Platelets are the most frequently transfused blood component. Therefore, learning how to stimulate endogenous platelet production through the control of megakaryocytopoiesis and how to better preserve platelets during storage in the blood bank are of major clinical importance. In this proposal we aim to clarify the role of the adhesive interactions of megakaryocytes and platelets with other vascular cells during platelet generation, storage, circulation and clearance. The adhesion receptors, whose function we will particularly scrutinize, belong to the selectin family of receptors whose ligands are specific carbohydrate, the beta/2 integrins and molecules of the immunoglobulin superfamily. We will take advantage of newly developed animal models deficient in a single adhesion receptor or double mutations. This proposal can be divided into three interrelated specific aims. In the first aim, we will evaluate the role of P-selectin in the development of the platelet storage lesion which may occur through interactions with contaminating leukocytes mediated by P- selectin. We will determine whether the expression of P-selectin induces clearance of activated platelets by examining the half-life of P-selectin- positive and negative platelets after transfusion. In the second aim, we will study the interaction of platelets and endothelium. Through intravital microscopy we have observed platelets rolling on the vessel wall, a behavior similar to that of leukocytes. We will examine what stimuli induce platelet rolling and which adhesive molecules are responsible for this interaction. In animal models of injury and vessel leakage we will investigate the biological function of platelet rolling. In the last aim, we will characterize the expression of P-selectin and other adhesion molecules by permanent human megakaryocytic cell lines and primary marrow megakaryocytes in response to inflammatory or physiological stimuli. The role of P-selectin and other adhesion molecules such as LFA- 1 in mediating megakaryocyte interactions with endothelial cells and marrow stroma cells will be investigated. Following these in vitro studies, we will investigate megakaryocytopoiesis in CD43, LFA-1, ICAM-2, and double-mutant P- and E-selectin null mice. In addition, we will explore regulation of protein tyrosine kinases and their substrates as an parameter of the platelet activation state during platelet storage. We believe that our studies should bring us closer to understanding the role of adhesion molecules in platelet generation and function in both hemostasis and inflammation, and in platelet clearance from circulation. This work may lead to the development of agents that will improve platelet survival during blood banking and novel therapeutics that may enhance or block platelet function depending on clinical needs.
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