THROMBIN ENZYMATIC-ACTIVITY INCREASES PERMEABILITY OF ENDOTHELIAL-CELL MONOLAYERS

Human α-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of α-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included α-thrombin and two digestion products, ζ-thrombin (chymotryptic product with 89% clotting activity) and γ-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) α-thrombin and diisopropylphosphorofluoridate-(DIP) α-thrombin. Cell binding activity of α- and PPACK-α-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-α-thrombin. γ-Thrombin, on the other hand, did not compete for binding of 125I-labeled α-thrombin. All enzymatic forms of α-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with α- or γ-thrombin prevented the increase in permeability, further indicating that α-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of α-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of α-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion -binding site of α-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with α-thrombin. Addition of hirugen (which inhibited binding of 125I-α-thrombin comparable to competition of 125I-α-thrombin with unlabeled α- or PPACK-α-thrombin) to α-thrombin did not prevent the increase in permeability. These findings indicate that the thrombin-induced increase in endothelial permeability requires thrombin's enzymatic activity but not high-affinity binding activity.