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.
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.
