Toll-like receptor 2 and 7 mediate coagulation activation and coagulopathy in murine sepsis

SUMMARY

Background:

Sepsis is a life-threatening condition often manifested as marked inflammation and severe coagulopathy. Toll-like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction, and mortality in animal sepsis.

Objectives:

Here we investigated the role of TLR signaling in mediating sepsis-induced coagulopathy (SIC) in a mouse model.

Methods:

Polymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed using rotational thromboelastometry (ROTEM) and presented as maximum clot firmness (MCF): 1) EXTEM to test tissue factor (TF)-initiated clot formation and 2) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified by ELISA. TF gene and protein expression was determined by qRT-PCR and flow cytometry, respectively.

Results:

Between 4 and 24 hours after CLP surgery, WT mice exhibited significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by a marked thrombocytopenia and a significant increase in plasminogen activator inhibitor-1, plasma TF, and d-dimer. In comparison, TLR2 ^−/− and TLR7 ^−/− CLP mice exhibited preserved MCF, platelet counts, and near normal plasma TF concentration. Bone marrow-derived macrophages treated with TLR2 agonist (Pam3cys) or TLR7 agonist (R837) had a marked increase in TF gene and protein expression. microRNA-146a, a newly identified proinflammatory mediator upregulated during sepsis, induced TF production via a TLR7-dependent mechanism.

Conclusions:

Murine sepsis leads to increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 plays an important role in procoagulant production and in SIC.