Coagulation Factor XI and Factor XII in DNA-Induced Thrombin Generation

Components of the blood coagulation mechanism are activated during infections as part of the innate immune response to invading microorganisms. This process is crucial for limiting spread of infection; however, excessive activation of coagulation can contribute to thrombotic tissue ischemia and systemic inflammation. During a variety of conditions associated with leukocyte activation and tissue damage, including sepsis, levels of cell free DNA rise in plasma. Neutrophils are one source of free DNA, releasing chromatin upon activation to form neutrophil extracellular traps (NETs). DNA in NETs form a physical barrier that contains microorganisms, while histones and antimicrobial proteins associated with DNA have bactericidal properties. NETs trigger platelet activation and coagulation, and likely contribute to inflammation, thrombosis and disseminated intravascular coagulation in sepsis. In a murine venous thrombosis model, neutrophils and NETs appear to contribute to thrombus formation through a process involving factor XII (fXII) (von Bruhl et al. J Exp Med 2012;209:819). When plasma fXII binds to anionic surfaces it is converted to the protease fXIIa. The results with the mouse model suggest that DNA, an anionic polymer, contributes to thrombosis by enhancing fXII activation.

FXIIa contributes to thrombin generation and thrombosis by converting zymogen factor XI (fXI), to the protease factor XIa (fXIa). FXI has been implicated in the pathology of sepsis and thrombosis in animal models. Mice lacking fXI have better survival than wild type mice after ligation and puncture of the cecum, and are more resistant to Listeria infection. Inhibition or deficiency of fXI/fXIa confers an antithrombotic phenotype in rodents, rabbits and primates. Furthermore, epidemiologic data support a role for fXI in arterial and venous thrombosis in humans. FXI is activated in plasma by fXIIa or thrombin; however, the reactions proceed slowly in the absence of anionic substances. This suggests that a cofactor with anionic properties is required for fXI activation in vivo. For example, polymers of inorganic phosphate (polyphosphate or polyP) released by platelets enhance fXI activation by thrombin 3000-fold and by fXIIa 30-fold. PolyP also induces fXI and fXII autoactivation. The backbone of DNA is a type of phosphate polymer. We examined the capacity of DNA to enhance fXI and fXII activation and to contribute to fXI/fXII-dependent plasma coagulation.

We isolated DNA from human peripheral blood leukocytes. Like polyP, DNA induced fXII autoactivation. Similarly, fXI underwent autoactivated in the presence of DNA, with the rate of activation increasing as DNA concentration increased. For reactions with either fXII or fXI, the enhancing effect of DNA was neutralized by treatment with DNAse. DNA, but not DNAse treated DNA, enhanced fXI activation by fXIIa 4-fold and by thrombin 30-fold compared to reactions without DNA. FXI is a dimeric protein composed of two identical subunits. PolyP binds to each fXI subunit through two anion binding sites (ABS), one on the fXI apple 3 domain (ABS1) and the other on the fXI catalytic domain (ABS2). These interactions are required for polyP-mediated enhancement of fXI activation by thrombin and fXIIa. Using DNA as a cofactor, the rate of activation of fXI lacking ABS1 by thrombin or fXIIa was 4-fold slower than for wild type fXI, while DNA failed to enhance activation of fXI lacking ABS2. This indicates that binding of fXI to DNA through the ABSs is required for expression of the DNA cofactor effect. Addition of DNA (50 ug/ml) induced thrombin generation in normal plasma (Endogenous Thrombin Potential 356 nM.min) through a process that was blocked by a fXIIa inhibitor (corn trypsin inhibitor) or a monoclonal antibody to fXI. Thrombin generation is dependent on fXI in normal plasma triggered to clot with a low (0.2 pM) concentration of tissue factor. Addition of DNA (50 ug/ml) to this system increased peak thrombin generation and endogenous thrombin potential by ~50%, while shortening time to peak thrombin potential. This effect was completely neutralized by an antibody to fXI. In conclusion, fXII and fXI have a propensity to bind to, and become activated on, anionic polymers and surfaces. Our results suggest that DNA released from leukocytes or damaged tissues could enhance activation of these protease zymogens, facilitating their contribution to hypercoagulability and inflammation.