Murine platelet production is suppressed by S1P release in the hematopoietic niche, not facilitated by blood S1P sensing

Abstract

The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P1 receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)–derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P1. Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by S1pr1 deletion selectively in MKs. Moreover, S1P1 expression and signaling were both undetectable in mature MKs in situ, and MK S1pr1 deletion did not affect platelet aggregation or spreading. When S1pr1 deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted Sphk2 deletion and was reversed by compound deletion of either Sphk1 or S1pr2, suggesting that this phenotype arises from increased S1P export and S1P2 activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P1 in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P1, and can further suppress thrombopoiesis through S1P2 when aberrantly secreted in the hematopoietic niche.

Deletion of Mfsd2b impairs thrombotic functions of platelets

“…We do not observe any significant reduction in number of platelets at the steady state or the recovery state after induction of platelet depletion from global knockout or platelet-specific knockout of Mfsd2b. Thus, our observations suggest that Mfsd2b-S1P pathway is not required for platelet biogenesis, consistent with a recent report 15 . Why is there a thrombotic phenotype in Mfsd2b knockout platelets?…”

“…S1P synthesis in megakaryocytes was shown to play a role in platelets biogenesis in global deletion of SphK2 knockout mice 32 . In contrast, specific deletion of SphK2 in platelets did not affect platelet biogenesis 15 . We do not observe any significant reduction in number of platelets at the steady state or the recovery state after induction of platelet depletion from global knockout or platelet-specific knockout of Mfsd2b.…”

“…The impaired functions of Mfsd2b knockout platelets are unexpected given the recent report of dispensable roles of S1P signaling in platelets 15 . However, we noted that Mfsd2b KO platelets had altered morphology in complete blood count analysis (Supplementary Fig.…”

“…Platelet biogenesis and production from megakaryocytes were also reported to be reduced in S1P1 knockout mice 14 . However, the signaling roles of S1P from platelets requires further investigations as some parts of the phenotypes were not recapitulated in a recent study 15 . Nevertheless, S1P produced by platelets is essential for several important physiological conditions including anaphylactic shock 16 .…”

Sphingosine-1-phosphate modulates PAR1-mediated human platelet activation in a concentration-dependent biphasic manner

“…Previous studies showed that impaired platelet S1P synthesis in SphK2 deficient mice protected mice from in vivo arterial thrombosis, without associated bleeding, indicating a role for S1P in thrombosis, likely through an effect on S1PR1 13 . However, other studies suggest that S1PR1 is not expressed in murine platelets and that S1P has little effect on murine platelet function 53 . Our findings of biphasic effects of S1P on human platelet function raises the intriguing question of how this translates into a physiological role in platelet aggregation and thrombus formation in humans.…”