Examining phosphatidylethanolamine externalization on cells and microparticles in hemolysis and hemolytic anemia

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are aminophospholipids located primarily on the inside face of healthy human cell membranes. The overarching hypothesis of this proposal is that the aminophospholipid probe, duramycin, is a useful biological tool for examining PE exposure in hemolysis seen in diverse biological systems and models.;Tis dissertation demonstrated the overlap between calcium-phosphate precipitates and MPs in size when examined by flow cytometry. The microprecipitates bound non-specifically to fluorescently-labeled antibodies, further mimicking membrane vesicles. A solution to this problem was proffered by the addition of a calcium chelator. Also, contrary to the common practice of using the slowest possible cytometer flow rate while examining MPs, increasing the flow cytometer flow rate was shown to enhance detection of membrane vesicles.;We investigated the utility of duramycin, a PE-binding peptide, for the direct labeling and detection of MPs. Duramycin was also compared to other aminophospholipid probes in the labeling of RBCs, RBC-derived MPs, and MPs from numerous cellular sources. Duramycin was shown to be equivalent, if not superior in the case of cancer MPs, to labeling MPs at optimal conditions with other aminophospholipid probes.;Also detailed were the effects of dilution and centrifugation on MP concentrations in the resulting supernatant, suggesting that RBC MPs are much more abundant than previously thought. A novel approach to gently isolating MPs from their parent RBCs was used to characterize large vesicles (greater than 3im in diameter), suggesting there exists a size continuum between RBC fragments and RBC MPs in hemolysis. This discovery challenges the current practice of excluding RBC vesicles from analysis based on their size being larger than 1mum.;This dissertation also demonstrated the exposure of PE on RBC units over the duration of their storage. Previous studies using the calcium-dependent aminophospholipid probe, annexin V, demonstrated less than 1% to 6% of donor unit RBCs have deranged membrane bilayers at the time of expiration. Duramycin use demonstrated that nearly 18% of RBCs exposed appreciable PE at the time of expiration. This was roughly the same percentage of RBCs that are rapidly cleared after transfusion of blood at the end of its shelf-life, providing new insights into the possible mechanism of RBC clearance and transfusion reactions.;Also demonstrated, using duramycin to probe PE at the cellular level, was the damage done to RBCs following routine laboratory manipulations, including washing and centrifugation/resuspension. Washing RBCs from donor units near their expiration date resulted in significantly elevated PE exposure. Centrifugation alone damaged both aged and fresh RBCs sufficient to cause cell-free hemoglobin release. The results from this may have implication on the standard operating procedures in blood banks world-wide.;Lastly, radiolabeled duramycin was used to examine PE exposure on the tissue level in murine models of hemolytic anemia. Mice expressing human sickle hemoglobin were injected, and the areas of duramycin uptake examined using dual 3D imaging technologies: SPECT, to determine the radiotracer distribution, and microCT to co-localize anatomical locations. This provided a novel approach to objectively identifying ischemic injury in sickle cell disease. Additionally, mice exhibiting severe hereditary spherocytosis and spontaneous thrombosis were also examined as an alternative (nonsickling) hemolytic anemia model. Duramycin localized to areas of known thrombi, highlighting radiolabeled duramycin as a means to assess upstream occlusions (thrombi, as seen in the spherocytosis mice) or downstream ischemic damage (as demonstrated in the sickle cell mice). (Abstract shortened by UMI.).