Anti human cd62p phycoerythrin labeled murine antibodies

Platelet functionality was assessed in 27 RA patients and 15 healthy individuals in parallel experiments (one control for one or more RA samples analyzed at a time) by expression of P-selectin (CD62p) and active integrin αIIbβ3 (determined by its fibrinogen-binding capacity) before and after activation with the PAR1-specific thrombin receptor-activating peptide 6 (TRAP-6)(Bachem Americas Inc., Torrance, CA, USA). TRAP-6 was added to isolated platelets at 50 μM and incubated for 3 min at room temperature. Then platelets (200,000 in 50 μL in Tyrode’s buffer) were mixed with 0.045 μg/mL anti-human-CD62p phycoerythrin-labeled murine antibodies (BD Biosciences, San Jose, CA, USA) or 5 μg/mL Alexa Fluor 488-labeled human fibrinogen (ThermoFisher Scientific, Waltham, MA, USA) and incubated for 10 min. After incubation with the labeled ligands, the platelets were analyzed using a FacsCalibur flow cytometer equipped with BD Cell-Quest software (Table 1 and Figure S1). Platelets were gated based on their size and granularity and 5000 platelets were counted in each sample. FlowJo X software was used for data analysis.

Platelet functionality was analyzed in 11 VTE patients and 11 healthy individuals in parallel by expression of P-selectin (CD62p) and active integrin αIIbβ3 (determined by its fibrinogen-binding capacity) before and after activation with a thrombin-receptor activation peptide (TRAP-6, the PAR-1-specific hexapeptide Ser-Phe-Leu-Leu-Arg-Asn; Bachem Americas Inc., Torrance, California, United States). TRAP-6 was added to isolated platelets at 50 µM for 3 minutes at room temperature. Then platelets (200,000 in 50 µL) were incubated for 10 minutes with anti-human-CD62p phycoerythrin-labeled murine antibodies (BD Biosciences, San Jose, California, United States) (0.045 µg/mL) or Alexa fluor 488-labeled human fibrinogen (ThermoFisher Scientific, Waltham, Massachusetts, United States) (5 µg/mL). After incubation with the labeled ligands, the platelets were analyzed using a FacsCalibur flow cytometer equipped with BD CellQuest software. Platelets were gated based on their size and granularity and 5,000 platelets were counted in each sample. FlowJo X software was used for data analysis.