Non-Muscle Myosin II Mediates Microparticle Release From Endothelial Cells Activated by Antiphospholipid Antibodies,

Abstract 3343

The antiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLA). The majority of pathogenic APLA are actually directed against β₂-glycoprotein I (β₂GPI), an abundant plasma phospholipid binding protein. Microparticles are small phospholipid-rich vesicles that may express tissue factor and are thought to play an important role in thrombosis. Increased release of microparticles may be stimulated by cellular activation or apoptosis, and previous work from our laboratory and others has demonstrated increased levels of circulating microparticles in patients with APS, even far removed from thrombotic events. Though several mechanisms have been proposed for mediating release of microparticles from cells, this process is poorly understood and there is little information available concerning the mechanisms of microparticle release from endothelial cells stimulated by APLA/anti-β₂GPI antibodies. To address this issue we assessed the role of non-muscle myosin II motor protein in mediating the release of microparticles from endothelial cells activated by anti-β₂GPI antibodies. We observed that incubation of endothelial cells with β₂GPI and anti-β₂GPI antibodies caused significant increases in the release of microparticles compared to cells treated with β₂GPI and control antibodies. Proteomic and biochemical studies demonstrated that anti-β₂GPI antibodies caused increased phosphorylation of endothelial cell Myosin II light chain (MLC), an essential step in the activation of Myosin II motor protein. Moreover, examination of endothelial cells stimulated by anti-β₂GPI antibodies using confocal microscopy revealed a dramatic increase in the formation of actin-myosin II filaments. To assess the effects of these changes on microparticle release, we inhibited MLC phosphorylation in anti-β₂GPI-treated endothelial cells using ML-7, a highly specific inhibitor of MLC. ML-7 caused a significant decrease in the release of microparticles from anti-β₂GPI-treated cells. In conclusion, our results demonstrate that non-muscle myosin II motor protein activation by anti-β₂GPI antibodies is necessary for the increased release of microparticles from activated cells. These microparticles may play important roles in the propagation of thrombosis and other clinical sequelae observed in patients with APS.