EspP, An Extracellular Serine Protease From Enterohemorrhagic E. Coli, Induces Coagulopathy In Human Whole Blood and Plasma
Bibliographic record
Abstract
Abstract Abstract 4416 EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7. Brunder et al. (Mol Microbiol 1997, 24:767–78) have shown that EspP cleaves, amongst other proteins, human coagulation factor V, and the authors hypothesized that it may contribute to the mucosal hemorrhage in patients with EHEC infection. We have since shown that EspP also cleaves factor VIII. Since the mechanism by which EHEC induces diarrhea-associated Hemolytic Uremic Syndrome (D+HUS) has not been fully elucidated, and EspP has been cited as a putative virulence factor in D+HUS, we investigated the role of EspP in primary and secondary hemostasis in the pathogenesis of D+HUS. Wild type EspP (EspPwt) and EspPS263A, where the serine at the active site was mutated to an alanine thereby abolishing its proteolytic activity, were expressed in the non-pathogenic E. coli host BL21(DE3) and purified by hydrophobic interaction and size-exclusion chromatography. EspPwt at 1.0 mg/mL was incubated for 0.5, 2.0 and 4.0 hours ex vivo with citrated plasma from 6 healthy adults. EspPS263A, bovine serum albumin (BSA) and phosphate buffer saline-glycerol (PBS-G) served as negative controls. PT, aPTT and TT were found to be significantly prolonged and activity of factors V, VII, VIII and XII were reduced in a time- and concentration-dependent manner (Figures 1 and Figure 2). When citrated plasma was incubated with 1 mg/mL EspPwt at 37°C for 4 hours, PT was prolonged by 23.2 +/− 3.8 s, aPTT by 41.6 +/− 8.3 s and TT by 6.1 +/− 0.6 s, relative to the negative controls. Factor V activity decreased by 0.82 +/− 0.14 U/mL, factor VII by 0.72 +/− 0.28 U/mL, factor VIII by 0.69 +/− 0.31 U/mL and factor XII by 0.36 +/− 0.09 U/mL, relative to the negative controls. Prothrombin activity was significantly reduced (0.16 +/− 0.08 U/mL) compared to all negative controls but remained above 0.75 U/mL. Factors IX, × and XI activity, and fibrinogen concentration were not significantly different from the controls. To determine whether any cellular components in whole blood contribute to EspP's effect on the coagulation cascade, the experiment was repeated using citrated whole blood in place of plasma during the incubation phase. Plasma was then recovered and analyzed. Similar results were observed. The results suggest that EspP has proteolytic activity against specific coagulation factors at least in an ex vivo setting. In patients with EHEC infection, EspP may contribute to the hemorrhagic diarrhea by impairing the coagulation cascade. Further studies are needed to determine whether EspP is able to induce coagulopathy in vivo and if so, whether induction of such a coagulopathic state may favour the entry of Shiga toxin into systemic circulation in patients with D+HUS. Figure 1 EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 1. EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 2 EspP reduces coagulation factor activity in a time-dependent manner. Figure 2. EspP reduces coagulation factor activity in a time-dependent manner. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".