Platelet ?IIb?3 has been considered the prototypic integrin whose quintessential feature is its nearly instantaneous conversion from an inactive bent conformation on circulating platelets to an extended ligand binding conformation following vascular trauma. This global reorganization is initiated by platelet agonist-stimulated biochemical reactions that disrupt an intramolecular clasp composed of portions of the ?IIb and ?3 cytosolic, transmembrane, and extracellular stalk domains. Recent work suggests that ?IIb?3 is not representative of all integrins and that various integrins differ in the stringency of their regulation. Unlike ?IIb?3, some integrins may be constitutively active. Project 2 addresses topics related to the protein-protein interactions that maintain integrins in their basal states, intra-molecular interactions in Specific Aim 1 and inter- molecular interactions in Specific Aim 2.
In Specific Aim 1, intramolecular constraints located in the interface between the ?IIb and ?3 extracellular stalks will be identified using a novel structural bioinformatics method to predict interacting interfacial ?hot spots?. The relative importance of the predicted hot spots will then be determined by expressing hot spot mutants in CHO cells and in iPSC-derived human megakaryocytes produced in collaboration with Project 4. This experimental approach will then be used to compare ?IIb?3 to the integrins ?v?3, ?2?1, and ?v?8 and in collaboration with Project 1, to characterize the interaction between the PH and BEACH domains of Nbeal2 in studies designed to understand the pathogenesis of ? granule defect in the gray platelet syndrome. A second set of integrin constraints located in the transmembrane domain interface will be studied based on preliminary data indicating that ?3 uses different motifs to interact with ?IIb and ?v. Novel computational methods will then be used to derive two- dimensional kinetic parameters for these interactions in collaboration with Project 3. Lastly, we will use high-resolution cryo-electron microscopy to correlate our computational and experimental results with the global conformation of full-length integrins.
Specific Aim 2 will the identify and quantitively evaluate the protein-protein interactions responsible for ?IIb?3-mediated fibrin clot contraction. The studies are based on the observation that agonist stimulation causes platelet calpain activation and the degradation of platelet cytosolic proteins, in particular the proteins talin and vinculin that link ?IIb?3 to the actin cytoskeleton. Proposed studies will test the hypothesis that talin cleavage by calpain enables vinculin binding, thereby generating sufficient traction force to contract ?IIb?3-bound fibrin clots. This hypothesis will also be tested in vivo using mouse thrombosis models and calpain-deficient mice in collaboration with Project 3.
Project 2: Narrative Integrin-mediated cell-cell and cell-matrix adhesion is essential for many fundamental biologic processes and is an important contributor to human diseases such as thrombosis and cancer metastasis. Although the major platelet integrin ?IIb?3 has been a model for integrin function, its behavior may not be typical for integrins in general. The objective of this project is to compare the behavior of ?IIb?3 with that of the other integrins present in platelets to gain a more general understanding of integrin function, thereby enabling the future generation of more effective and safer anti-integrin therapeutics.