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Platelet-derived nitric oxide (NO) has been shown to play conflicting roles in platelet function, although it is accepted that NO mediates its actions through soluble guanylyl cyclase (sGC). This confusion concerning the roles of platelet NO may have arisen because of an uncharacterized mechanism for activation of sGC.To examine the ability of the novel platelet agonist globular adiponectin (gAd) to stimulate the NO-independent cGMP-protein kinase G (PKG) signaling cascade.We used three independent markers of NO signaling, [3H]L-citrulline production, cGMP accrual, and immunoblotting of vasodilator-stimulated phosphoprotein (VASP), to examine the NO signaling cascade in response to gAd.gAd increased platelet cGMP formation, resulting in a dose- and time-dependent increase in phospho-VASP157/239. Phosphorylation of VASP in response to gAd was mediated by both protein kinase A and PKG. Importantly, cGMP formation occurred in the absence of NO synthase (NOS) activation and in the presence of NOS inhibitors. Indeed, inhibition of the NOS signaling cascade had no influence on gAd-mediated platelet aggregation. Exploration of the mechanism demonstrated that NO-independent cGMP formation, phosphorylation of VASP and association of sGCα1 with heat shock protein-90 induced by gAd were blocked under conditions that inhibited Src kinases, implying a tyrosine kinase-dependent mechanism. Indeed, sGCα1 was reversibly tyrosine phosphorylated in response to gAd, collagen, and collagen-related peptide, an effect that required Src kinases and downstream Ca2+ mobilization.These data demonstrate activation of the platelet cGMP signaling cascade by a novel tyrosine kinase-dependent mechanism in the absence of NO.