Platelets provide for primary hemostasis by aggregating at sites of vascular injury. Moreover, platelet aggregation on abnormal vessels is responsible for the thrombosis characteristic of arterial vascular disease. Fibrinogen and von Willebrand factor binding to glycoprotein IIb- IIIa (GPIIb-IIIa) complexes on agonist-stimulated platelets is a prerequisite for platelet aggregation. However, the nature of the intracellular processes that activate GPIIb-IIIa to expose its ligand binding sites is not known, but they likely do so by interacting with one or both of its cytoplasmic domains. We have developed an in vitro expression system using Epstein-Barr virus transformed human B lymphocytes in which recombinant GPIIb-IIIa can be induced to interact specifically with fibrinogen by stimulating the lymphocytes with phorbol esters. Consequently, this expression system permits the use of molecular techniques to study the process of GPIIb-IIIa activation.
In Specific Aim 1 of this project, we will use in vitro mutagenesis to examine the structural features of the cytoplasmic and transmembrane domains of GPIIb and GPIIIa that contribute to GPIIb-IIIa activation. We will also explore the similarities and differences in the signaling pathways required to activate endogenous lymphocyte beta2 integrins and recombinant GPIIb-IIIa to provide clues as to the nature of the intracellular signaling pathways that might be operative in platelets.
In Specific Aim 2, we will use in vitro mutagenesis to identify the domains of the extracellular portion of GPIIb-IIIa that are involved in ligand binding and in the conformational change that accompanies GPIIb-IIIa activation. These studies will make use of the observation that BiP, a molecular chaperone that resides in the endoplasmic reticulum (ER), interacts with a limited number of sites on nascent GPIIb and GPIIIa. Because BiP binding bites are predominantly hydrophobic, they will either be folded into the interior of mature GPIIb and GPIIIa or reside at the interface between subunits. The latter are of particular interest because they may be involved in the allosteric change that accompanies GPIIb-IIIa activation.
In Specific Aim 3, we will examine the role of GPIIb cleavage in GPIIb-IIIa function. GPIIb is synthesized as a single chain precursor that undergoes cleavage into disulfide-linked heavy and light chains in a post-ER compartment. The functional consequences of this post-translational modification of GPIIb are not known. We will characterize the enzyme in megakaryocytes responsible for GPIIb cleavage and examine the consequences of this cleavage on GPIIb-IIIa activation using our lymphocyte model. The information gained from these studies will add to our understanding of normal hemostasis and will aid in the design of rational therapy for arterial vascular disease. Moreover, this information will provide a paradigm for a general understanding of the relationship of integrin structure and function.
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| Loh, E; Qi, W; Vilaire, G et al. (1996) Effect of cytoplasmic domain mutations on the agonist-stimulated ligand binding activity of the platelet integrin alphaIIbbeta3. J Biol Chem 271:30233-41 |
