Fig. 3. Role of pulmonary endothelial cell health and microvascular structure on platelet generation.

a, b Pulmonary endothelial cells (ECs) were isolated from perfused lungs under air- (black) or pure nitrogen-ventilation (red) or without ventilation (blue) for ~2 h. ECs from fresh lung tissue served as control (grey). ECs were stained with FITC-conjugated anti-CD31/PECAM-1 or anti-CD102/ICAM-2 antibodies. N = 5 independent experiments for each group. Two-tailed Mann–Whitney test. a Viability of pulmonary ECs were determined by Calcein Deep Red retention and displayed as mean ± SEM. b Mitochondrial membrane potential was determined by accumulation of Tetramethyl rhodamine methyl ester (TMRM) in active mitochondria and displayed as mean fluorescence intensity ± SEM. Air-ventilated lung vs unventilated lung: p = 0.0159 and 0.0159 for CD102 and CD31, respectively. Air-ventilated lung vs nitrogen-ventilated lung: p = 0.0317 and 0.0317 for CD102 and CD31, respectively. c Design of microfluidic chamber simulating a physiological pulmonary vascular system (details shown in Methods), including a photomicrograph of the smallest channels in the system and indication of the flow direction by arrows (red). Dimensions indicated on the figures are width of channels. d, e Mouse megakaryocytes (MKs) prelabelled with CD41-PE were repeatedly pumped through the microfluidic chamber. d The viability of generated platelets from the microfluidic chamber was determined by Calcein AM staining (Generated platelets: CD41+/Calcein AM+ in upper right quadrant, Q3 in green). All generated platelets identified in this way showed no DNA content (DRAQ5 -ve staining). e Quantification of generated platelets per MK in perfusates under air (purple circles) or pure nitrogen conditions (green circles), measured by FACS. For comparison, numbers of platelets generated in the unventilated lung-heart system (blue squares), from Fig. 2a, are shown. N = 4 (for air-chamber or nitrogen-chamber) and 5 (for unventilated lung-heart model) independent experiments. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. f Mouse MKs prelabelled with CD41-FITC were repeatedly pumped through the microfluidic chamber under air 18 times. Generated platelets were washed and then integrin αIIbβ3 activation and P-selectin expression, induced by 2 U/mL thrombin (Thr) or 5 µg/mL CRP-XL, were measured by FACS. N = 10 independent experiments and data are mean ± SEM. Two-tailed Mann–Whitney test, p < 0.0001. Source data are provided in the Source Data file.