Regulation of Platelet Glycoprotein (GP) Ib-IX Complex Function by Membrane Lipid

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DESCRIPTION (provided by applicant): The formation of platelet thrombi at shear stress is initiated by the binding of the platelet receptor, glycoprotein (GP) Ib-IX complex, to its ligand, von Willebrand factor (VWF). This receptor-ligand interaction is essential for tethering platelets to the injured vessel wall as a prerequisite for integrin-mediated firm arrest.Amalfunction in this interaction causes either Bernard-Soulier Syndrome (BSS) or platelet-type von Willebrand disease (VWD). It has long been thought that the GP Ib-IX complex/VWF interaction only provided the physical force to decelerate the flowing platelets. Recently, however, we have begun to realize that upon interacting with VWF, the GP Ib- IX complex can initiate transmembrane signaling events for integrin activation, leading to platelet firm adhesion and aggregation. Signaling molecules, such as Src family kinase, 14-3-3> and PI-3-Kinase, through association with the cytoplasmic domains of individual polypeptides, mediate these events. Lipid domains, also known as glycosphingolipid-enriched membranes (GEMs), can act as a platform for the assembly of downstream signaling molecules of the GP Ib-IX complex. Dissociation of the GP Ib-IX complex from the GEMs by membrane cholesterol depletion abolishes platelet activation and adhesion to VWF. Nevertheless, basic inquiries as to what the structural elements of the GP Ib-IX complex for GEMs association are, how such interaction is regulated, and what the physiological relevance of GEMs association in the GP Ib-IX function is, have never been answered. Our preliminary data demonstrate that GP Ib1 association with the GEMs domain is primarily mediated by GP Ib2/GP IX. Removal of disulfide linkage between GP Ib1 and GP Ib2/GP IX not only inhibits GP Ib1 association with GEMs domain, but also inhibits GP Ib-IX complex-expressing CHO (Chinese Hamster Ovary) cells interaction with VWF under high shear. In addition, we found that protein disulfide isomerase (PDI) associates with the GP Ib-IX complex in both platelets and the GP Ib-IX complex-expressing CHO cells, an interaction only being seen in GEMs domain. Furthermore, we demonstrated that alteration of platelet membrane lipid composition inhibits both GP Ib-IX complex association with GEMs domain and complex- mediated platelet interaction with VWF. Based on these results and previously reported evidence, we hypothesize that 1) GP Ib2/GP IX contains the structural determinants to mediate the GEMs interaction with the GP Ib-IX complex; 2) redox regulation by protein disulfide isomerase plays a role in the formation of disulfide linkage between GP Ib1 and GP Ib2/GP IX for GP Ib1 association with GEMs domain; 3) specific GEMs lipid composition is critical for complex association; and 4) specific disruption of GP Ib1 association with GEMs domain abolishes GP Ib-IX complex function. Overall, this proposed study will help to elucidate the structural basis for the proper localization of the GP Ib-IX complex on platelet surface, explore a novel mechanism for GP Ib-IX complex function regulation, and finally, to provide a mechanistic guide for developing novel therapeutic strategies to combat various complex related bleeding disorders and cardiovascular disease. PUBLIC HEALTH RELEVANCE: Platelet membrane lipid domain is important for platelet function in thrombosis and haemostasis. The platelet glycoprotein GP Ib-IX complex is one of the proteins regulated by membrane lipid domain. In this project, we sight to investigate this regulatory mechanism.