Fluorescence microscopy of washed platelets adherent on collagen (COL)-, FN-, or VN-coated coverslips.

Platelets were placed on the coverslips for 15 minutes and then incubated with 20 mg/L FITC-FN, 2 mM Ca⁺⁺ in HEPES-Tyrode buffer supplemented with 1 g/L FA-free albumin in the absence (−) or presence (+) of LPA, 1 μM. Platelets were washed and fixed in 3% paraformaldehyde in PBS, pH 7.3, for 30 minutes; permeabilized with 0.4% octylglucopyranoside in PHEM buffer for 2 minutes; incubated with 0.1 mg/L rhodamine-labeled phalloidin; and photographed for FITC-FN, rhodamine, and phase contrast. Comparison of the fluorescence associated with platelets on the 3 substrates shows the heaviest deposition of FITC-FN by platelet aggregates adherent on collagen without or with additional agonist. Deposition of FITC-FN by spread platelets adherent to FN or VN was enhanced by LPA. Bar = 10 μm.