Page 268 - Haematologica May 2020

J. Köhler et al.
Role of contact factors in bacterial-triggered fibrinolysis
S. pyogenes activates plasminogen to escape from the local site of infection;4 however, knockdown of PPK decel- erated bacterial spreading in mice, implicating a role of PK in bacteria-induced fibrinolysis. To further explore the human relevance to our findings, we employed in vitro plas- ma clot-lysis assays in normal human plasma and congen- ital FXII- or PPK-deficient plasma, followed by incubation
with streptokinase or the host plasminogen activators uPA and tPA. Clot lysis in PPK-deficient plasma was significant- ly longer, regardless of the fibrinolysis activator used (Figure 5A). In contrast, clot lysis in FXII-deficient plasma was significantly shorter compared to normal plasma. When corn trypsin inhibitor (CTI), a FXII inhibitor, was added to normal plasma, the clot lysis, induced by strep- tokinase, was again significantly shorter compared to nor- mal plasma (Figure 5B). Similarly, the addition of the PK
AB
CD
EF
Figure 1. Decreased mRNA levels of F12 and Klkb1 in vitro and in vivo after infection with S. pyogenes. (A and B) HepG2 cells (2x105 cells/mL) were incubated with IL6 (50 ng/mL) or S. pyogenes [2x106 colony forming units (CFU)/mL] for 6 hours (h). After incubation, cells were washed and the medium was replaced with fresh
medium containing 1% PenStrep. After 6 and 24 h, cells were harvested, total RNA was isolated, and real-time polymerase chain reaction (PCR) TaqMan® gene expression assays were performed. N≥9. (*P≤0.05; **P≤0.01; ***P≤0.001). (C-F) Groups of mice (n=8-10) were subcutaneously (sc.) infected with 2x107 CFU/mouse of S. pyogenes AP1. Animals were killed 6 and 24 h after infection, and liver tissue was collected for total RNA isolation (C-E) and real-time PCR TaqMan® gene expression assays were performed. (F) Spleen and liver were homogenized and the number of CFU was quantified 6 h after infection. *P≤0.05; ***P≤0.001.
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