Thrombin's enzymatic activity increases permeability of endothelial cell monolayers.
Human alpha-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 alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
DeMichele
M A
MA
Department of Physiology, Albany Medical College 12208.
Moon
D G
DG
Fenton
J W
JW
Minnear
F L
FL
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004798:Enzymes / Q000494:pharmacology*; D006629:Hirudins / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D012709:Serum Albumin / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D004798:Enzymes; D006629:Hirudins; D010446:Peptide Fragments; D012709:Serum Albumin; C062510:hirugen; D013917:Thrombin; C027962:diisopropylphosphoryl-thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin