Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

Abstract

It has been postulated that thrombin binds to

Modulation of glycosaminoglycan production and antithrombin III binding by cultured aortic endothelial cells treated with 4-methylumbelliferyl-beta-D-xyloside.

“…14 Cells were placed into gelatin-precoated 35-mm Petri dishes containing RPMI-1640 medium, supplemented with 30% fetal calf serum and antibiotics (100 U/ml penicillin, 100 /ng/ml streptomycin, and 1.25 Mg/ml amphotericin B), and were incubated at 37° C in a humidified atmosphere of 5% CO 2 in air. At confluence, usually 7 to 9 days after seeding, endothelial cells were subcultured by employing 0.125% trypsin and 0.02% ethylenediaminetetraacetic acid in phosphate-buffered saline at a split ratio of 1:4.…”

“…14 Once it was established that results were qualitatively the same for primary and passaged cells, cells in the second to third passage were used in the present experiments.…”

“…The radioactivity eluted by heparin solution accounted for approximately 80% of the total radioactivity initially associated with the cells and was comparable to that dissociated by trypsin treatment. 14 Hence, it was considered to represent reversible antithrombin binding to the cell surface in the present study. The radioactivity in heparin and NaOH solutions was counted in a y-scintillation counter (Packard Instrument Co., Downers Grove, Illinois).…”

“…At the end of the radiolabelling period, medium was taken and applied to Pharmacia PD-10 column equilibrated with 0.01 M Tris-HCI (pH 7.4) containing 0.15 M NaCI for removal of free ^S-sulfate. Labelled macromolecules in a portion (0.5 of 3.5 ml) of excluded fractions were then precipitated with cetylpyridinium chloride ("medium" fraction) before or after treatments with 15 U/ml of chondroitin ABC or AC lyase for 4 hours at 37° C, or 0.6 U/ml of heparitinase for 3 hours at 43° C. 14 The cell layers were washed with Hanks' balanced salt solution 10 times, followed by incubation with 0.5 ml of 0.25% trypsin to lift the cells from the plates. The cell suspensions were centrifuged to obtain a supernatant ("cell surface" fraction) and a cell pellet ("cell" fraction).…”

“…The radioactivity recovered in "cell" and "plate" fractions was considerably less compared with that in other fractions. 14 The ^S-labelled glycosaminoglycans in "cell surface" fractions were isolated before or after treatments with chondroitin ABC lyase, following proteolytic digestion with pronase as described previously. 14 Glycosaminoglycans or proteoglycans isolated from medium and cells were dissolved in 0.4 ml of water, and their radioactivity was measured by liquid scintillation counting.…”