EspP, an Extracellular Serine Protease from Enterohemorrhagic E. coli, Reduces Coagulation Factor Activities, Reduces Clot Strength, and Promotes Clot Lysis

Abstract

Background: EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7, a causative agent of diarrhea-associated Hemolytic Uremic Syndrome (D+HUS). The mechanism by which EHEC induces D+HUS has not been fully elucidated.

Objectives: We investigated the effects of EspP on clot formation and lysis in human blood.

Methods: Human whole blood and plasma were incubated with EspP(WT )at various concentrations and sampled at various time points. Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT), coagulation factor activities, and thrombelastgraphy (TEG) were measured.

Results and conclusions: Human whole blood or plasma incubated with EspP(WT) was found to have prolonged PT, aPTT, and TT. Furthermore, human whole blood or plasma incubated with EspP(WT) had reduced activities of coagulation factors V, VII, VIII, and XII, as well as prothrombin. EspP did not alter the activities of coagulation factors IX, X, or XI. When analyzed by whole blood TEG, EspP decreased the maximum amplitude of the clot, and increased the clot lysis. Our results indicate that EspP alters hemostasis in vitro by decreasing the activities of coagulation factors V, VII, VIII, and XII, and of prothrombin, by reducing the clot strength and accelerating fibrinolysis, and provide further evidence of a functional role for this protease in the virulence of EHEC and the development of D+HUS.

Fig 1. Concentration- and time-dependent prolongation of PT, aPTT and TT by EspP^WT.

(A) Fresh-frozen plasma from donor B was incubated (37°C, 4 h) with buffer alone (PBS-G) or with 0.1, 0.5, or 1.0 mg/mL of BSA, EspP^WT, or EspP^S263A. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were then determined for each sample. Shown are mean ± 95% CI values derived from three parallel experiments. Note that most error bars are smaller than the height of the data point markers due to the small variability between measurements. (B) Fresh-frozen plasma from 6 donors (A to F) was incubated (37°C) with buffer alone (PBS-G) or with 1.0 mg/mL of BSA, EspP^WT (WT), or EspP^S263A (S263A) for 0.5, 2, or 4 h. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) were then determined for each sample. Shown are values obtained from single measurements.

Fig 2. Selective concentration- and time-dependent reduction in coagulation factor activities by EspP^WT.

(A) Fresh-frozen plasma from donor B was incubated (37°C, 4 h) with buffer alone (PBS-G) or with 0.1, 0.5, or 1.0 mg/mL of BSA, EspP^WT, or EspP^S263A. Residual activities of coagulations factors II, V, VII, VIII, IX, X, XI, and XII were then determined for each sample. Graphically shown are mean ± 95% CI values derived from three parallel experiments. Note that these error bars are shorter than the height of the data point markers for many of the data points. (B) Fresh-frozen plasma was incubated (37°C) with buffer alone (PBS-G) or with 1.0 mg/mL of BSA, EspP^WT, or EspP^S263A for 0.5, 2, or 4 h. Residual activities of coagulation factors II, V, VII, VIII, IX, X, XI, and XII were then determined for each sample. Shown are values obtained from single measurements. Blood samples from donor A were not analyzed for residual activity of factors II, IX, X, XI, and XII. Blood samples from donor B were not analyzed for any factor activity.

Fig 3. Cleavage of human coagulation factor VIII by EspP.

Purified human coagulation factor VIII (10 μg) was incubated for 16 h at 37°C with buffer alone (50 mM TEA, pH 7.4 and 500 mM NaCl; lane 1) or with 0.1 μg EspP^WT (lane 2). EspP^WT incubated with buffer alone was loaded in lane 3. Molecular weight markers were loaded in lane M. Protein bands were visualized by SYPRO Orange staining.

Fig 4. Clot formation kinetics of whole blood treated with EspP.

Fresh citrated whole blood from 6 donors (A to F) was incubated with EspP^WT (1.0 mg/mL) or with EspP^S263A (1.0 mg/mL) for 0.5, 2, or 4 h, then analyzed by TEG. Blood from donors B and D were additionally incubated with buffer alone (PBS-G) as a negative control. Blood from donors B, C, E, and F were additionally incubated with BSA (1.0 mg/mL) as a negative control. Shown are the observed reaction time (R-time), clot formation time (K-time), and α-angle. These results can be explained by LPS contamination.

Fig 5. EspP accelerates fibrinolysis in only five of six additional donors.

Fresh citrated whole blood from 6 donors (A to F) was incubated with EspP^WT (1.0 mg/mL) or with EspP^S263A (1.0 mg/mL) for 0.5, 2, or 4 h, then analyzed by TEG. Blood from donors B and D were additionally incubated with buffer alone (PBS-G) as a negative control. Blood from donors B, C, E, and F were additionally incubated with BSA (1.0 mg/mL) as a negative control. Shown are the observed maximum amplitude of clot (MA) and percent clot lysis (LY30). Blue lines represent the MA and LY30 values obtained for donor A, who did not show an increased LY30 upon incubation with EspP^WT.

Fig 6. Thrombelastograph of whole blood from 6 donors after treatment with EspP^WT, EspP^S263A, or BSA.

Shown are the thrombelastographs, monitored for a duration of 70 min following reconstitution with calcium chloride, of blood samples treated with EspP^WT (*), EspP^S263A (×), and BSA (+).