Pa5 53814

Platelets were washed with 10 mM HEPES buffer, pH 6.5, containing 150 mM NaCl, 3 mM EDTA, 1M PGE1, and 0.3 units/mL apyrase. The washed platelets were re-suspended in modified Tyrode’s buffer (see STAR Methods, Method details, section isolation and enumeration of platelets). Platelets were activated by incubation with 1 U/mL thrombin and 3 mM CaCl₂ for 15 min. Subsequently, five hundred-microliter aliquots of isolated washed resting and activated platelets (2x10⁸ cells/mL) were lysed with 125 μL of 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer containing 1% NP-40, 150 mM NaCl, 4 mM EDTA, and protease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich). Proteins were then separated in 4-12% NuPAGE Bis-Tris gels (Life Technologies), and transferred to nitrocellulose membranes (IB301001, Life Technologies) for immunoblotting. Septin-2 was immunoblotted with the primary rabbit polyclonal antibodies against Septin-2 (Invitrogen, PA5-53814, 1:450). Immunoblotted protein bands were visualized using horseradish peroxidase-conjugated anti-rabbit IgG (SouthernBiotech, 4051-05, 1:1000) and ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences).

Glass-bottom microwell dishes (MatTek) were coated with human fibrinogen (Sigma-Aldrich) in PBS (20 μg/mL) overnight at 4°C, then rinsed with PBS and air-dried. Gel-filtered platelets in Tyrode’s buffer (pH 7.4) were layered onto fibrinogen-coated surfaces and platelets were allowed to spread for 60 min. For fluorescent microscopy, adherent platelets were fixed with 2% paraformaldehyde (PFA) for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 5 min and washed 3 times with PBS. For fluorescent staining, platelets were incubated in the blocking buffer for 60minat a room temperature, followed by overnight incubation with primary antibodies against septin 2 (Invitrogen, PA5-53814, 1:50) at 4°C. Platelets were washed with PBS 3 times, 5 min each, followed by 90-min incubation at room temperature with secondary Alexa Fluor 488-conjugated anti-rabbit antibodies (Invitrogen, A21441 1:1000) and phalloidin-iFLuor 594 (1:1000, Abcam) added to each well with fixed platelets. Finally, platelets were washed in PBS three times for 5 min and imaged with a Zeiss LSM 880 microscope.