Interaction of Human Platelets with Enterohemorrhagic E. coli O111, Induces an Increase in the Procoagulant Activity, Thrombin Generation and Adhesion to Endothelial Cells

Acute inflammation in response to severe bacterial infection, results in hemostatic abnormalities ranging from subclinical to sustained systemic clotting activation leading to massive thrombin and fibrin formation and microvascular thrombosis. Endothelial activation and dysfunction are critical determinants of the host response and provide an explanation for the different abnormalities involved in the pathophysiology of sepsis.

Infection with pathogenic E. coli may present with a wide spectrum of clinical manifestations, from no symptoms or mild non-bloody diarrhea to severe cases, such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, which is characterized by hemolytic anemia and low platelet counts.

Although the understanding of the mechanisms that are involved in blood coagulation abnormalities in sepsis has gradually progressed, the role of platelets (Plts) on the procoagulant state during a severe infection remains to be addressed. Human platelets contain functional tissue factor (TF) (Panes et al. 2007) and TFPIa, but it is unknown if bacteria-platelet interaction affects platelet-TF procoagulant or platelet TFPI anticoagulant activities. Moreover, the effects of bacterial activation of platelets on thrombin generation (TG) in platelet rich plasma (PRP) or adhesion to endothelial cells have not been explored.

Aims: We assessed the effect of platelet-E. coli interaction on platelet TF-dependent procoagulant activity (PCA), the changes induced by this interaction on platelet TFPI, in TG in PRP and in the adhesive capacity of platelets on cultured HUVEC.

Plts activation by E. coli was demonstrated by a significant increase of p-selectin exposure on platelet surface compared to control Plts after 30 min of interaction with this microorganism harvested at exponential phase of growth and incubated in a ratio Plts/bacteria 1:10.

Platelet TF-dependent PCA was assessed by FXa generation in washed Plts exposed to E. coli, with addition of exogenous FVIIa and FX, with no extra source of TF. Using the same ratio Plts/bacteria, we observed an increase in FXa after 30 min of incubation, compared with control platelets (p=0.0002, n=12). This enhancement in TF-PCA was concomitant with a decreased expression of TFPI in Plts surface after exposure of PRP to E. coli for 30 min. (p=0.0012, n=9).

TG was measured in PRP, previously stimulated by E. coli for 30 min. We observed a shortening in the Lag time and time to peak and a higher thrombin peak in stimulated than in control PRP (p=0.0001, p=0.005 and p=0.0342, respectively, n=12). The reduction in lag time and time to peak was more pronounced than that obtained after eliciting platelet activation with Ristocetin. Preincubation of Plts with E. coli also increased the velocity index of TG compared to PRP alone (p=0,005; n=12).

Static adhesion of Pts to endothelium was studied by stimulating fresh washed Plts with E. coli for 30min and then co-incubating them with HUVEC. After 20 min, an increased number of bacterial-activated Plts were adhered to HUVEC, compared with unstimulated Pts. Moreover, visible Plts aggregates were observed, which were positive for fibrin immunostaining, suggesting clot formation during the interaction of Plts with E. coli O111.

Our findings show that Plts activated by bacteria results in an enhanced platelet procoagulant activity and adhesion to endothelium. By extension, these in vitro results suggest that platelets play an important role in the prothrombotic state associated with bacterial infections.

This work was supported by FONDECYT-Chile 1130835