The role of Sphingomyelin synthase 2 (SMS2) in platelet activation and its clinical significance

Abstract

Background: Sphingomyelin (SM) is an essential component of biological lipid rafts, and it plays an indispensable role in maintaining plasma membrane stability and in mediating signal transduction. The ultimate biosynthesis of SM is catalyzed by two sphingomyelin synthases (SMSs) namely SMS1 and SMS2, which are selectively distributed in the trans-Golgi apparatus and the plasma membrane. It has been demonstrated that SMS2 acts as an irreplaceable molecule in the regulation of transmembrane signaling, and loss of SMS2 has been reported to worsen atherosclerosis and liver steatosis. However, the function of SMS2 in platelet activation and its association with the pathological process of thrombosis in acute coronary syndrome (ACS) and portal hypertension (PH) remain unclear.

Methods: In this study, we tested the role of SMS2 in platelet activation and thrombosis using SMS2 knockout (SMS2 -/-) mice and SMS2-specific inhibitor, D609. Furthermore, we detected SMS2 expression in patients with ACS and PH.

Results: SMS2 -/- platelets showed significant reduction in platelet aggregation, spreading, clot retraction and in vivo thrombosis. Similar inhibitory effects on platelet activation were detected in D609-treated wild-type platelets. PLCγ/PI3K/Akt signaling pathway was inhibited in SMS2 -/- platelets and D609-treated wild-type platelets. In addition, we discovered that platelet SMS2 expression was remarkably increased in patients with ACS and PH, compared with healthy subjects.

Conclusions: Our study indicates that SMS2 acts as a positive regulator of platelet activation and thrombosis, and provides a theoretical basis for the potential use of D609 in anti-thrombosis treatment.

Keywords: D609; Platelet; SMS2; Thrombosis.

Fig. 1

Human, mouse and rat platelets express SMS2. a Western blot detection of SMS2 in platelets from WT mice, SMS2 –/– mice, SH-SY5Y cell line, rats and human subjects. b PCR detection of monocyte-specific marker CD14. c, d Platelet count and mean volume in the peripheral blood of WT (black) and SMS2 –/– (red) mice (n = 8, n.s. P > 0.05). e, f Morphology of WT and SMS2 –/– platelets observed by transmission electron microscope. The average number of alpha and dense particles were calculated and no significant difference was found between WT and SMS2 –/– platelets (n = 10, n.s. P > 0.05)

Fig. 2

SMS2 deficiency reduced platelet activation in aggregation, spreading and clot retraction experiments. a, b Platelets from WT and SMS2 –/– mice were re-suspended with Tyrode’s buffer. Thrombin- and collagen-induced aggregation were recorded in a platelet aggregator (black, WT, red, SMS2 –/–, n = 4, n.s. P > 0.05, * P < 0.05, *** P < 0.001). c, d WT and SMS2 –/– platelets spreading on fibrinogen-coated slides. Spreading areas were quantified at indicated time points (n = 4, ** P < 0.01). e, f WT and SMS2 –/– platelets were stimulated with thrombin (0.2 U/mL) to coagulate. Images were pictured at indicated timepoints (n = 4, ** P < 0.01). g, h Thrombin- and collagen-induced human platelet aggregation by adding D609 (red curve, 100 µM, purple curve, 30 µM, gray curve, 10 µM, black curve, DMSO control, n = 4, ** P < 0.01, *** P < 0.001). i, j Human platelets spreading on fibrinogen with or without D609 (100 µM). Fully spread ratios were quantified (n = 4, *** P < 0.001). k, l Washed platelets added with thrombin (0.2 U/mL) and D609 at indicated concentration to coagulate (red, 100 µM, gray 10 µM, black, DMSO control, n = 4, n.s. P > 0.05, ** P < 0.01, *** P < 0.001)

Fig. 3

SMS2 deficiency decelerated arteriolar thrombosis. a Representative images of thrombus formation in mesenteric arterioles induced by ferric trichloride injury in WT and SMS2 –/– mice (upper row, WT, lower row, SMS2 –/–). Thrombus size and occlusion time were analyzed as (c) and (d) showed (n = 6, ** P < 0.01). b Tail bleeding time of WT and SMS2 –/– mice (n = 8, ** P < 0.01). e Representative images of thrombus formation in mice with or without D609 (10 mg/kg) treatment. Statistic result of thrombus size was showed in (h) (n = 6, ** P < 0.01). f Arteriovenous shunt thrombosis experiment showed D609 treatment diminished thrombus weight (n = 5, ** P < 0.01). g D609 prolonged tail bleeding time (n = 8, *** P < 0.001)

Fig. 4

PLCγ/PI3K/Akt phosphorylation mediated reduction effects of SMS2 deficiency on platelet activation. a SMS2 –/– diminished thrombin- and collagen-induced PLCγ/PI3K/Akt phosphorylation. Results shown are representative images of at least 3 experiments using WT and SMS2 –/– platelets. b D609 weakened PLCγ/PI3K/Akt phosphorylation in human platelets after stimulated by thrombin (0.03 U/mL) in a dose-dependent manner. Results shown are representative images of at least 3 experiments. c-f SMS2 expression in healthy people and patients with SAP, ACS and PH (Health = 27, SAP = 12, ACS = 12, PH = 15, n.s. P > 0.05, *** P < 0.001)