Mechanisms of platelet stimulation in hemolysis

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ABSTRACT While sickle cell disease (SCD) is instigated by a mutant hemoglobin that polymerizes in hypoxia to cause hemolysis and vaso-occlusion, inflammation plays a fundamental role in propagating this pathogenesis. SCD patients are at significantly higher risk of thrombosis, which contributes to severe life-threatening complications such as pulmonary hypertension and stroke. On a cellular level, it is well established that in SCD patients, platelet activation is elevated and correlates with hemolysis. However, the contribution of inflammatory signaling to platelet activation and its cross-talk with hemolysis remain unknown. High-mobility group box 1 (HMGB1), an inflammatory mediator released by cells, has been shown to be elevated in the blood of SCD patients and to stimulate platelet activation. Notably, the ability of HMGB1 to mediate platelet activation is dependent on the oxidation state of critical cysteine residues in the protein. Preliminary data shows that HMGB1-mediated platelet activation is significantly enhanced in the presence of cell-free hemoglobin (Hb), a major component of hemolysis. Further, Hb and HMGB1 each independently increase platelet mitochondrial reactive oxygen species (mtROS) generation, a known stimulus for platelet activation. Based on these data, I hypothesize that in SCD, Hb oxidizes HMGB1 to increase platelet mtROS production leading to platelet activation and secondary release of additional thrombotic stimulants including HMGB1 from platelets. Aim 1 will determine whether Hb oxidizes HMGB1 to potentiate its activation of platelets using human healthy and sickle Hb and platelets in ex vivo experimental models. Aim 2 will determine the mechanism by which HMGB1-induced mtROS stimulates platelet activation. Aim 3 will utilize murine models of hemolysis to determine whether neutralizing HMGB1 attenuates platelet activation and thrombus formation. Successful completion of these aims will elucidate a novel mechanism of synergy between hemolysis and inflammatory signaling in potentiating thrombosis. This work will reveal novel therapeutic targets and potential therapeutics for the treatment of thrombosis in SCD. In addition, this project, along with the guidance of my multi-disciplinary mentorship team, career development plan, and cutting-edge research environment at the Vascular Medicine Institute at the University of Pittsburgh, will serve as a strong training vehicle to facilitate my independence as a researcher focused on mechanisms of thrombosis in hemolytic disease.