Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent.

Coagulation factor XIa is a candidate target for anticoagulants that better separate antithrombotic efficacy from bleeding risk. We report a co-crystal structure of the FXIa protease domain with DEF, a human monoclonal antibody that blocks FXIa function and prevents thrombosis in animal models without detectable increased bleeding. The light chain of DEF occludes the FXIa S1 subsite and active site, while the heavy chain provides electrostatic interactions with the surface of FXIa. The structure accounts for the specificity of DEF for FXIa over its zymogen and related proteases, its active-site-dependent binding, and its ability to inhibit substrate cleavage. The inactive FXIa protease domain used to obtain the DEF-FXIa crystal structure reversed anticoagulant activity of DEF in plasma and in vivo and the activity of a small-molecule FXIa active-site inhibitor in vitro. DEF and this reversal agent for FXIa active-site inhibitors may help support clinical development of FXIa-targeting anticoagulants.

Factor XIa-specific IgG and a reversal agent to probe factor XI function in thrombosis and hemostasis.

Thrombosis is a major cause of morbidity and mortality. Current antithrombotic drugs are not ideal in that they must balance prevention of thrombosis against bleeding risk. Inhibition of coagulation factor XI (FXI) may offer an improvement over existing antithrombotic strategies by preventing some forms of thrombosis with lower bleeding risk. To permit exploration of this hypothesis in humans, we generated and characterized a series of human immunoglobulin Gs (IgGs) that blocked FXIa active-site function but did not bind FXI zymogen or other coagulation proteases. The most potent of these IgGs, C24 and DEF, inhibited clotting in whole human blood and prevented FeCl3-induced carotid artery occlusion in FXI-deficient mice reconstituted with human FXI and in thread-induced venous thrombosis in rabbits at clinically relevant doses. At doses substantially higher than those required for inhibition of intravascular thrombus formation in these models, DEF did not increase cuticle bleeding in rabbits or cause spontaneous bleeding in macaques over a 2-week study. Anticipating the desirability of a reversal agent, we also generated a human IgG that rapidly reversed DEF activity ex vivo in human plasma and in vivo in rabbits. Thus, an active site-directed FXIa-specific antibody can block thrombosis in animal models and, together with the reversal agent, may facilitate exploration of the roles of FXIa in human disease.

Oxidized low density lipoprotein blocks lipopolysaccharide-induced interferon beta synthesis in human macrophages by interfering with IRF3 activation.

In response to lipopolysaccharide (LPS) exposure, macrophages activate the transcription of a large number of pro-inflammatory genes by way of signaling pathways downstream of the LPS receptor, Toll-Like Receptor 4. Many of these genes are expressed sequentially in time, with early synthesis events resulting in the secretion of soluble factors that drive the transcription of genes expressed later in the activation cycle. In this study we show that human blood-derived macrophages pretreated with oxidized low density lipoprotein (OxLDL) fail to transcribe and secrete interferon beta (IFNbeta) immediately following LPS stimulation. As such, the normal downstream activation of Stat1 is blocked, and numerous IFNbeta/Stat1-activated genes, including the chemokines IP10 and ITAC, are weakly expressed or not expressed at all in these cells. Inspection of the LPS-induced activation state of several transcription factors known to play a prominent role in IFNbeta transcription reveals that, although NFkappaB, c-Jun, and ATF-2 activation appears normal, the LPS-induced activation of IFNbeta regulatory factor 3 (IRF3), as measured by DNA-binding activity and association with the coactivator CBP, is inhibited in the OxLDL pre-treated cells. These IRF3 activities have been shown to be essential for the initiation of transcription of the IFNbeta gene, and the loss of these activities presumably accounts for the lack of LPS-induced IFN beta transcription seen in the OxLDL pre-treated cells.