First we detected the amounts of apoptotic proteins in the platelets. the anti-GPIb monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIb antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA RTA-408 inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIb antibody-induced platelet apoptosis. We further demonstrate that this antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic RTA-408 platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIb autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic RTA-408 factors. Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count (1,2) which is usually caused primarily by autoantibodies against two major receptors of platelets, the fibrinogen receptor glycoprotein (GP) IIb/IIIa and the von Willebrand factor (VWF) receptor GPIb-IX complex (35). The autoantibody-bound platelets are thought to be removed by Fc-dependent phagocytosis in the spleen (1,2,6). Therefore, the main therapeutic strategies for ITP are immune suppression, immune modulation, and splenectomy (1,2,7). However, ITP patients with antiGPIb-IX autoantibodies present more severe decreases in platelet count (4) and are less responsive to conventional therapies such as steroid treatments (8), i.v. IgG (IVIG) (5,9), and even splenectomy (10,11), suggesting that a different pathogenic mechanism may be involved in antiGPIb-IX autoantibody-induced platelet clearance. Anti-GPIb monoclonal antibodies were found to activate platelets in vitro (1216) and induce platelet clearance in vivo (12,1720). More recent studies exhibited that anti-GPIb antibodies induced phagocytosis of platelets in the liver through an Fc-independent mechanism (12,17,20). Anti-GPIb antibodies targeting the N terminus of the receptor cause it to cluster, resulting in phagocytosis of platelets by microphages in the liver (12). On the other hand, GPIb desialylation was demonstrated to contribute to platelet clearance in an hepatocyte AshwellMorell receptor-dependent manner (20). Moreover, shear-induced unfolding of the GPIb mechanosensory domain name by Rabbit Polyclonal to TIE2 (phospho-Tyr992) anti-GPIb monoclonal antibodies was found to trigger signaling, leading to platelet clearance (21). Therefore, while increasing evidence suggests that anti-GPIb autoantibodies may induce platelet clearance via an Fc-independent manner, the mechanism for anti-GPIb antibody-induced thrombocytopenia remains elusive. GPIb, the main subunit of the GPIb-IX complex, contains binding sites for several important ligands including VWF and thrombin at the N-terminal extracellular domain name (16,22,23). The conversation of the VWF multimer with GPIb induces translocation and cross-linking of GPIb-IX complexes in lipid rafts (2427), triggering signaling cascades (28,29) and leading to platelet activation and thrombus formation (30,31). Interestingly, we found that the GPIbVWF conversation could also induce platelet apoptosis, but the mechanism remains unknown (32). We recently reported that protein kinase A (PKA)-mediated platelet apoptosis occurs extensively in pathophysiological conditions (33). Moreover, accumulating evidence suggests that various pathological stimuli lead to thrombocytopenia in many common diseases, such as infection, malignancy, diabetes, and heart and circulation RTA-408 diseases (3437). However, little is known about the pathogenesis leading to thrombocytopenia. In this study, we find that anti-GPIb monoclonal antibodies induce Akt activation and Akt-mediated platelet apoptosis. We demonstrate that.