Suppression of platelet aggregation by Bordetella pertussis adenylate cyclase toxin

Abstract

The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 microM)-induced aggregation of rabbit platelets at 3 micrograms/ml and strongly suppressed thrombin (0. 2 U/ml)-induced aggregation at 10 micrograms/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.

FIG. 1

ACT and ACTK58Q preparations used in this study. ACT and ACTK58Q were prepared as described in Materials and Methods by DEAE-Sepharose Fast Flow chromatography. The preparations (20 μg of protein/lane) were loaded on an SDS–8% polyacrylamide gel and visualized by Coomassie brilliant blue staining. Lanes: 1, ACT; 2, ACTK58Q; 3, molecular weight markers.

FIG. 2

Effects of ACT on platelet intracellular cAMP accumulation. A rabbit platelet suspension (1.2 × 10⁸/ml) was incubated with indicated concentrations of ACT, ACTK58Q, or buffered 8 M urea for 5 min at 37°C in the presence or absence of calcium chloride as described in Materials and Methods. A cAMP radioimmunoassay was performed as described in Materials and Methods. Bars indicate standard errors from triplicate assays.

FIG. 3

Suppression of ADP-induced platelet aggregation by ACT. Platelets (1.5 × 10⁸/ml) were treated with ACT (3 μg/ml) or ACTK58Q (added as 2.4 μl of a 625-μg/ml solution in buffered 8 M urea), 10 μM forskolin (added as 2.0 μl of a 2.4 mM solution in dimethyl sulfoxide), 1 mM dibutyryl-cAMP (dbcAMP; 10 μl of a 50 mM aqueous solution), or 2.4 μl of buffered 8 M urea in 500 μl of Na,K-Tris (137 mM NaCl, 5.4 mM KCl, 11 mM glucose, 25 mM Tris-HCl [pH 7.4]) containing 1 mM CaCl₂ at 37°C for 5 min. Then 50 μl of 100 μM ADP was added, and changes in absorbance were recorded.

FIG. 4

Blocking of ACT-mediated suppression of platelet aggregation by anti-ACT antibody. Anti-ACT monoclonal antibody B7E11 or control mouse immunoglobulin G (IgG; Sigma) (6.6 μg/ml; added as 20 μl of a 165-μg/ml solution in Tris-buffered saline) and ACT (7.5 μg/ml; added as a 1.25 μl of a 3-mg/ml solution in buffered 8 M urea) were sequentially added to 500 μl of platelet suspension (1.5 × 10⁸/ml) in Na,K-Tris in the absence of calcium chloride and incubated for 8 min at 37°C; 25 μl of Na,K-Tris containing 20 mM CaCl₂ was then added; after a further 5 min of incubation at 37°C, 50 μl of 100 μM ADP was added to initiate platelet aggregation and changes in absorbance were recorded.

FIG. 5

ACT dose dependency in suppression of platelet aggregation. Platelets (10⁸/ml) were treated with indicated concentrations of ACT (added as 2.4 μl of solutions of appropriate concentrations in buffered 8 M urea) or buffered 8 M urea solution (2.4 μl) in 500 μl of Na,K-Tris containing 1 mM CaCl₂ at 37°C for 5 min. Then 50 μl of 100 μM ADP was added, and changes in absorbance were recorded. Percent suppression was calculated from the results of ACT-urea pairs in a two-chamber platelet aggregometer analyzed in parallel as follows: % suppression = 1 − (maximum aggregation of ACT-treated platelets/maximum aggregation of urea-treated platelets) × 100.

FIG. 6

Suppression of thrombin-induced platelet aggregation by ACT. Platelets (1.7 × 10⁸/ml) were treated with ACT (3 or 10 μg/ml; added as 2.4 μl of a 625-μg/ml solution or 2.9 μl of a 1.7-mg/ml solution in buffered 8 M urea, respectively) or ACTK58Q (10 μg/ml; added as 2.9 μl of a 1.7-mg/ml solution in buffered 8 M urea) or 2.9 μl of buffered 8 M urea in 500 μl of Na,K-Tris (137 mM NaCl, 5.4 mM KCl, 11 mM glucose, 25 mM Tris-HCl [pH 7.4]) containing 1 mM CaCl₂ at 37°C for 5 min. Then 50 μl of thrombin (2 U/ml) was added, and changes in absorbance were recorded.