Collectively, our findings uncover the molecular pathway that enables the final steps of thrombopoiesis. S1P navigates selleck Vandetanib PP extensions into BM sinusoids and initiates platelet release Mature MKs form intravascular PP extensions that grow from the MK cell body at a mean speed of 10 ��m/min under shear conditions in vivo (Fig. 4 J), with the DMS functioning as the membrane reservoir for PP elongation (Schulze et al., 2006). During elongation, PPs are equipped with specific proteins associated with platelets, including von Willebrand factor (vWF) and fibrinogen receptors. Microtubules, assembled from ��/��-tubulin dimers, are the primary structural component of the engine that drives the elongation of PPs. Correspondingly, PPs fail to form when cultured MKs are exposed to agents that inhibit microtubule assembly (Italiano et al.
, 1999) or sliding (Patel et al., 2005b), and mice lacking ��1-tubulin, the most abundant ��-tubulin in platelets, develop thrombocytopenia (Schwer et al., 2001). Cultured MKs form PPs that elongate into random directions (Italiano et al., 1999; Dunois-Lard�� et al., 2009). In contrast, we show here by MP-IVM that PPF occurs in a highly polarized fashion in the BM in vivo. This suggests that PPs integrate a previously unknown guidance signal, which navigates them into the intravascular compartment and avoids the interstitial BM compartment. Our study has uncovered this guidance signal and shows that a transendothelial S1P gradient in the BM controls the directionality of PPF and elongation.
We observed that the interstitial S1P concentrations in the BM are low, whereas the S1P blood concentration is orders of magnitude higher. Residing at the vascular interface, mature MKs are located in a particularly strategic position for integrating the guidance cues provided by the transendothelial S1P gradient. Equipped with S1pr1, they sense the steep vascular S1P gradient and extend dynamic PP protrusions into microvessels along increasing concentrations of this lipid mediator. A similar S1P gradient also exists in the lymph node between lymph node interstitium and the lymph fluid, where it drives the migration of T cells into efferent lymph vessels (Matloubian et al., 2004). Recent observations showing that the S1P pathway controls the egress of lymphocytes from the BM into the blood stream emphasize the biological relevance of the transendothelial S1P gradient for BM homeostasis (Allende et al.
, 2010). Once MKs have successfully extended their PPs into the blood, they release fragments from the tips of their intravascular projections (Video 7; Junt et al., 2007). These new PP fragments break down further in the circulation giving rise to mature platelets of 2�C3-��m diameter within the circulation (Stenberg and Levin, 1989). Blood shear stress contributes to the shedding Batimastat of PPs (Junt et al., 2007; Dunois-Lard�� et al., 2009; Thon et al.