All animal studies were approved by the institutional review boards of the Instituto de Salud Carlos III and the Centro de Biología Molecular Severo Ochoa (Madrid, Spain). Cells were treated with Fc-Block (BD Biosciences, San Diego, CA) and 10% normal mouse serum in phosphate-buffered saline before incubation (4°C, 30 minutes) with fluorescein isothiocyanate (FITC), biotin-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated antibodies (Abs) (as indicated in Supporting Table 1). Cell debris and dead cells were excluded by light-scattering parameters and propidium

iodide staining, and cell suspensions were analyzed in a FACSCalibur with the CellQuest (BD Biosciences) and FlowJo (Tree Star Inc., Stanford University, Roxadustat chemical structure Stanford, CA) software packages. Cells were purified under sterile conditions by fluorescence-activated cell sorting (FACS) BMS-907351 cell line on a FACSAria (BD Biosciences), and the purity of the cells recovered was over 98%. For cell-culture experiments, staining with Abs directed against c-Kit/FITC, CD49f/PE, CD45/biotin and Ter-119/biotin (visualized with streptavidin/APC) allowed c-KitDCD45−Ter119−, c-KitDCD45−Ter119−CD49fH, and c-KitDCD45− Ter119−CD49fD cells to be purified (forthwith referred to as c-KitDCD45−, CD49fH, and CD49fD, respectively). Because purified

CD49fH and CD49fD cells were CD41HCD42c+CD45− and CD41− CD42c−CD45−, respectively, for some experiments, CD49fHCD41H and CD49fD cells were purified after staining with Abs directed against CD41/FITC, CD45/APC, and CD49f/PE. Cells were collected, activated with adenosine diphosphate (ADP), thrombin, and the PAR4 thrombin receptor-activating peptide, and analyzed by flow cytometry VAV2 (as indicated in the Supporting Methods). RNA was extracted and oligo(dT)-primed complementary DNA (cDNA) was prepared as previously described.14 Polymerase chain reaction (PCR) amplification was performed with the primers and conditions indicated in Supporting Table 2 and Supporting Methods. For quantification

of ALB and kinase domain region (KDR) expression, quantitative real-time PCR was performed as previously described.15 The relative amount of specific cDNA on each sample was determined by the 2−ΔΔCt method using G-protein subunit αs (GαS) expression as an internal control. Purified E11.5 FL cells (1-2 × 105 cells/cm2) were cultured for 1-7 days under the general conditions detailed in the Supporting Methods. In some experiments, the following soluble factors were added: murine TPO (50 ng/mL; PeproTech, London, UK); VEGF-A (1-10 ng/mL; PeproTech); serotonin (1 μM; Sigma-Aldrich, St. Louis, MO); and cytochalasin B (CytoB; 20 μM; Sigma-Aldrich). When indicated, purified Leaf antimouse VEGF-A or Leaf isotype-matched Abs (ISO; clones 2G11-2A05 and RTK2758, respectively; BioLegend, San Diego, CA) were added to cultures.