HlyA cause platelet activation and neutrophil/ monocyte interaction during urosepsis

The urinary tract is frequently a primary infection site in patients with severe sepsis or septic shock . Urinary tract infections are most commonly caused by E.coli sub‐strains that produce several virulence factors – , of which α‐haemolysin (HlyA) is the most abundant . Our previous data demonstrate that the biological effects of HlyA are intimately associated with extracellular ATP signalling secondarily to ATP release from cells through the HlyA‐pore. Our previous data support that HlyA is largely responsible for the septic symptoms observed in response to bacteraemia with uropathogenic E.coli . ATP and its degradation products are aggressive activators of immune cells and platelets – , and prime candidates for promoting the intravascular coagulation observed during septic shock. We tested the effect of antagonising platelet P2Y receptors (P2Y 12 and P2Y 1 ) during sepsis with uropathogenic E.coli (ARD6, O6:K13:H1) in anaesthetised mice. In aggreement with the literature, infusion of the P2Y 12 antagonist cangrelor (8.6 or 86mg/hour) did not change survival or any tested sepsis parameters. However, the P2Y 1 inhibitor MRS2500 reduced platelet activation by pore‐forming bacterial toxins (70–80%) in vitro , measured as surface exposure of fibronectin, DC 63 and P‐selectin. I n vivo , infusion of MRS2500 (624μg/hour) increased the survival in mice exposed to uropathogenic E. coli . Moreover, infusion of MRS2500 partially prevented the 60% fall in platelet count otherwise seen in response to sepsis in control mice. Interestingly, targeting of P2Y 2 receptors, which is highly expressed on neutrophils and monocytes but not in platelets, showed that lack of P2Y 2 receptors entirely prevented the fall in circulating platelets in response to sepsis. Moreover, P2Y 2 receptor‐deficient mice die much earlier in response to intravenous application of uropathogenic E. coli compared to controls. By flow cytometry, we are able to detect increased complex formation between thrombocytes (CD 42 ) and neutrophils (Ly 6 ‐G) /monocyte (Ly 6 ‐G/CD11B). These data could potentially suggest that complex formation between thrombocytes and neutrophils and/or monocytes are important for the observed reduction in free thrombocyte numbers during sepsis and that the complexes could dampen the septic reaction to the circulating bacteria and improve survival. Support or Funding Information This project is funded by The Independent Research Foundation ‐ Denmark The Augustinus Foundation