By serving as an activation dependent receptor for adhesive proteins, alphaIIbbeta3 mediates platelet aggregation, an essential event in thrombus formation. This function depends upon the capacity of alphaIIbbeta3 to transmit an """"""""inside-out"""""""" signal from its cytoplasmic domain to render its extracellular domain competent to bind fibrinogen. At the same time, platelets play a central role in the coagulation arm of the hemostatic/thrombotic response by providing a surface for prothrombin activation. A role of alphaIIbbeta3 in thrombin formation has been established; and a potential mechanism for this participation has been provided: prothrombin is a ligand for alphaIIbbeta3. The overall objective of this proposal is to develop a molecular understanding of ligand binding to alphaIIbbeta3 and the consequences of such interactions.
Three Aims will be explored with address specific aspects of the ligand binding function of the integrin. First, the capacity of alphaIIbbeta3 to bind prothrombin and accelerate thrombin formation will be explored. The hypothesis will be tested that platelets circulate in blood pre-armed with prothrombin bound via the RGD sequence in its thrombin domain to unactivated alphaIIbbeta3. Bound prothrombin is more readily activated to thrombin by Factor Xa, and thrombin is released from the receptor to stimulate platelets and activate alphaIIbbeta3, which then functions as an adhesion receptor to mediate platelet aggregation. Second, the nature of the ligand binding pocket(s) in alphaIIbbeta3 will be analyzed. The hypothesis will be tested that two very similar cyclic ligand peptides, cRGD and cHarGD, are high affinity surrogates for the RGD and fibrinogen gamma-chain recognition peptides and can bind to distinct, but allosterically linked sites in the receptor. These sites will be localized and characterized, and the reactivity of therapeutic GPIIb-IIIa blockers with these two sites will be analyzed. Third, the mechanism by which the cytoplasmic tails of the alphaIIb and beta3 subunits control activation of the ligand binding function of the receptor will be assessed. Based on a NMR structure, a """"""""molecular on-off switch"""""""" model for integrin activation by the cytoplasmic tail of alphaIIb has been proposed. This model will be tested using synthetic peptide, molecular biology, molecular modeling and further NMR analyses. The existence of a similar molecular switch in the beta3 subunit also will be evaluated. Taken together, these studies will provide fundamental insights into the ligand binding function of alphaIIbbeta3 and, by extrapolation, to that of other integrins as well. At the same time, these studies may have direct bearing on the use and future development of the GPIIb-IIIa blockers as antithrombotic agents.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL054924-06
Application #
6195325
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1995-08-01
Project End
2005-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
6
Fiscal Year
2000
Total Cost
$370,000
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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Podolnikova, Nataly P; Yakubenko, Valentin P; Volkov, George L et al. (2003) Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen. J Biol Chem 278:32251-8
Cierniewska-Cieslak, Aleksandra; Cierniewski, Czeslaw S; Blecka, Kamila et al. (2002) Identification and characterization of two cation binding sites in the integrin beta 3 subunit. J Biol Chem 277:11126-34
Plow, E F; Cierniewski, C S; Xiao, Z et al. (2001) AlphaIIbbeta3 and its antagonism at the new millennium. Thromb Haemost 86:34-40
Forsyth, C B; Solovjov, D A; Ugarova, T P et al. (2001) Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides. J Exp Med 193:1123-33
Yakubenko, V P; Solovjov, D A; Zhang, L et al. (2001) Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2. J Biol Chem 276:13995-4003
Byzova, T V; Kim, W; Midura, R J et al. (2000) Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein. Exp Cell Res 254:299-308
Cierniewski, C S; Byzova, T; Papierak, M et al. (1999) Peptide ligands can bind to distinct sites in integrin alphaIIbbeta3 and elicit different functional responses. J Biol Chem 274:16923-32
Forsyth, C B; Plow, E F; Zhang, L (1998) Interaction of the fungal pathogen Candida albicans with integrin CD11b/CD18: recognition by the I domain is modulated by the lectin-like domain and the CD18 subunit. J Immunol 161:6198-205
Byzova, T V; Plow, E F (1998) Activation of alphaVbeta3 on vascular cells controls recognition of prothrombin. J Cell Biol 143:2081-92

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