Preexisting proteins are activated by x-irradiation and contribute to tissue repair and/or tissue injury. These proteins are sequestered in storage reservoirs, and where they are available for rapid dispensing to accomplish hemostasis and leukocyte activation. Weibel Palade bodies (WPB) within the cytoplasm of endothelial cells translocate to the vascular lumen following irradiation of blood vessels. We utilized immunohistochemistry to study the histologic pattern of WPB components and observed that several of these proteins translocate to the vascular lumen in response to ionizing radiation. These proteins included CD62P (P-selectin), CD63, Interleukin-8, and von Willebrand factor (vWF). We have initiated our studies of the biological consequences of WPB exocytosis by use of the P-selectin knockout mouse. We observed two phenotypes in the P-selectin knockout mouse. We observed our studies of the biological consequences of WPB exocytosis by use of the P-selectin knockout mouse. We observed two phenotypes in the P-selectin -/-, which include edema and hemorrhage into the irradiated intestine and lung. Secondly, the absence of absence of platelet aggregation in P-selectin knockouts, whereas aggregation occurred immediately following x- irradiation in wild type mice. We will study physiological mechanisms of these phenotypes in Aims 3 and 4 of this proposal. We will determine whether P-selectin within platelets within platelets is sufficient to selectin -/- mice will revert these phenotypes. We will determine whether bone marrow transplantation from P-selectin within platelets is sufficient to restore the wild type phenotype in these knockouts. We will also transplant bone marrow from P-selectin -/- mice into wild type mice to determine whether platelet P-selectin is required to prevent edema and hemorrhage in irradiated tissues. The mechanisms by which ionizing radiation induces exocytosis of storage reservoirs is through membrane transport. Using immunofluorescence confocal microscopy, we observed that P-selectin, CD63, and vWF are translocated to the cell membrane following irradiation of endothelial cells. P-selected remains tethered to the cell membrane, whereas vWF is secreted into medium or vascular lumen. Membrane transport requires motor proteins that move organelles over the cytoskeleton. We observe that microtubule depolymerizing agents inhibit exocytosis, whereas the antagonist of actin (cytochalazin-B) did not prevent x-ray induced exocytosis of WPB. Calcium-dependent signal transduction has been proposed to initiate exocytosis of storage reservoirs. We utilize the intracellular chelator BAPTA to inhibited x-ray-induced translocation of WPB. Calmodulin is a protein that is activated by increased intracellular calcium and participates in the activation of membrane transport. The calmodulin inhibitor W13 abrogated the translocation of WPB to the cell membrane following irradiation. We will study the roles of calcium and calmodulin in the activation of motor proteins that are required for microtubule- dependent translocation of WPB to the cell membrane.
