The objectives of this proposal are to understand the structure-function relationships and molecular pathology of platelet septins, a family of cytoplasmic proteins involved in cellular events where dynamic membrane movements occur. Originally identified in yeast, the septin family extends from yeast to humans and has been associated with events ranging from cytokinesis to vesicle trafficking. In Preliminary Studies we characterize a prototypic human septin, termed CDCrel-1, expressed in brain, heart and megakaryocytes. Work from neurobiology labs has linked CDCrel-1 to the exocytic complex of neurons with implications that CDCrel-1 regulates neurotransmitter release. We have studied a mouse colony with a targeted deletion of the murine homologue to CDCrel-1. Most strikingly, platelets from CDCrel-1null animals aggregate and release 14C-serotonin in response to subthreshold levels of agonist compared to their wild-type littermates. Thus, an in vivo role for CDCrel-1 in regulating the platelet release reaction is established. All septins contain a conserved central core domain flanked by terminal ends unique to each protein. Relative to CDCrel-1, studies are proposed to examine the in vivo regulatory role of GTP and PtdIns(4,5)P2 binding motifs in the core domain (Aims 1 and 2) and examine the functional specificity provided by the NH2 and COOH termini (Aim 3). These aims will be achieved via an integrated approach of heterologous cell expression and transgenic models of septin expression. Preliminary studies have also identified additional platelet septins. We propose to examine the role of an uncharacterized platelet septin that interacts with CDCrel-1 (Aim 4) and define the complete repertoire of platelet septins (Aim 5). These studies will provide new information on the molecular events regulating platelet secretion and are directly relevant to mechanisms controlling hemostasis and thrombosis. These studies will also provide new insights on the causes of platelet secretion disorders and other aspects of megakaryocyte/platelet biology where active membrane movement is critical. Our results will be relevant to any number of disease processes where controlled secretion is relevant. A platelet model to examine septin function exploits the fundamental importance of secretion for the platelet response at sites of vascular injury.

R01HL069951

2002-04-01

2007-03-31