Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

Abstract

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)

Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis *

“…Thrombin, the ultimate product of these reactions, is an extremely potent platelet activator (6,7) and converts fibrinogen to a fibrin clot (8), thus plugging damaged vessels. Besides its central role in hemostasis, thrombin also enhances vascular permeability (9), induces leukocyte chemotaxis (10,11), and potentiates lipopolysaccharide-stimulated interleukin-1 production by macrophages (12). These data, and the fact that prothrombin activation in vivo is known to be associated with inflammatory conditions, implicate thrombin as a major player in inflammation.…”

Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury

“…In bovine pulmonary arterial endothelial cell monolayers, ␣-thrombin causes a dose-dependent increase in endothelial permeability to albumin (6,7,58,59,102,145,153). Interestingly, ␥-thrombin, which lacks the fibrinogen recognition site, also causes a dose-dependent increase in endothelial permeability to albumin (6,39,174). In bovine pulmonary arterial endothelial cell monolayers, the ␣-thrombin-active catalytic site is required for the increase in transendothelial permeability to albumin (7).…”

Identification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1

“…The various activities are either dependent or independent of the active site. For instance, the procoagulant activity, the mitogenic activity on fibroblasts (Perdue et al, 1981) and the effect on the permeability of endothelial cell monolayers (DeMichele et al, 1990) is dependent on an intact active site, whereas the chemotactic activity (Bar-Shavit et al, 1983) and the mitogenic activity for vascular smooth muscle cells (Bar-Shavit et al, 1990) is expressed also by active-site-blocked thrombin. Cleavage of thrombin by RMCP-1 leads to destruction of the active site of thrombin and would thus inhibit the active-sitedependent activities of thrombin.…”