Interaction of integrin alpha2b-beta3 with talin and with mouse connective tissue growth factor or CYR61

Integrin alphaIIbbeta3 plays an important role in hemostasis and thrombosis. Following fibrinogen binding and platelet aggregation, alpha IIbbeta3 becomes associated with the platelet cytoskeleton, and this process has been implicated in alphaIIbbeta3 -dependent post-occupancy function. Talin and vinculin have been proposed to mediate the attachment of alphaIIbbeta3 to actin filaments. Using a solid-phase binding assay, we demonstrated that purified alpha IIbbeta3 bound to both talin and vinculin in a dose-dependent manner. Furthermore, alphaIIbbeta3 bound to talin captured by immobilized vinculin. These findings support the hypothesis that alpha IIbbeta3 interacts with talin, which binds through vinculin to actin filaments. However, in the absence of talin, vinculin may serve as an alternative linker mediating alphaIIbbeta3-cytoskeleton association. To further characterize alphaIIbbeta3-talin interaction, we found that alphaIIbbeta3 bound dose-dependently to talin fragments corresponding to the 47-kDa head or the 200-kDa tail domains of talin, indicating that an integrin-binding site is present in each domain. In addition, over-expression of a recombinant fusion protein consisting of the talin N-terminal head domain and Enhanced Green Fluorescent Protein (rTalin-N/EGFP) in Chinese hamster ovary (CHO) cells expressing alphaIIbbeta 3 resulted in disruption of cell spreading on fibrinogen. These results suggest that talin requires the vinculin and/or actin binding sites within its tail domain in mediating cell spreading and focal adhesion formation. To localize the talin binding site within alphaIIbbeta 3, we found that talin failed to interact with peptides containing the AKWDTANNPLYK sequence, but bound to peptides containing the membrane proximal HDRKEFAKFEEERARAK sequence of the beta3 cytoplasmic tail. Moreover, the conserved basic amino acid pair (R724K725) in this region of beta3 is essential for talin binding.;Connective tissue growth factor (CTGF) and Cysteine-rich (CYR)61, members of the CCN family of extracellular matrix-associated signaling molecules, are present in normal blood vessel walls. Additionally, CTGF has been reported to be over-expressed in advanced atherosclerotic lesions. Both CTGF and CYR61 are adhesive ligands for integrin alphavbeta3 on endothelial cells. In the present study, we examined whether mouse CTGF (mCTGF) and CYR61 could serve as substrates for platelet adhesion. Agonist (ADP, thrombin or U46619)-stimulated but not resting platelets adhered to both mCTGF and CYR61, and this process was completely inhibited by prostacyclin I2, which prevents platelet activation. The specificity of platelet adhesion to mCTGF and CYR61 was demonstrated by specific inhibition of this process with polyclonal anti-mCTGF and anti-CYR61 antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent-cation dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha IIbbeta3), but not by LM609 (anti-alphavbeta 3), indicating that the interaction is mediated through integrin alpha IIbbeta3. In a solid-phase binding assay, activated alpha IIbbeta3, purified by RGD affinity chromatography, bound to immobilized mCTGF and CYR61 in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alphaIIbbeta3 failed to bind either protein. Collectively, these findings identify mCTGF and CYR61 as two novel activation-dependent adhesive ligands for the integrin alphaIIbbeta3 on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.