Platelet Adhesion and Spreading on Fibrinogen

Platelet spreading on fibrinogen was assayed as previously described [4 (link)]. Briefly, glass cover slips were coated with fibrinogen overnight at 4 °C. Non-specific binding was blocked by incubating cover slips with bovine serum albumin (BSA, 1%) in Tyrode’s-HEPES buffer at 37 °C. Cover slips were rinsed with Tyrode’s-HEPES buffer after removing BSA. Aspirin (1 mM)-treated washed human platelets containing apyrase (3 U Ml⁻¹) were layered over cover slips in the presence or absence of Phox-I. After 10 min incubation at 37 °C the cover slips were rinsed with phosphate buffered saline (PBS) to remove free platelets. Platelets on cover slips were then fixed with 4% paraformaldehyde for 10 min, rinsed with PBS twice and permeabilized with 0.1% Triton X-100 for 60 s. After two rinses with PBS, platelets were stained with Alexa 594-phalloidin to visualize F-actin. A Carl Zeiss LSM-510 confocal Axioplan 200 microscope and a Plan-Neofluar 100×/1.45 oil objective were used to generate platelet images. Digital images were processed using Zen 2007 software from Carl Zeiss (Thornwood, NY, USA).