When stimulated with thrombin, platelets assemble and reorganize actin filaments, adhere to subendothelium or each other, and contract an actomyosin network. The ability of platelets to arrest bleeding is heavily reliant on the maintenance of stable adhesion to the substratum and firm attachment of the cytoskeleton to the plasma membrane. This proposal is directed at understanding the molecular mechanism by which these associations are achieved and regulated. Specifically, the function of talin, an abundant 235 kD platelet protein, will be investigated. Talin is a component of adhesion plaques, sites of cell-substratum interaction in fibroblasts. At these sites talin is thought to associate with integrin, a transmembrane receptor for extracellular matrix, as well as another adhesion plaque protein, vinculin. Thus, talin appears to participate in establishing the transmembrane connection between an extracellular ligand and the cytoskeleton. The role of talin in platelet adhesion and actin-membrane interaction will be investigated by a combination or biochemical, immunochemical, and morphological approaches. Talin will be localized in normal and thrombasthenic platelets by high resolution immunoelectron microscopy. Binding studies will be performed to analyze the mechanism by which talin associates with the platelet membrane. Specifically, the ability of talin to interact with transmembrane glycoproteins that serve as extracellular matrix receptors in platelets will be investigated. Modification of talin by proteolysis or phosphorylation may represent a mechanism by which talin function is regulated in living platelets. Calcium- dependent proteolysis of talin occurs upon platelet aggregation. The effect of such proteolysis on talin's biochemical properties will be investigated in an attempt to determine the functional significance of this cleavage event. In addition, talin is a substrate for protein kinase C in vitro. The ability of protein kinase C to phosphorylate platelet talin in vivo will be determined. If talin is phosphorylated in response to platelet activation, we will examine whether such phosphorylation regulates the protein's ability to associate with the plasma membrane and participate in linking the cytoskeleton to extracellular ligands at sites of platelet adhesion.
| Macalma, T; Otte, J; Hensler, M E et al. (1996) Molecular characterization of human zyxin. J Biol Chem 271:31470-8 |
| Kosa, J L; Michelsen, J W; Louis, H A et al. (1994) Common metal ion coordination in LIM domain proteins. Biochemistry 33:468-77 |
| Bertagnolli, M E; Beckerle, M C (1994) Regulated membrane-cytoskeleton linkages in platelets. Ann N Y Acad Sci 714:88-100 |
| Bertagnolli, M E; Beckerle, M C (1993) Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets. J Cell Biol 121:1329-42 |
| Bertagnolli, M E; Locke, S J; Hensler, M E et al. (1993) Talin distribution and phosphorylation in thrombin-activated platelets. J Cell Sci 106 ( Pt 4):1189-99 |
| Michelsen, J W; Schmeichel, K L; Beckerle, M C et al. (1993) The LIM motif defines a specific zinc-binding protein domain. Proc Natl Acad Sci U S A 90:4404-8 |
| Beckerle, M C; Yeh, R K (1990) Talin: role at sites of cell-substratum adhesion. Cell Motil Cytoskeleton 16:7-13 |
| Beckerle, M C; Miller, D E; Bertagnolli, M E et al. (1989) Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets. J Cell Biol 109:3333-46 |
