The glycoprotein (GP) IIb-IIIa complex mediates the binding of fibrinogen to platelets and thus plays a major role in inducing the formation of platelet aggregates. On unstimulated platelets, GPIIb-IIIa cannot bind fibrinogen. Upon activation of platelets, unidentified intracellular events cause a change in the extracellular domain of GPIIb-IIIa that allows it to bind fibrinogen. This is known as """"""""inside-out"""""""" signaling. Binding of fibrinogen leads to the induction of intracellular events such as phosphorylation of specific proteins on tyrosine residues, activation of calpain and hydrolysis of specific cytoskeletal proteins. This is referred to as """"""""outside-in"""""""" signaling. The way in which the two way signaling across GPIIb-IIIa occurs is not understood. GPIIb-IIIa is a member of the integrin family of adhesion receptors. In cultured cells, evidence has been provided that integrins can associate with the cytoskeleton and that this interaction regulates functional activities of the integrins. Little is known about the possibility that the cytoskeleton regulates the functional activities of integrins in other cells. In preliminary experiments, the investigators have provided evidence that 1) GPIIb-IIIa is associated with the membrane skeleton in unstimulated platelets, 2) the tyrosine kinase, pp60c-src, appears to be associated with the membrane skeleton of platelets that are not yet competent to bind fibrinogen, and several of the proteins that become rapidly phosphorylated on tyrosine residues when platelets are activated co-isolate with the membrane skeleton in detergent lysates; 3) binding of fibrinogen drives GPIIb-IIIa and associated membrane skeleton proteins into association with cytoplasmic actin filaments, GPIIb-IIIa clusters, additional cytoskeletal proteins become phosphorylated on tyrosine residues, and additional signaling molecules (e.g., protein kinase C and phosphoinositide 3-kinase) are recruited to the integrin- rich skeletal structure; 4) incubation of platelets with cytochalasins inhibits the two way signaling across the integrin. These findings are consistent with the hypothesis that the cytoskeleton regulates functional activities of GPIIb-IIIa in platelets. Experiments in this proposal are designed to identify the mechanism(s) by which GPIIb-IIIa associates with the cytoskeleton, and to use this information to directly test the hypothesis that the interaction(s) is essential for regulating functional activities of the GPIIb-IIIa complex.
The Specific Aims of the proposed research are 1) to characterize interactions between GPIIb-IIIa and cytoskeletal proteins with which the integrin is known to interact in vitro; 2) to identify additional proteins that interact with the cytoplasm domains of GPIIb-IIIa; and 3) to elucidate the role of cytoskeletal proteins in regulating two way signaling across GPIIb-IIIa. Dr. Fox anticipates that these studies will provide insight into mechanisms involved in signaling across a variety of integrins, and may lead to understanding of ways in which integrin functions could be inhibited.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056264-03
Application #
2445341
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1995-08-04
Project End
2000-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
O'Toole, Timothy E; Bialkowska, Katarzyna; Li, Xiaohong et al. (2011) Tiam1 is recruited to ?1-integrin complexes by 14-3-3? where it mediates integrin-induced Rac1 activation and motility. J Cell Physiol 226:2965-78
Bialkowska, Katarzyna; Saido, Takaomi C; Fox, Joan E B (2005) SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes. J Cell Sci 118:381-95
Bialkowska, Katarzyna; Zaffran, Yona; Meyer, Sylvie C et al. (2003) 14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta. J Biol Chem 278:33342-50
Austin, Richard C; Fox, Joan E B; Werstuck, Geoff H et al. (2002) Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion. J Biol Chem 277:47106-13
Kulkarni, Sucheta; Goll, Darrel E; Fox, Joan E B (2002) Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J Biol Chem 277:24435-41
Fox, J E (2001) Cytoskeletal proteins and platelet signaling. Thromb Haemost 86:198-213
Reddy, K B; Bialkowska, K; Fox, J E (2001) Dynamic modulation of cytoskeletal proteins linking integrins to signaling complexes in spreading cells. Role of skelemin in initial integrin-induced spreading. J Biol Chem 276:28300-8
Zaffran, Y; Meyer, S C; Negrescu, E et al. (2000) Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system. J Biol Chem 275:16779-87
Kulkarni, S; Fox, J E (2000) Localization of calpain by immunofluorescence in adherent cells. Methods Mol Biol 144:151-9
Bialkowska, K; Kulkarni, S; Du, X et al. (2000) Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active. J Cell Biol 151:685-96

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