6 C), consistent with a central role of the S1P�CS1pr1 pathway for PP fragmentation and release of kinase inhibitor Volasertib platelets. Short-term treatment with W146 also reduced platelet counts in CD1 mice (Fig. 6 D), suggesting that S1pr1 controls thrombopoiesis across different strains of mice. W146 maintains an adequate in vivo receptor blockade for only 5�C6 h (Sanna et al., 2006), and shedding reoccurred 6 h after a single dose of W146, suggesting that S1pr1 inhibition does not affect the viability of MKs (Fig. 6 A and Video 8). In rare instances, where PP shedding occurred in the presence of the S1pr1 inhibitor W146, the time required until an intravascular fragment dissociated from its MKs stem was significantly prolonged (Fig. 6 E). The failure to properly shed PPs resulted in the formation of abnormal, thick intravascular PP processes (Video 8).

In line with this observation, the few PP fragments that were released despite the presence of W146 were significantly bigger compared with those in vehicle-treated control mice (Fig. 6 F), reminiscent of the large platelets observed in S1pr1-null chimaeras (Fig. 6 G and Table S2). S1pr1 agonists enhance platelet production Modulation of S1P receptors by FTY720 (fingolimod) has become a promising strategy for the treatment of patients with multiple sclerosis (Kappos et al., 2006). Here, we show that treatment of mice with a single dose of FTY720 leads to a prompt and transient increase in circulating platelets (Fig. 7 A). When we used MP-IVM to examine MKs before and after treatment with a single dose of FTY720, we found that FTY720 accelerates the shedding of intravascular PP extensions into the blood stream.

As a consequence, the number of MKs carrying intravascular PPs significantly decreased immediately after a single dose of FTY720 compared with vehicle (Fig. 7, B and C). This suggests that FTY720 represents an agonist for megakaryocytic S1pr1 receptors and has the potential to rapidly mobilize PPs into the blood, most likely by supporting fragmentation of intravascular PPs (Fig. 7, B and C). Treatment with the S1pr1-specific agonist SEW2871 also caused an increase in circulating blood platelets (Fig. 7 D), further supporting that activation of S1P�CS1pr1 receptor signaling enhances thrombopoiesis. Figure 7. The S1P analogue FTY720 and SEW2871 trigger rapid release of platelets. (A) Mice were treated with a single dose of FTY720 (3 mg/kg i.

p.) or DMSO (vehicle). Open bars indicate baseline of platelet counts assessed before drug administration; closed bars … DISCUSSION Our results assign a new role for S1P and its receptor S1pr1 as master regulators of thrombopoiesis. Entinostat In a dose-dependent and sequential manner, S1P controls two key steps in the cascade of thrombopoiesis by BM MKs: (1) the polarized development of PP extensions into the blood stream and (2) the subsequent shedding of PPs from their transendothelial stems.