Sphingomyelin encrypts tissue factor: ATP-induced activation of A-SMase leads to tissue factor decryption and microvesicle shedding.

A majority of tissue factor (TF) on cell surfaces exists in an encrypted state with minimal to no procoagulant activity. At present, it is unclear whether limited availability of phosphatidylserine (PS) and/or a specific membrane lipid in the outer leaflet of the plasma membrane contributes to TF encryption. Sphingomyelin (SM) is a major phospholipid in the outer leaflet, and SM metabolism is shown to be altered in many disease settings that cause thrombotic disorders. The present study is carried out to investigate the effect of SM metabolism on TF activity and TF(+) microvesicles (MVs) release. In vitro studies using TF reconstituted into liposomes containing varying molar ratios of SM showed that a high molar ratio of SM in the proteoliposomes inhibits TF coagulant activity. Treatment of macrophages with sphingomyelinase (SMase) that hydrolyzes SM in the outer leaflet results in increased TF activity at the cell surface and TF(+) MVs release without increasing PS externalization. Adenosine triphosphate (ATP) stimulation of macrophages that activates TF and induces MV shedding also leads to translocation of acid-sphingomyelinase (A-SMase) to the plasma membrane. ATP stimulation increases the hydrolysis of SM in the outer leaflet. Inhibition of A-SMase expression or activity not only attenuates ATP-induced SM hydrolysis, but also inhibits ATP-induced TF decryption and TF(+) MVs release. Overall, our novel findings show that SM plays a role in maintaining TF in an encrypted state in resting cells and hydrolysis of SM following cell injury removes the inhibitory effect of SM on TF activity, thus leading to TF decryption.