Platelets play a critical role in the development of blood clots. This is an important physiological event in normal hemostasis, but the pathological development of blood clots can lead to heart attacks and strokes. During the formation of a blood clot, platelets become activated and rapidly assemble their actin cytoskeletons. These become linked to the major transmembrane integrin alphaIIb/beta3, which mediates adhesion to fibrin on the outside of the platelet. The coupling of alphaIIb/beta3 to the cytoskeleton allows platelets to contract clots.
The first aim of this proposal is to determine how the cytoplasmic domains of alphaIIb/beta3 interact with specific cytoskeletal components. Several strategies will be used to identify cytoskeletal proteins that associate with alphaIIb/beta3 in vivo. The interactions of alphaIIb/beta3 mutated in the cytoplasmic domains will be investigated, as will the interactions of integrin chimeras in which the cytoplasmic domains are ligated onto the transmembrane and extracellular domains of other proteins. A scheme is proposed to permit analysis of the cytoskeletal links to individual cytoplasmic domain chimeras as well as to chimeras that have been induced to dimerize. The roles of specific proteins, such as talin, vinculin, and alpha-actinin, will be explored in cells from which vinculin has been deleted and in which talin or alpha-actinin have been functionally disrupted. Many agents which prevent platelet activation raise cyclic nucleotide levels and stimulate protein kinase A or G. A prominent substrate for these kinases in platelets is the vasodilator-stimulated phosphoprotein (VASP). The applicant will explore the function of VASP and the consequences of its phosphorylation in relation both to actin polymerization and the activation of alphaIIb/beta3. In response to platelet activation and aggregation, several tyrosine kinases become activated. The applicant will investigate the regulation of platelet tyrosine phosphorylation by tyrosine phosphatases. Specifically, he will look for tyrosine phosphatases that associate with and regulate tyrosine kinases, and those that associate with alphaIIb/beta3.
