Mutagenesis studies toward understanding the intracellular signaling mechanism of antithrombin

Abstract

Summary background: Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties.

Objectives: The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site.

Methods: Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P(1)). Furthermore, the roles of prostacyclin and nuclear factor (NF)-kappaB in modulating these effects were investigated.

Results: Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-kappaB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P(1) was inconsequential.

Conclusions: The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P(1)-related G(i)-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.

Figure 1

Crystal structure of AT. The P3-P4’ residues of RCL in AT/Proth-2 (Ala-391 to Pro-397, colored in black) were replaced with corresponding residues of prethrombin-2 (Pre-2). The D-helix residues of AT (Thr-115-Arg-132) are colored in black. The side chains of Lys-114 in the beginning of D-helix and Lys-125 are marked. The coordinates (Protein Data Bank accession code 2ANT) were used to prepare the figure [28].

Figure 2

Barrier protective activity of AT derivatives in endothelial cells in response to LPS. (A) The effect of increasing concentrations of LPS (x-axis) on the permeability of EA.hy926 cells was measured as described under “Materials and methods”. (B) The same as (A) except that the activities of different concentrations of AT-WT were monitored in cells stimulated with LPS (10 ng/mL). (C) The same as (B) except that the activities of AT derivatives (150 µg/mL each) were measured in the absence (white bars) and presence (black bars) of indomethacin. (D) Effect of AT derivatives on the induction of PGI₂ synthesis was analyzed by measuring the enhancement in the concentration of 6-keto-prostaglandin F_1α in cell culture media in the absence (white bars) and presence (black bars) of indomethacin. Data are expressed as mean ±SEM (n=3).

Figure 3

Comparison of the barrier protective activities of APC and AT. (A) The barrier protective activity of APC (20 nM, 4h) or AT-WT (150 µg/mL, 4h) in LPS-stimulated endothelial cells (10 ng/mL for 4h) was measured before and after treatment of cells with PTX (100 ng/mL, 16h) or with specific siRNA for S1P₁ (0.2 µg/mL, 3h) and MβCD (10 mM, 1h prior to treatment with agonists). (B) The same as A except that the activity of AT-WT alone or in complex with pentasaccharide or heparin (5 µM) was measured. For the experiments in the presence of siRNA, the cell monolayer was treated with non-specific (NS) or specific siRNA for syndecan-4 (0.2 µg/mL, 3h) prior to treatment with AT. (C) The same as A except that the concentration-dependence of barrier protective activity of AT-WT (open circles) was compared with that of cleaved (filled circles) and latent AT (open squares). Data are expressed as mean ±SEM (n=3). (D) SDS-PAGE (10%) and Western-blotting of non-treated (Cont), non-specific siRNA (NS) and syndecan-4 siRNA treated EA.hy926 cells developed with an anti-syndecan-4 antibody.

Figure 3

Comparison of the barrier protective activities of APC and AT. (A) The barrier protective activity of APC (20 nM, 4h) or AT-WT (150 µg/mL, 4h) in LPS-stimulated endothelial cells (10 ng/mL for 4h) was measured before and after treatment of cells with PTX (100 ng/mL, 16h) or with specific siRNA for S1P₁ (0.2 µg/mL, 3h) and MβCD (10 mM, 1h prior to treatment with agonists). (B) The same as A except that the activity of AT-WT alone or in complex with pentasaccharide or heparin (5 µM) was measured. For the experiments in the presence of siRNA, the cell monolayer was treated with non-specific (NS) or specific siRNA for syndecan-4 (0.2 µg/mL, 3h) prior to treatment with AT. (C) The same as A except that the concentration-dependence of barrier protective activity of AT-WT (open circles) was compared with that of cleaved (filled circles) and latent AT (open squares). Data are expressed as mean ±SEM (n=3). (D) SDS-PAGE (10%) and Western-blotting of non-treated (Cont), non-specific siRNA (NS) and syndecan-4 siRNA treated EA.hy926 cells developed with an anti-syndecan-4 antibody.

Figure 4

Protective effect of AT derivatives in the LPS-mediated neutrophil adhesion assay. (A) Adhesion of neutrophils to LPS-treated EA.hy926 cells (10 ng/mL, 4h) was analyzed after treating monolayers with increasing concentrations of AT-WT for 4h. (B) The same as (A) except that adhesion was monitored in cells pre-treated with AT derivatives (150 µg/mL) in the absence (white bars) and presence (black bars) of indomethacin. (C) LPS-mediated expression of adhesion molecules VCAM-1 (white bars), ICAM-1 (gray bars) and E-selectin (black bars) in confluent EA.hy926 cells in AT-treated cells (150 µg/mL). (D) The same as (C) except that analysis of the expression of adhesion molecules was conducted in the presence of indomethacin. Data are expressed as mean ±SEM (n=3).

Figure 5

Regulation of the activation of NF-κB by AT derivatives in EA.hy926 cells. (A) LPS-mediated (10 ng/mL, 1h) activation of NF-κB by AT derivatives (150 µg/mL) was analyzed by Western-blotting using specific antibodies as described under “ Materials and methods”.

Figure 6

Apoptotic activity of AT derivatives in LPS-stimulated and non-stimulated EA.hy926 cells. (A) Confluent monolayers of EA.hy926 cells were treated with AT derivatives (150 µg/mL) for 24h followed by induction of apoptosis with LPS (10 ng/mL, 4h). Cells were fixed with paraformaldehyde and incubated with the TUNEL reaction mixture followed by Hoechst 33342 to stain apoptotic cells (green) and the total number of nuclei (blue), respectively. (B) The same as A except that the apoptotic activities were monitored without stimulation of cells with LPS. (C) The same as B except that the concentration-dependence of the apoptotic activity of AT-WT (open circles), AT-K114E (filled circles) and AT-K125E (open squares) was measured and the number of apoptotic cells was expressed as the percentage of TUNEL-positive cells of the total number of nuclei. (D) The same as C except that the concentration-dependence of the apoptotic activity of AT-WT (open circles), cleaved AT (filled circles) and latent AT (open squares) was measured. The number of TUNEL-positive cells in the absence of treatments was 10–15%. Data are expressed as mean ±SEM (n=3).

Figure 7

Comparison of the apoptotic activity of AT derivatives in EA.hy926 cells. The apoptotic activity of AT-WT, latent AT (AT-L) and AT-K114E (20 µg/mL) was measured and the number of apoptotic cells was expressed as the percentage of TUNEL-positive cells. The symbols in successive order are: AT alone (white bars); AT+pentasaccharide (gray bars); AT+heparin (dark gray bars); AT+MβCD (black bars); AT+non-specific siRNA (vertical hatched bars); AT+siRNA specific for syndecan-4 (horizontal hatched bars). Data are expressed as mean ±SEM (n=3).