Summary. Background: Although platelets are anucleated cells, they express several transcription factors that exert nongenomic functions, including the positive and negative regulation of platelet activation. NF-jB is a major transcriptional regulator of genes involved in survival, proliferation and inflammation. Objective: Because platelets play a critical role not only in hemostasis, but also in inflammation and tumor progression, we evaluated the role of NF-jB in platelet physiology. Results: Immunofluorescence, Western blotting and ELISA studies revealed that platelets express IjBa and NF-jB, and that stimulation with thrombin triggers IjBa phosphorylation and degradation and the binding of platelet NF-jB p65 subunit to synthetic olignoucleotides containing the consensus sequence for NF-jB. Two specific unrelated inhibitors of NF-jB activation, BAY 11-7082 and Ro 106-9920, reduced PAC-1 and fibrinogen binding to integrin a IIb b 3 and restricted platelet spreading on immobilized fibrinogen. Both inhibitors impaired aggregation mediated by ADP, epinephrine, collagen or thrombin, but not arachidonic acid. ATP release, TXB 2 formation, P-selectin expression, ERK phosphorylation and cPLA 2 activity stimulated by thrombin were reduced in BAY 11-7082-or Ro 106-9920-treated platelets. Although bleeding time was not affected, ADP-induced platelet aggregation was impaired in mice treated with BAY 11-7082. Conclusions: Our results suggest that NF-jB may be a novel mediator of platelet responses. The blockade of platelet function by NF-jB inhibitors might be relevant in those clinical situations where these drugs are being considered for antitumor and/or anti-inflammatory therapy.

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmann's thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin-1 (Gal-1), a β-galactoside-binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal-1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti-α(IIb) subunit ofα(IIb)β(3) integrin or platelets from patients with Glanzmann's thrombasthenia syndrome (α(IIb)β(3) deficiency), identified this integrin as a functional Gal-1 receptor in platelets. Binding of Gal-1 to platelets triggered the phosphorylation of β(3)-integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA(2)) release, and Ca(2+) mobilization. Not only soluble but also immobilized Gal-1 promoted platelet activation. Gal-1-deficient (Lgals1(-/-)) mice showed increased bleeding time (P<0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1(-/-) platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of α(IIb)β(3) was also observed in Lgals1(-/-) platelets, indicating a failure in the "outside-in" signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin-integrin interactions, for regulating platelet activation.