Pitstop 2

The following reagents were obtained: human fibronectin (EMD Millipore, Burlington, MA), poly-L-lysine solution (Sigma Life Science, St. Louis, MO), SPHERO Ultra Rainbow fluorescent particles (Spherotech, Inc, Lake Forest, IL), phorbol 12-myristate 13-acetate (PMA) (Enzo Life Sciences, Farmingdale, NY), human fibrinogen (Haematologic Technologies, Essex Junction, VT), and Pitstop 2 (Cayman Chemical, Ann Arbor, MI). Small interfering RNAs (siRNAs) against RUNX1, CAV1, FLOT1, IFITM3 interferon induced transmembrane protein 3, and CLTC clathrin heavy chain, and control siRNAs, were purchased from Santa Cruz Biotechnology Inc, (Dallas, TX). Supplemental Table 1 presents the antibodies and other reagents used.

Protein uptake and retention (up to 24 hours) was studied in washed platelets, PMA-treated HEL cells and CD34⁺ cell–derived MKs suspended in buffer using flow cytometry. Platelet suspensions (1 × 10⁶/mL) were incubated with fluorescence conjugated Cy3-albumin (10 μg/mL), Cy3-fibrinogen (50 μg/mL), or Cy3-IgG (20 μg/mL) at 37°C for different times. HEL cells or MK (1.5 × 10⁵) were incubated with fluorescent-conjugated albumin Alexa Fluor 488 (30 μg/mL), fibrinogen Alexa Fluor 647 (10 μg/mL), or IgG-Alexa Fluor 488 (30 μg/mL) at 37°C. Cells were fixed (2% paraformaldehyde) and uptake-retention was evaluated using a BD LSRII flow cytometry (San Jose, CA) and analyzed with FlowJo software, v10.5.3. The LSRII flow cytometer was calibrated using standard beads, SPHERO Ultra Rainbow Fluorescent Particles (Spherotech, Inc) on each experimental day. Where indicated, cells were incubated (15 minutes, RT) before uptake studies with 30 μM Pitstop 2 (Cayman Chemical).⁴ (link)