In this study, we show that hydrodynamic forces alone are not sufficient to allow the release of new platelets from MK PP extensions. Instead, selleck chem ARQ197 we found that high concentrations of the bioactive lipid S1P prevailing in the sinusoidal blood, but not in the BM interstitium, are mandatory for the release of new platelets from MKs. From a teleological point of view, the S1P-dependent sequential guidance of thrombopoiesis comprising (a) directional PPF along a transendothelial S1P gradient and (b) subsequent S1P-dependent intravascular PP shedding leads to the introduction of naive platelets into the circulating blood and prevents aberrant platelet production within the BM interstitium. S1P guidance of intravascular PPF, elongation, and shedding therefore provides grounds for efficient thrombopoiesis, which seems instrumental given the relative paucity of MKs.
S1P controls thrombopoiesis via megakaryocytic S1pr1 receptors Our study shows that MKs robustly express three different S1P receptors, S1pr1, S1pr2, and S1pr4. Loss of S1pr1 on hematopoietic cells and also conditional deficiency of S1pr1 in MKs were associated with severe thrombocytopenia. Moreover, gain of S1pr1 function in S1pr1?/? MKs rescued their defect in platelet production. These results clearly demonstrate that S1pr1 expressed by the MK lineage intrinsically controls platelet homeostasis. It has been shown previously that signaling via S1pr1 activates Rac GTPases in multiple hematopoietic lineages, including T cells (Matsuyuki et al., 2006; G��rard et al., 2009).
Consistent with this, we observed here that Rac activation is triggered in MKs by S1pr1 agonists. Rac GTPases are known to regulate actin dynamics and induce the formation of membrane extensions (Aspenstr?m et al., 2004). In MKs, the turnover of actin filaments is known to control platelet formation (Bender et al., 2010). Correspondingly, we found here that Rac GTPase activation leading to cytoskeletal reorganization is indispensable for S1P�CS1pr1-driven PPF and fragmentation. Although we observed that simultaneous pharmacological inhibition of all Rac GTPases by NSC23766 virtually abolishes S1P-driven thrombopoiesis in vitro, loss of Rac1 alone does not lead to thrombocytopenia in vivo (McCarty et al., 2005), suggesting that other Rac GTPase family members, including Rac2, Rac3, and RhoG, may have redundant functions in thrombopoiesis and its control by S1P.
In addition, we cannot rule out that other small Rho GTPases, including cdc42 and RhoA (Pleines et al., 2010; Pleines et al., 2012), also contribute to S1P-driven platelet generation by MKs. Unlike the loss of S1pr1, genetic disruption of S1pr2 or S1pr4 on hematopoietic cells was not Carfilzomib associated with thrombocytopenia. Moreover, loss of S1pr2 or S1pr4 did not result in any gross defect of MK development, directional PPF or PP fragmentation in vitro or in vivo.