A novel mechanism ofthrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency

Abstract Sphingomyelin synthase 1 (SMS1) contributes to the generation of membrane sphingomyelin (SM) and affects SM-mediated physiological functions. Here, we describe the hematological phenotypes, such as reduced circulating platelets and dysfunctional hemostasis,in SMS1-deficient (SMS1-KO) mice. SMS1-KO mice display pathologic manifestations related to idiopathic thrombocytopenia (ITP),includingrelatively high amounts of peripheral bloodreticulated platelets, enhancedmegakaryopoiesis in the bone marrow and spleen, and splenomegaly. Deficiency of SMS1, but not SMS2, prevented SM production and enhanced phosphatidylserine (PS) externalization on the plasma membranes of platelets and megakaryocytes. Consequently, SMS1-KO platelets were excessively clearedbymacrophages in the spleen. Multimer formation in the plasma membrane of TMEM16F, a known calcium (Ca2+)-activated nonselective ion channel and Ca2+-dependent PS scramblase, wasenhanced, resultingin PS externalization to outer-leaflets through increasedCa2+ influx in immortalized mouse embryonic fibroblasts established from SMS1-KO mice(SMS1-KO tMEFs), as seen with SMS1-KO platelets. Thus, SMS1 deficiency changedthe TMEM16F distribution on the membrane microdomain, regulatingCa2+ influx-dependent PS exposure. SMS1-KO tMEFsin which TMEM16F was knocked out using the CRISPR-Cas9 system lacked both the Ca2+ influx and excess PS exposure seen in SMS1-KO tMEFs. Therefore, SM depletion on platelet membrane microdomains due to SMS1 deficiency enhanced PS externalization via a Ca2+ influx through TMEM16F activation, leading to elevated platelet clearance and causing hemostasis dysfunction through thrombocytopenia. Our current findings show that the SM-rich microdomain generated by SMS1 is a potent regulator of thrombocytopenia through TMEM16F, suggesting that its dysfunction may be a novel additional mechanism of ITP.