A549 LUNG EPITHELIAL-CELLS SYNTHESIZE ANTICOAGULANT MOLECULES ON THE CELL-SURFACE AND MATRIX AND IN CONDITIONED MEDIA

To investigate mechanisms regulating intra-alveolar coagulation, we studied monolayers of the A549 human lung epithelial cell line. The surface of A549 cells delayed the onset of prothrombin-to-thrombin conversion and prevented total prothrombin consumption in normal plasma compared to plastic cell-free wells. Similar results were achieved with bovine pulmonary endothelial (CPAE) and rat intestinal epithelial (IEC-6) cell lines, whereas Madin-Darby canine kidney renal epithelial cell line accelerated thrombin formation. The A549 surface catalyzed antithrombin III-thrombin complex formation with no significant increase in thrombin inactivation from heparin cofactor II. The A549 cell surface effects were largely, but not completely, reversed to values obtained for plastic when protein C-deficient plasma was used. Pretreatment of the cell surface with chondroitinase ABC plus heparitinase prior to thrombin generation experiments had no effect on the total prothrombin consumed but decreased the initial delay. Heparan sulfate as well as dermatan sulfate and other chondroitin sulfates were detected on the A549 surface using alcian blue staining. Conditioned media from A549, CPAE, and IEC-6 cells delayed the clot time of recalcified plasma. Use of chondroitinase ABC and heparitinase were both required to obliterate the A549 conditioned media activity. After growing A549 cells in (SO4)-S-35-O2--containing medium, the resultant conditioned medium was found to contain 2,000 kD and 300- to 1,000-kD proteoglycans that yielded chains of less-than-or-equal-to 100 kD on reductive elimination with base. Active fractions from the conditioned medium were shown to increase the heparin cofactor II-thrombin complex formation in plasma reacted with [I-125]thrombin and analyzed by densitometry of autoradiograms made after sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was concluded that A549 cells inhibit prothrombin consumption and active thrombin formation on the cell surface via a combination of mechanisms involving inactivation of Va and VIIIa by active protein C generated by thrombomodulin-thrombin complexes and catalysis of antithrombin III-thrombin formation by heparan sulfate glycosaminoglycans. Also, the A549 cells accelerate thrombin inhibition by secretion of dermatan sulfate and heparan sulfate proteoglycans into the surrounding media.