Prostaglandin e1 pge1

The platelets were isolated from C57B6J or P-selectin^−/− mice whole blood and anti-coagulated with sodium citrate 3.8%. Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood at 200 g for 20 min. Platelets were washed with prostaglandin E1 (PGE1) (Sigma, Italy) and resuspended in an ice-cold, HEPES-Tyrode buffer (pH 7.4) containing 129 mM NaCl, 8.9 mM NaHCO₃, 2.8 mM KCl, 0.8 mM KH²PO⁴, 56 mM dextrose, 10 mM HEPES. Immediately before platelet injection, 1 mmol/L MgCl², 1 mmol/L CaCl² and RGD peptide (GRGDNP) (Sigma, Italy) were added to the cells [43 (link)].

The whole blood was drawn from human subjects and centrifuged at 250g for 20 mins at room temperature to obtain platelet-rich plasma (PRP). The PRP was shifted to a new collection tube, followed by second centrifugation to absolutely spin down the residual blood cells. After treating with 100nM Prostaglandin E1 (PGE1, Sigma), the supernatant was centrifuged at 1000g for 5 mins to segregate plasma and platelets. The platelets were resuspended to 10⁸ platelets/ml in HEPES-Tyrode’s buffer. Platelet markers (CD41>95% positive) were used to determine the purity of platelets. The extraction method of platelet microparticles (PMPs) is described in previous studies (28 (link)). PMPs were extracted from the same amount of blood. Briefly, the platelet resuspension was slowly shaken at 10 rpm at room temperature for 4 hours. Next, platelets were centrifuged at 1000g for 5 mins. The supernatant was then centrifuged for 90 mins at 20,000 g at 18°C to obtain PMPs precipitates, and be resuspended in HEPES Tyrode’s buffer in the same volume as the platelets described above. Platelet markers (CD41>90% positive) were used to determine the purity of PMPs. The supernatant containing platelet-releasate was collected and stored at -80°C.

The PKA regulatory and catalytic subunits were transiently expressed [30 (link)]. Fluorometry was done using Förster/Fluorescence Resonance Energy Transfer (FRET) based biosensors for cytosolic Ca²⁺ and cAMP. For Ca²⁺ the troponin C-based sensor, Twitch-2B was used [31 (link)]. Additionally, for cAMP the Epac-based sensors Epac-S^H187 and Epac-S^H189 were used [32 (link)]. The compounds bradykinin, forskolin, 3-isobutyl-1-methylxanthine (IBMX) and ionomycin were from Calbiochem-Novabiochem Corp. (La Jolla, USA). Prostaglandin E1 (PGE1) was from Sigma-Aldrich (Zwijndrecht, The Netherlands). The PKA inhibitor H-89 was obtained from (Biolog, Bremen, Germany). The Epac-selective cAMP analogue 007-AM (8-pCPT-2-O-Me-cAMP-AM) was from Cayman Chemical (Michigan, USA). CaCl₂ salt was from Merck (Darmstadt, Germany). Fluorescent Ca²⁺ indicators Oregon Green 488 BAPTA-1-AM and Fura Red-AM were from Invitrogen, Life Technologies (Waltham, MA, USA).

Acid Citrate Dextrose (ACD)-A Vacuette tubes (455055) were purchased from Greiner Bio-One (Stonehouse, United Kingdom). The purinergic receptor agonist, ADP (01905), the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) (F3506) and prostaglandin E₁ (PGE₁) were purchased from Sigma Aldrich (Poole, United Kingdom). The HTS Transwell 96-well plates (3-μm pore size) (10077792) and RPMI 1640 cell media with l-glutamine (12004997) were purchased from Fisher Scientific (Loughborough, United Kingdom). The P2Y₁ antagonist, MRS2500 (2,159/1), and the P2Y₁₂ antagonist, AR-C66096 (3,321/1), were purchased from Bio-Techne (Minnieapolis, MN). Stromatol (321200S) was purchased from Mascia Brunelli (Milan, Italy). Tuerk’s Solution (93770) was purchased from Merck (Darmstadt, Germany).

Vacutainer tubes containing acid citrate dextrose (ACD) anticoagulant, anti-CD63 antibody-conjugated magnetic beads (catalog no.: 10606D), magnetic rack (catalog no.: 12321D), TaqMan^® miRNA ABC purification kit–Human Panel A, TaqMan^® microRNA reverse-transcription kit, MicroBCA^™ Protein Assay Kit, phosphate-buffered saline (PBS), RIPA buffer, and proteinase inhibitor were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cel-miR-238-3p and miR-126-3p were purchased from Genomics (New Taipei City, Taiwan). Lyophilized bovine serum albumin (BSA) and prostaglandin E1 (PGE1) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iodixanol (OptiPrep) was obtained from Axis-Shield (Dundee, Scotland, UK). Sodium cacodylate buffer and 300-mesh Formvar/carbon-coated grids were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).

Sanguinarine (13-methyl(1,3) benzodioxolo (5,6-c) -1,3-dioxolo(4,5-i) phenanthridinium), (HPLC ≥ 98%, SA, Figure 1) (Absin, Shanghai, China), was dissolved in 0.1% DMSO (final concentration). The EDTA, prostaglandin E1(PGE1) and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The CHRONO-LUME reagent and collagen were acquired from Chrono-Log Corp. (Havertown, PA, USA). The collagen-related peptide was obtained from Dr. Newman’s lab (Blood Center of Wisconsin, Milwaukee, WI, USA). The FITC-conjugated anti-CD62P (P-selectin) antibody and FITC-conjugated anti-PAC-1(αIIbβ3) antibody were purchased from Biolegend (San Diego, CA, USA). The Fluo-3AM was purchased from MCE (Shanghai, China). The CellTrace Calcein Green was purchased from Invitrogen (Carlsbad, CA, USA). The anti-phospho-PI3K(Ser1070), anti-PI3K, anti-phospho-Akt(Ser473), anti-phospho-GSK3β (Ser9), anti-phoshpo-PLCγ2 (Tyr1217), anti-β3, anti-phospho-β3 (Tyr474), anti-Src, anti-phospho-Src (Tyr416) and anti-phospho-Syk (Tyr525/526) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-Syk, anti-PLCγ2, anti-Akt and antil-GSK3β were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).