Thrombin receptor activating peptide 6 trap 6

Platelets were obtained from one day expired human platelet-rich plasma concentrates (anticoagulated with citrate) by centrifugation at 670× g for 10 min. Isolated platelet pellets were resuspended in platelet buffer (10 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose) and the concentration was adjusted to 4 × 10⁸ platelets/mL. CaCl₂ was added to a concentration of 1 mM. Prior to activation, in some cases preincubation of platelets was carried out with UFH (UFH, Ratiopharm GmbH, Ulm, Germany) and Enoxaparin (Sanofi, Paris, France) for 30 min. The anticoagulant exposure was performed in platelet buffer at a final concentration of 1 IU/mL which corresponds to therapeutic concentrations in anticoagulated patients. Platelets were stimulated with 42.5 µM Thrombin Receptor Activating Peptide-6 (TRAP-6) (Tocris Bioscience, Bristol, UK) or 1 × 10⁴ tumor cells/mL for 12 min at 37 °C. Activation by tumor cells was carried out with PC-3 and AsPC-1 cells in presence or absence of anticoagulants. Platelet supernatants/releasates were obtained by sample centrifugation for 20 min at 1000× g.

Diluted human whole blood or isolated human platelets were treated with SVEP1 as described previously, pre-incubated with FITC-PAC-1 and P-selectin-PE antibodies (BD, Franklin Lakes), and stimulated with either ADP (10 μM) (Chronolog, Harverton); Thrombin receptor-activating peptide-6 (TRAP-6; 10 μM) (Tocris, Bristol, UK); Protease-activated receptor-4 activating peptide (PAR4-AP; 100 μM) (Abcam, Cambridge, UK) or Thrombin (0.1 U/mL) (Chronolog, Harverton) for 15 min. Samples were immediately fixed and run using a CytoFlex analyzer and (Beckman Coulter, Pasadena) and analysis was performed with Kaluza software (Beckman Coulter, Pasadena).
For murine whole blood flow cytometry, whole blood was collected from the retro-orbital plexus using heparinized capillary tubes. Diluted whole blood was pre-incubated with fluorescently conjugated JON/A-PE and CD62P-FITC antibodies (Emfret, Eibelstadt, Germany) and stimulated with either ADP (10 μM), PAR4-AP (100 μM) or Thrombin (0.1 U/mL) for 15 min. After incubation, samples were immediately fixed and read on a CytoFlex analyzer.