Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouseconnective tissue growth factor is mediated through integrin alpha(IIb)beta(3)

Cyr61 and connective tissue growth factor (CTGF) members of a newly identif ied family of extracellular matrix-associated signaling molecules, are foun d to me mediate cell adhesion, promote cell migration and enhance growth fa ctor-induced cell proliferation in vitro, and induce angiogenesis in vivo, We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha(v)beta (3). Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atheros clerotic lesions, In the present study, we examined whether Cyr61 and Fisp1 2/mCTGF could serve as substrates for platelet adhesion, Agonist (ADP, thro mbin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostagland in I-2, which prevents platelet activation. The specificity of Cyr61- and F isp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibit ion of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibo dies, respectively. The adhesion of ADP-activated platelets to both protein s was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or e chistatin, but not by RGES, Furthermore, this process was specifically inhi bited by the monoclonal antibody AP-2 (anti-alpha(IIb)beta(3)), but not by LM609 (anti-alpha(v)beta(3)), indicating that the interaction is mediated t hrough integrin alpha(IIb)beta(3), In a solid phase binding assay, activate d alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to immo bilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable mann er. In contrast, unactivated alpha(IIb)beta(3) failed to bind to either pro tein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two n ovel activation-dependent adhesive ligands for the integrin alpha(IIb)beta( 3) On human platelets, and implicate a functional role for these proteins i n hemostasis and thrombosis.