Keith A. Cockerill

Domain V of β2-Glycoprotein I Binds Factor XI/XIa and Is Cleaved at Lys317-Thr318

The fifth domain (DV) of β2-glycoprotein I (β2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). β2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, β2GPI retains binding to FXI but not to phospholipids. Native β2GPI, but not cleaved β2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on β2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of β2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves β2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of β2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of β2GPI.

In vivo characterization of bioconjugate B cell toleragens with specificity for autoantibodies in antiphospholipid syndrome.

This study investigated the use of well-defined bioconjugate molecules to suppress antigen-specific B cell responses to domain I (DI) of human beta(2)-glycoprotein I (beta(2)GPI) in rats. DI is the dominant target of pathogenic autoimmune antibodies in patients with antiphospholipid syndrome (APS), a disease characterized by antibody-mediated thromboembolic events. Rats primed with DI conjugated to keyhole limpet hemocyanin (DI-KLH) were rendered tolerant to subsequent antigen challenge by treatment with multivalent conjugates of DI. Antibodies to DI were suppressed 89-96% with intravenous doses of 500 micro g, and reductions were paralleled by decreases in splenic antigen-specific antibody-forming cells (AFC). Suppression was achieved with a variety of conjugates having two to four copies of DI and circulating half-lives of 2.6-8.7 h. Antibodies to KLH were not suppressed, indicating the specificity of the approach. These results establish the basis for further development of therapeutic B cell toleragens to suppress pathogenic antibodies in APS and other autoimmune diseases.

Therapeutic Potential of Toleragens in the Management of Antiphospholipid Syndrome

Autoantibodies to β2-glycoprotein I (β2GPI) are believed to be the primary cause of coagulation abnormalities in patients with antiphospholipid syndrome (APS). Clinical features include a range of life-threatening thrombotic events and microangiopathies affecting multiple organ systems. Current standard of care relies on long-term, high-intensity anticoagulation and is associated with a high risk for serious bleeding events. The relation between autoantibodies and the pathophysiology of APS is not clearly understood, but numerous in vitro studies have characterized the effects of antiphospholipid autoantibodies on various components of the coagulation cascade, including tissue factor and the protein C pathway. The fine specificity of autoantibodies to β2GPI is a subject of considerable debate; however, a body of evidence may offer resolution by integrating concepts of antibody affinity and assay sensitivity with carefully designed molecular studies. An investigational new therapy for APS is based on the approach that pathogenic antibodies may be reduced via depletion of circulating autoantibodies and induction of immune tolerance at the B-cell level. Preliminary results from a phase I/II clinical trial with LJP 1082, a B-cell toleragen, indicate the drug was well tolerated and may warrant further development for reduction of thrombotic events in patients with APS.