Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation

Background: Histones are basic proteins that contribute to cell injury and tissue damage when released into the extracellular space. They have been attributed a prothrombotic activity, because their injection into mice induces diffuse microvascular thrombosis. The protein C-thrombomodulin (TM) system is a fundamental regulator of coagulation, particularly in the microvasculature, and its activity can be differentially influenced by interaction with several cationic proteins. Objective: To evaluate the effect of histones on the protein C-TM system in a plasma thrombin generation assay and in purified systems. Methods: The effect of histones on plasma thrombin generation in the presence or absence of TM was analyzed by calibrated automated thrombinography. Protein C activation in purified systems was evaluated by chromogenic substrate cleavage. The binding of TM and protein C to histones was evaluated by solid-phase binding assay. Results: Histones dose-dependently increased plasma thrombin generation in the presence of TM, independently of its chondroitin sulfate moiety. This effect was not caused by inhibition of activated protein C activity, but by the impairment of TM-mediated protein C activation. Histones were able to bind to both protein C and TM, but the carboxyglutamic acid domain of protein C was required for their effect. Histones H4 and H3 displayed the highest activity. Importantly, unlike heparin, DNA did not inhibit the potentiating effect of histones on thrombin generation. Conclusions: Histones enhance plasma thrombin generation by reducing TM-dependent protein C activation. This mechanism might contribute to microvascular thrombosis induced by histones in vivo at sites of organ failure or severe inflammation.