in the post-resuscitation MAP ( p = 0.8573). Conclusion: This study demonstrates that resuscitation with RcoM supports the blood pressure in a murine traumatic hemorrhagic shock model, suggesting its potential application as a non-globin artificial oxygen carrier for hemorrhagic shock.