Clb gran 12

In order to analyze platelet activation, CSP were stimulated with thrombin receptor-activating peptide-6 (TRAP-6, 10 µM, HART Biologicals, Hartlepool, UK) and incubated with CD62-P (CLB-Thromb/6, Beckman Coulter, Krefeld, Germany) or CD63 (CLB-Gran/12, Beckman Coulter) and measured by flow cytometry (FC) (Navios, Beckman-Coulter, Krefeld, Germany). Light transmission platelet aggregation assay was performed using a four-channel aggregometer (LABiTec, LAbor BioMedical Technologies, Ahrensburg, Germany) to investigate the aggregation ability in response to 20 µM TRAP-6, 1.0 mg/mL ristocetin (HART Biologicals, Hartlepool, UK) or NaCl (Braun, Melsungen, Germany). Next, the adhesion ability of CSP was assessed as previously described using coverslips (Corning, New York, USA) coated with 100 mg/mL fibrinogen (Sigma Aldrich, Munich, Germany) or 5% human serum albumin (Grifols, Berlin, Germany). 9 (link) CSP were allowed to seed on coverslips in the presence of 10 µM TRAP-6. Platelet clot retraction was assessed upon incubation with 7.4 µM CaCl 2 (Sigma Aldrich, Darmstadt, Germany) and thrombin 10 U/mL (Roche, Mannheim, Germany). Thromboelastography was performed using the ClotPro analyzer (Haemonetics, Munich, Germany) according to the manufacturer's instructions. See the Online Supplementary Appendix for further information concerning each assay.