PP shedding from MKs was a frequent event in naive (nontransplanted) S1pr1+/+ CD41-YFPki/+ transgenic mice (Junt et al., 2007) but also in S1pr1+/+ CD41-YFPki/+ BM chimaeras (Video 7). Most MKs shed PP fragments that consist of beaded platelet-like structures (Fig. 6, A and B; and Video 7), which generate mature platelets choose size by undergoing consecutive fragmentation steps (Behnke and Forer, 1998; Junt et al., 2007). More than 60% of the S1pr1+/+ MKs carrying intravascular PP processes showed fragmentation within 1 h (Fig. 6, A and B; and Video 7). We did not find any defect in PP fragmentation in S1pr2?/? or S1pr4?/? mice (Fig. 6 A), whereas this process was severely impaired in S1pr1 mutants. In S1pr1?/? chimaeras, we barely observed intravascular PP processes because of the aberrant interstitial PPF reported above (Fig.

4, H and I). However, 70�C100% of the PP processes that had eventually made their way into BM sinusoids remained firmly attached to their MK stems; only in rare instances did MKs release PP fragments (Fig. 6, A and B; and Video 7). Together, these data indicate that S1pr1 is critical for both directional PPF and for proper intravascular PP fragmentation. Defective PP shedding is likely to explain the increase in size of S1pr1?/? MKs (Fig. 3 E). Interestingly, the frequency of intravascular PP shedding was only moderately reduced in CD41-YFPki/+ S1pr1+/? mice (Fig. 6 A), suggesting that a single S1pr1 allele is sufficient to maintain intravascular PP shedding and that the mild thrombocytopenia observed in S1pr1+/? mice is mostly caused by a defect in navigating PP processes into BM sinusoids (Fig.

4, H and I). Figure 6. The effect of S1P on PP fragmentation in vivo. (A) Percentage of PP fragmentation events observed by MP-IVM over 1 h in the indicated groups. n = 13�C33 per group. Data are pooled from three to seven independent experiments. (B) Role of S1pr1 for … To examine whether S1pr1 regulates the dynamic process of PP shedding independently from its effects on PP invasion into BM sinusoids, we next tested the consequences of short-term pharmacological inhibition of S1pr1. We treated naive (nontransplanted) S1pr1+/+ CD41-YFPki/+ mice with a single dose of the selective S1pr1 antagonist W146 and visualized PP shedding immediately thereafter. In contrast to protracted inhibition or genetic Entinostat ablation of S1pr1 (Fig. 4, H and I), this did not affect the overall number of MKs with intravascular PP protrusions. However, <20% of MKs with established intravascular protrusions managed to release PP fragments into the blood stream within 6 h after administration of W146; the vast majority of the intrasinusoidal processes remained attached to their MK stems (Fig. 6, A and B; and Video 8).