Unique disulfide bonds in epidermal growth factor (EGF) domains of ß3 affect structure and function of αIIbß3 and αvß3 integrins in different manner.

The ß3 subunit of αIIbß3 and αvß3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbß3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbß3 and αvß3. We created ß3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbß3 and αvß3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbß3 and αvß3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbß3 and αvß3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbß3. Activation of C523S/C544S αvß3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ß3 in αIIbß3 but not in αvß3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ß3 differently affect the function of αIIbß3 and αvß3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbß3 and αvß3 and for Cys-523-Cys-544 only in αvß3.

Disulfide bond exchanges in integrins αIIbß3 and αvß3 are required for activation and post-ligation signaling during clot retraction.

BACKGROUND: Integrin αIIbß3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbß3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbß3 and αvß3 activation. METHODS: In order to investigate the role of integrin activation and disulfide bond exchange during αIIbß3- and αvß3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human ß3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. RESULTS: Cells expressed WT or mutated human αIIbß3 as well as chimeric hamster/human αvß3. The αIIbß3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvß3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvß3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. CONCLUSIONS: Both αIIbß3 and αvß3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbß3, but not of αvß3 enhances clot retraction. Both αIIbß3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.