U46619

An amount of 100 μL murine blood was collected in 300 μL heparin (20 U/mL), then 500 μL murine Tyrode’s buffer was added and samples were centrifuged at 650× g for 5 min. The remaining pellet was resuspended in 500 μL Tyrode’s buffer and washed again two times as described above. Afterwards, the remaining pellet was resuspended in 500 μL Tyrode’s buffer supplemented with CaCl₂ (0.1 M). Platelets were activated with one of the following agonists: collagen-related peptide (CRP)-XL (University of Cambridge, United Kingdom), Adenosine 5’-diphosphate (ADP) (Sigma-Aldrich, Burlington, MA, USA, # 01905), U46619 (Tocris Bioscience, Bristol, United Kingdom, # 1932), recombinant human biglycan (R&D Systems, Minneapolis, MI, USA, # 2667-CM-050), protease-activated receptor 4 (PAR-4) peptide (JPT Peptide Technologies GmbH, Berlin, Germany # 16035374). FITC-labeled rat anti-mouse P-selectin (JON/A-PE) monoclonal antibody (M130-1), PE-labeled rat anti-mouse integrin α_IIbβ₃, monoclonal antibody (M023-2), and FITC-labeled CD61 antibody (M031-1) (all purchased from Emfret Analytics, Würzburg, Germany) were used for labeling. The mean fluorescence intensity (MFI) was measured by flow cytometry.

General chemicals and pharmacological agents were purchased from Sigma-Aldrich, St. Louis, MO, USA, including; insulin (1–10 nM), H₂O₂(0.01–1 mM), tetraethylammonium (1 mM) (KCa channels inhibitor), iberiotoxin (100 nM) (BKCa channels inhibitor), genistein (50 μM) (tyrosine kinases inhibitor) and wortmannin (30 nM) (phosphatidylinositol 3-kinase inhibitor). U46619 (0.0001–1 μM; thromboxane A2 analog) was obtained from Tocris Bioscience, Bristol, UK.

Platelet-rich plasma (PRP) was collected from Pf4-Cre⁺Gucy1b1^+/LoxP and Pf4-Cre⁺Gucy1b1^LoxP/LoxP mice as stated in the manuscript and thrombocyte count was measured using an automated hematology analyzer (XP-300; Sysmex Corp). PRP was centrifuged at 700g for 10 min to receive PPP used for blanking. Samples were incubated at 37 °C in glass cuvettes with constant stirring on an 8-channel personal computer-controlled platelet aggregation profiler (PAP-8, Biodata Corp) either in the presence of sodium nitroprusside (final concentration 10 μmol l⁻¹, catalog no. HN34.1; Carl Roth), BAY-747 (150 ppm) or vehicle (dimethyl sulfoxide (DMSO), 0.4%) for 2 min. Subsequently, thrombocyte aggregation was induced by the addition of ADP (final concentration 2 μmol l⁻¹, catalog no. 0203001; mölab), Ala-Tyr-Pro-Gly-Lys-Phe-NH₂ (final concentration 75 μM, catalog no. A3227; Sigma-Aldrich), U46619 (final concentration 5 μM, catalog no. 1932; Tocris Bioscience) and collagen (final concentration 1 μg ml⁻¹, catalog no. 0203009). Platelet aggregation was recorded over 5 min to measure the area under the curve and displayed as a.u. min.

PAH induction was performed twice, at baseline and following RDN or the sham procedure, depending on the allocation group, using a synthetic analogue of TXA2 (U46619; 10 mg/mL; Tocris Bioscience, MN, USA). During induction, the mean PA pressure was targeted at 40 mmHg. Continuous administration of TXA2 was gradually increased every 5 min according to a previously established protocol [13 (link)]. Electrocardiography (ECG) and both systemic pressure and PAP were continuously recorded. After the target PA pressure level was achieved, PWCP, PA pressure, RV, and RA pressures were measured and recorded; CO, SVR, and PVR were calculated before induction, at the target mean PA pressure, and after spontaneous reversal of mean PA pressure to baseline.

The GST-Rhotekin Rho-binding domain (RBD) fusion protein (obtained from Prof. Chris Marshall, Cambridge University) was bound to prewashed Glutathione-coated Sepharose beads (GE Healthcare, Buckinghamshire, UK). A negative control was also set-up using GST-only ‘bait’ protein. Meanwhile, serum-starved cells were treated with TBXA2R agonist U46619 (Tocris, Abingdon, UK) at a concentration of 10 nm for 10 min and then lysed. The pre-washed GST-bound beads were then resuspended in 100 μl PBS to which 500 μg of cell lysate was added. This was rotated at 4°C for 30 min to allow binding of GST-Rhotekin RBD to GTP-Rho. The beads were pelleted and resuspended in protein sample buffer and levels of total Rho in whole cell lysate and active Rho in GST-Rhotekin RBD and GST-only pull-down samples were measured by Western blot analysis.

The thromboxane analog U46619 was purchased from Tocris; N^ω‐Nitro‐L‐arginine (L‐NAME), phenylephrine, and Streptozotocin from Sigma.