, 2009), neither positioning nor migration of MKs or their committed progenitors in marrow spaces is controlled by S1pr1. Figure 3. Loss of S1pr1 increases the size but has no effect on the positioning and motility of MKs in vivo. (A) Representative MP-IVM images of YFP+ or EGFP+ MKs (green) in BM. sellckchem BM microvasculature was visualized by intravenous injection of TRITC-dextran (red). … S1pr1 is essential for intravascular PP formation (PPF) During thrombopoiesis, mature MKs extend transendothelial protrusions, termed PPs, into BM microvessels (Junt et al., 2007). To test whether S1P/S1P receptor signaling plays a role during PPF, we cultured MKs in vitro (Lecine et al., 1998) and found that on average, 9 out of 100 WT MKs spontaneously formed PPs as assessed by phase-contrast microscopy.

MKs isolated from S1pr2?/? and S1pr4?/? mice generated similar number of PPs (unpublished data). In sharp contrast, in vitro PPF was reduced by >70% in S1pr1?/? MKs, as <2 out of 100 S1pr1?/? MKs formed PPs (Fig. 4 A). Importantly, lentiviral reexpression of GpIb�� promoter�Cdriven S1pr1 in S1pr1?/? MKs corrected PPF in vitro (Fig. 4 B). These results clearly indicate that S1pr1 plays a critical and intrinsic role for PPF by MKs. Figure 4. S1P regulates PPF. (A) The percentage of MKs displaying PPF. PPF is expressed as the percentage of MKs carrying PPs (8,000-10,000 MKs per experiment, five independent experiments with triplications). (B) The percentage of MKs displaying PPF in S1pr1+/+ ...

When we examined how S1pr1 might control PPF, we could exclude a primary lack of the invaginated demarcation membrane system (DMS), the predominant reservoir for PP membranes (Radley and Haller, 1982; Schulze et al., 2006), in S1pr1?/? MKs, as electron microscopy of S1pr1?/? BM MKs did not reveal abnormalities of the DMS when compared with S1pr1+/+ BM MKs (Fig. 4 C). Next we tested whether S1P serves as a chemoattractant for polarizing MKs and for inducing the formation of PP protrusions. Within the normal BM compartment, S1P is rapidly degraded by lyases and phosphatases expressed by most hematopoietic cells. Thus, the local S1P concentrations in the BM (with its densely packed hematopoietic cells) are exceedingly low (unpublished data), reflecting similar concentrations reported for other tissues such as lymph nodes (Schwab et al., 2005; Pappu et al., 2007).

In contrast, high Cilengitide S1P concentrations exist in the blood stream (Caligan et al., 2000; Berdyshev et al., 2005; Pappu et al., 2007). Because of their positioning at the vascular interface, MKs are therefore exposed to a steep transendothelial S1P gradient. To mimic the situation in the BM, we exposed cultured MKs to a gradient of S1P in vitro. Notably, PP extensions developed preferentially toward increasing concentrations of S1P but not of vehicle (Fig. 4 D). A similar result was also obtained with S1pr2?/? and S1pr4?/? MKs (Fig. 4 D). VPC23019, a previously described S1pr1 and S1pr3 antagonist (Davis et al.