Antigen and substrate withdrawal in the management of autoimmune thrombotic disorders

Abstract

Prevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia, antiphospholipid syndrome and thrombotic thrombocytopenic purpura, include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 in heparin-induced thrombocytopenia and β(2)-glycoprotein-I (β(2)GPI) in antiphospholipid syndrome. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In thrombotic thrombocytopenic purpura, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, that is, enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor. A corollary of these concepts is that disrupting platelet factor 4 and β(2)GPI conformation (or ultralarge multimer of von Willebrand factor oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.

Figure 1

Oligomerization of the HIT antigen. (A) PF4 and ULC formation and dissociation. PF4 tetramers exist in a dynamic equilibrium with dimers and monomers. Under normal circumstances, tetramerization is favored when PF4 is bound to cellular GAGs (right pointing arrow on left side of figure), leading to a high surface density of PF4 and a propensity to form oligomers that are capable of binding multiple HIT antibodies. Heparin promotes the formation of PF4 ULCs in solution and on cell surface GAGs, which clusters antibody as well. The addition of 2-O, 3-O desulfated heparin (ODSH) disrupts ULCs into smaller complexes that bind fewer antibodies (right side of figure). Similarly, PF4 antagonists impede PF4 tetramerization (upward pointing arrows on left side of figure), leading to a lower surface density of PF4 tetramers, less propensity to form ULCs, and fewer sites for antibody binding. (B) Schematic of pathogenic versus nonpathogenic antibody binding. Simplest model showing distinction between effects of heparin on binding of pathogenic (KKO) and nonpathogenic (RTO) anti-PF4 antibodies. Heparin (orange) binds to a circumferential band of cationic residues on the surface of each PF4 tetramer (blue); the interrupted line represents binding to the distal side of the tetramer. Heparin neutralizes cationic charge repulsion among PF4 tetramers forming oligomeric complexes (shown here as a dimer for simplicity), which approximates the binding sites for KKO (panel B 1A,1B). Epitope approximation increases the avidity of KKO through increased proximity to more than 1 binding site on PF4 (1B). Some KKO antibodies may bind to epitopes on neighboring tetramers stabilizing ULCs induced by heparin (1A). In contrast, heparin has no such effect or may partially inhibit exposure of the epitope recognized by RTO (2). (C) Disruption of PF4 tetramerization. Two PF4 dimers are shown as ribbon diagrams based on the published crystal structure. A PF4 antagonist (gray) is bound to the lower dimer (red/blue) preventing association with the upper dimer (purple/cyan).

Figure 2

Exposure of the APS antigen. The APLA epitope in β₂GPI domain 1, composed at least in part by Arg39-Arg43, may become exposed and recognized by anti-β₂GPI antibodies. Circulating β₂GPI to be largely in a circular conformation (top left), in which this epitope (red dot) is not exposed. Shielding of this epitope may result from interactions between domain 1 (DI) and domain 5 (DV), or possibly by steric effects of carbohydrate residues originating from domains 3 (DIII) and 4 (DIV) (represented by the blue triangle). (A) Depicts “unfolding” of circular β₂GPI to a fishhook-like shape after binding to anionic phospholipid. Binding to this surface is mediated by domain 5 and results in exposure of the domain 1 epitope. Binding of bivalent anti-β₂GPI antibodies to the exposed epitope may then promote functional β₂GPI dimerization. However, to stimulate unactivated cells, this complex would need to dissociate from phospholipid and subsequently bind cellular receptors. (B) Depicts the proposed effect of direct binding of β₂GPI to putative cellular receptors (annexin A2, apoER2, GPIb; green semicircles). Subsequent β₂GPI unfolding and cross-linking by anti-β₂GPI antibodies may activate cells directly through receptor oligomerization. (C) Shows how binding of β₂GPI to cellular receptors may lead to unfolding and subsequent cross-linking by PF4 tetramers depicted in blue. Cross-linking via PF4 might directly activate cells or facilitate the ability of anti-β₂GPI antibodies to cross-link β₂GPI. (D) Shows how partial epitope exposure may be induced by β₂GPI deglycosylation, oxidation, or interactions with proteins and/or proteases derived from infectious agents. Subsequent binding of anti-β₂GPI anti-bodies may then occur coincident with binding of β₂GPI to cellular receptors. Antibody binding may stabilize and promote the unfolded conformation of β₂GPI.

Figure 3

Nonimmunosuppression strategies to treat autoantibody-mediated TTP. VWF is secreted from activated endothelial cells as ULVWF multimers, which recruit flowing platelets and form thrombi. (A) In the presence of flow and/or platelet binding, VWF is stretched to expose its A2 domain that is cleaved by ADAMTS13, resulting in dissociation of platelet aggregates. (B) In the absence of ADAMTS13 activity because of hereditary mutations of ADAMTS13 or acquired autoantibodies that block ADAMTS13 function, a disulfide bond reducing agent, such as N-acetylcysteine, or the C-terminal TSP1 repeats of ADAMTS13^, may be able to reduce the disulfide bridges linking VWF polypeptide subunits disassembling the VWF multimers. (C) In the setting of insufficient ADAMTS13 activity as described in panel B, aptamer or nanobody may bind to the VWF-A1 domain, inhibit the interaction between ULVWF and platelet receptor GPIb, and eliminate thrombus formation under flow. (D) Anti-ADAMTS13 inhibitory autoantibodies may be bypassed through the use of novel recombinant ADAMTS13 preparations that have been engineered to retain VWF cleaving activity while deleting autoantibody-binding epitopes.