Currently, information about the assembly of the Gplb complex in megakaryocytes is fragmentary. Our hypothesis is that a number of coordinated processing events must occur during the terminal phases of megakaryocyte maturation that lead to the formation of a properly localized and functional Gplb complex. Megakaryocytes from patients with the Bernard Soulier syndrome are morphologically highly abnormal, and these patients suffer from thrombocytopenia in addition to the functional platelet disorder. Understanding the formation of the Gplb complex in normal and in Bernard Soulier megakaryocytes should provide new insight into megakaryopoiesis and megakaryocyte membrane assembly. These studies will be carried out in human megakaryocytes grown in liquid culture from the peripheral blood of normal volunteers as well as in megakaryocytes isolated from guinea pig bone marrow.
The aims of the proposal are to study the following potentially key events in the assembly of the Gplb complex in isolated guinea pig and human megakaryocytes at different phases of maturation prepared by the Celsep procedure: 1) To appraise the content of endogenous subunits of the Gplb complex and their surface orientation and association with the membrane skeleton: 2) To investigate the following: a) the expression of mRNA for subunits of the Gplb complex; b) the synthesis of these glycoproteins by metabolic labeling; c) the surface orientation and association with the cytoskeleton of newly-synthesized subunits of the Gplb complex: 3) To study the role of protein acylation in the assembly of the Gplb complex by: a) defining the relationship of the myristoylation and palmitoylation of subunits of the Gplb complex to megakaryocyte maturation; b) determining whether the acylation of subunits mediates the formation of the intact complex; c) determining whether the acylation of subunits mediates the surface orientation of the Gplb complex and its association with the membrane skeleton. To define the pathogenesis for the aberrant assembly of the platelet Gplb complex in the Bernard Soulier syndrome we will appraise these processes in megakaryocytes grown in liquid culture from the peripheral blood of patients with this disorder. We believe that this proposal fulfills the purpose of the RFA in that it will provide a better understanding of processes that appear to be vital for the development of megakaryocyte and platelet membranes.
