The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. (Blood. 2009;113: 1139-1148) IntroductionAside from its role in minimizing blood loss, the clotting system is also an effector arm of the immune system that modulates release of inflammatory mediators and prevents the spread of invasive microorganisms through extravascular fibrin deposition. This "quarantine through coagulation" strategy is evolutionarily primitive, as evidenced by the sensitivity of the horseshoe crab clotting system to activation by bacterial endotoxin. 1 Indeed, fibrin deposition is one of the hallmarks of inflammation in humans, and the induration accompanying delayed-type hypersensitivity skin reactions is caused by extravascular fibrin deposition. 2 Inflammatory responses to infection include expression of tissue factor on activated monocytes as well as increased acute phase proteins like fibrinogen and PAI-I, which contribute to hypercoagulable states. 3 Some bacteria modulate the host coagulation system to evade immune responses or facilitate dissemination through extravascular tissues. For example, staphylocoagulase and streptokinase form complexes with prothrombin or plasminogen, converting them into prothrombotic (thrombin) or profibrinolytic (plasmin) enzymes. 4 Likewise, plasminogen activator (Pla), a member of the omptin family of bacterial proteases, promotes dissemination of the plague agent, Yersinia pestis, from subcutaneous sites to the lymphatic and circulatory systems. 5 Proteolytic activation of plasminogen by Pla is thought to facilitate Y pestis migration through tissue barriers, because plasmin degrades fibrin clots and extracellular matrix components, activates procollagenases, and inactivates collagenase inhibitors. [6...

Activation of the clotting system is an effector function of the immune response, resulting in extravascular fibrin deposition whose purpose appears to be to limit the spread of invasive microorganisms. Activation of the clotting cascade may also modulate inflammatory reactions. Some bacteria are known to counteract the host coagulant response by modulating specific components of the clotting system, thereby promoting fibrinolysis or inhibiting coagulation activation. Pla, a member of the omptin family of Gram-negative outer membrane proteases, is a known virulence factor for the plague agent, Yersinia pestis. Pla’s ability to activate plasminogen by limited proteolysis is thought to facilitate the organism’s escape from fibrin meshworks, promoting its dissemination through extravascular tissues. We now show that Pla, as well as its homologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium, abrogate the function of the endogenous anticoagulant, tissue factor pathway inhibitor (TFPI). Using gene deletions, we found that bacterial inactivation of TFPI required the expression of these omptins. Consistent with prior studies of other omptin substrates, cleavage of TFPI by the Salmonella omptin was demonstrable only in rough LPS backgrounds. Western blotting experiments showed that proteolysis of TFPI correlated with loss of TFPI anticoagulant activity in clotting assays. The specificity of the omptin/TFPI interaction is supported by the finding that both common and distinct TFPI cleavage fragments are generated by the omptins in these three bacterial species. Furthermore, TFPI inactivation proceeds even in plasma where an abundance of competing protein substrates would be expected to render TFPI proteolysis almost non-existent if the reaction were not specific. We hypothesize that the sensitivity of TFPI to inactivation by bacterial omptins is a novel host-pathogen interaction that potentiates the procoagulant immune response to bacterial infection. With the heavy bacterial loads present in the circulation during the late stages of severe septicemia, this interaction may contribute to the development of disseminated intravascular coagulation and end organ failure.