Fig 2: LX‐2 derived EV contain renin activity that stimulates delayed platelet activation. (A) The aspartyl protease inhibitor pepstatin suppresses LX2 induced platelet activation. LX‐2 cell EV were preincubated with pepstatin (10 μg/mL, 10 min), or not, prior to assessing platelet activation induced by LX‐2 EV. HBSS buffer the EV were resuspended in was the negative control. ***p < 0.001 unpaired T test, N = 5 biologic replicates. (B) Renin enzymatic activity underlies platelet stimulation by LX‐2 EV. Freshly isolated human platelets were incubated (10 min, 1000 rpm) in aggregometer cuvettes with the stated concentrations of the DRI Aliskiren. Aggregation was then initiated by the addition of LX‐2 EV at t = 0. N = 7 biologic replicates. ***p < 0.001 by one way ANOVA. (C) The Direct Renin Inhibitor VTP23999 and Aliskiren inhibit HSC EV‐induced platelet aggregation. Platelets stirring in aggregometer cuvettes were pretreated, or not, with VTP23999 (40 μM) or Aliskiren (40 μM) prior to the addition of buffer or washed EV recovered from LX‐2 cell conditioned media. VTP23999 N = 15 biologic replicates ****p < 0.0001; Aliskiren N = 14 biologic replicates unpaired T test p < 0.0001. (D) Renin mRNA accumulates during iPSC maturation to initiate HSC. Transcript levels of the REN gene quantified by RT‐qPCR were assessed as human iPSC cells were differentiated to quiescent and then initiated HSC. mean ± SD using a two‐tailed unpaired t‐test with Welch's corrections to calculate exact p‐values from 4 independent replicates. (E) Renin enzymatic activity promotes platelet aggregation in response to EV from iPSC‐derived initiated HSC. Platelet aggregation induced by media conditioned by iPSC matured to initiated HSC in the presence or absence of 40 μM Aliskiren (N = 4 biologic replicates) or VTP23999 (N = 3 biologic replicates) **p < 0.01 with p = 0.0012. (F) LX‐2 cell derived EV contain prorenin while platelets abundantly express the prorenin receptor (p)RR. (left) Western blot for renin (37.5 kDa) and prorenin (~42–45 kDa) in EV isolated by ultracentrifugation from LX‐2 cell conditioned media, and (right) prorenin receptor (apparent ~42 kDa mobility) expressed by LX‐2 cells or washed human platelets. (G) The combination of platelets incubated with LX‐2 EV develops renin enzymatic activity. Renin enzymatic activity was assayed using a fluorogenic TF3/TQ3‐labeled peptide substrate (Abcam) to assess fluorescent resonant energy transfer over time as platelets were incubated with or without purified LX‐2 EV. N = 5 assay replicates. Unpaired T test p = 0.0172. (H) Renin knockdown in LX‐2 cells reduces EV‐associated renin and suppresses LX‐2 EV‐induced platelet aggregation. LX‐2 cells were incubated overnight with REN siRNA or a universal scrambled control siRNAs, the transfection media was removed and the cells were incubated with complete MEM overnight, this was removed and discarded and the cells incubated overnight for a second time before this conditioned media was collected, cleared by centrifugation, and concentrated by Centricon filtration. EV from knockdown or scrambled control cells were added to stirring platelets at t = 0 with aggregation assessed by clearing as before. The amount of aggregation after 35 min of incubation was quantitated with REN siRNA EV significantly less effective than EV from scrambled control‐treated cells. N = 5 biologic replicates ****p < 0.0001.