Tfllr

To assess the effect of glial cell activation on synapse parameters, spinal cords from litter-matched neonatal 5 day old homozygous PSD95-eGFP mice were obtained by dissection in artificial cerebrospinal fluid (aCSF, ~4 °C, equilibrated with 95% oxygen, 5% carbon dioxide) containing (in mM): 127 NaCl, 26 NaHCO₃, 10 glucose, 3 KCl, 2 CaCl₂, 1.25 NaH₂PO₄, and 1 MgCl₂. Isolated spinal cords were then kept in separate baths containing aCSF at room temperature, equilibrated with 95% oxygen, 5% carbon dioxide, for a total of 90 mins. The PAR1 receptor specific agonist, TFLLR (10 µM; Sigma-Aldrich), was bath-applied to a subset of spinal cords for either 15 mins or 60 mins prior to their fixation in ice cold PFA (4%). Histological processing of spinal cords was then conducted as described above.

Endothelial cells were washed and put into serum-free media for 1 h. To determine the role of TF-mediated signaling, EVs were pretreated with FVIIa and FX (10 and 150 nM, respectively, unless otherwise noted; Hematologic Technologies, Essex Junction, VT, USA). To inhibit TF, EVs were pretreated with 10 μg/ml HTF-1 (BD Bioscience, Franklin Lake, NJ, USA) or isotype control for 30 min. As positive controls, endothelial cells were treated with bovine thrombin (1 U/ml, Sigma-Aldrich, St. Louis, MO, USA), or FXa (80 nM, Hematologic Technologies). To inhibit FXa, FPRCK or EGRCK (40 μM, Hematologic Technologies) was added. To determine the role of endothelial PAR-1 and PAR-2 signaling, PAR-1 or PAR-2 agonist peptides (100 μM; TFLLR, Sigma-Aldrich, and SLIGKV, Abcam, Cambridge, UK, respectively) and scramble peptides (RLLFT, American Peptide Company, Sunnyvale, CA, USA, and VKGILS, Bachem, Torrance, CA, USA, respectively) were used with amastatin (10 μM, Santa Cruz Biotechnology, Dallas, TX, USA) to prevent peptide degradation (28 (link)). To inhibit PAR-1 and PAR-2, endothelial cells were pretreated with 0.1 μM E5555 (Axon Med Chem, Reston, VA, USA) and 50 μM FSLLRY (Bachem) for 30 min at 37°C. We did not use SCH79797, which induced cell death at the concentration required for PAR-1 inhibition (29 (link), 30 (link)). Untreated endothelial cells were used as negative controls.

The aCSF used for dissections and recordings contained (in mM) 127 NaCl, 26 NaHCO₃, 10 glucose, 3 KCl, 2 CaCl, 1.25 NaH₂PO₄, and 1 MgCl₂. Adenosine, 4-hydroxytamoxifen, and TFLLR were supplied by Sigma-Aldrich (Poole, UK); 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was supplied by Abcam (Cambridge, UK); 14–22 amide, SKF 38393, and tetrodotoxin (TTX) were supplied by Tocris Bioscience (Bristol, UK). TFLLR, 14–22 amide, and SKF 38393 were dissolved in reverse-osmosis water; 4-hydroxytamoxifen was dissolved in corn oil; TTX was dissolved in citrate buffer; and Adenosine and DPCPX were dissolved in DMSO. The concentration of DMSO in working solutions did not exceed 0.1% (vol/vol).