Opposites attract

In this issue of Blood, Sikara and colleagues link ever more closely 2 autoimmune disorders with remarkably similar target antigen characteristics.¹ 

Antiphospholipid syndrome (APS) and heparin-induced thrombocytopenia (HIT) are 2 prothrombotic syndromes in which antibodies against complexes of charged molecules are of fundamental importance. In both syndromes, IgG antibodies directed against positively charged endogenous proteins, β2 glycoprotein I (β2-GPI) in APS and platelet factor 4 (PF4) in HIT, are of major relevance. In APS, the antibodies are clearly autoantibodies, whereas in HIT, they are drug-induced. But at least a subset of these antibodies also shows features of autoreactivity.² 

Parallels between these 2 syndromes are well recognized,³  but the final link between them has remained enigmatic. In this issue of Blood, Sikara et al demonstrate in a series of in vitro experiments an interaction between β2-GPI and PF4.¹  This not only indicates a potential link between APS and HIT, but also raises a whole series of exciting new research questions. From a clinical perspective, these findings shed new light on the observation that APS patients often also have anti-PF4 antibodies in their plasma,⁴  raising issues of clinical relevance of these “PF4-only” autoantibodies. Potentially, binding of both types of antibodies (ie, anti–β2-GPI and anti-PF4) to PF4/β2-GPI–containing complexes could generate pathologic immune complexes, with 2 distinct mechanisms of cellular activation, one via the F(ab′)₂ part of the β2-GPI-IgG (as shown in the present paper) and one via the Fc part of the anti-PF4–IgG antibodies (as established for HIT).⁵ 

An alternative hypothesis, however, is that these anti-PF4 antibodies are protective—inhibiting complex formation of β2-GPI and PF4 by steric hindrance. The authors also show enhanced binding of IgG antibodies to β2-GPI/PF4 complexes compared with β2-GPI alone. This may not represent enhanced affinity as concluded by the authors, but more likely the effect of more β2-GPI protein being coated within the complexes compared with coating of β2GPI alone. It should be noted that all experiments reported by Sikara et al were performed using purified systems in vitro, and so whether these mechanisms also occur in a plasma-containing system or on cell surfaces needs to be determined. This is especially relevant, as interactions of charged molecules will be influenced by ions in the plasma.

These research findings could have treatment implications. β2-GPI and PF4 interact with each other because of opposite charges. Although β2-GPI exposes positive charges predominantly at domain V (by which it binds to phospholipids) and to a minor extent also at domain I (the major binding site for prothrombotic autoantibodies), it also contains negatively charged epitopes at domains III and IV.⁶  If the in vitro role of PF4 in stabilizing β2-GPI–containing complexes, and thereby mediating autoantibody binding to domain I (as proposed by the study of Sikara et al¹ ), is also relevant in vivo, interference with this charge-related interaction might be key to interrupting the prothrombotic process in acute severe APS. High-dose unfractionated or perhaps even low-molecular-weight heparin should be a good candidate for such an approach, as its charges are higher than β2-GPI's and should displace it through competitive binding of PF4, in addition to providing anticoagulant effects.

A strength of the study of Sikara et al is their molecular modeling of the interaction between β2-GPI and PF4: binding sites become exposed for the 2 F(ab) parts of the anti–β2-GPI IgG autoantibodies in an optimal distance for enabling high-affinity IgG binding.¹  This parallels previous work on the steric interactions of PF4 and heparin, and the resulting multimerized complexes.⁷ 

Both examples show the potential of using biophysical technologies to better understand the physical properties underlying the mechanisms turning endogenous proteins into autoantigens.

However, perhaps the most important perspective of the study of Sikara et al is that it indicates that β2-GPI and PF4 may be 2 representatives of an up-to-now unrecognized charge-related system in host defense. Isn't it striking that the biological roles of these 2 evolutionary highly preserved proteins are unknown? It is well recognized that negatively charged molecules derived from bacteria such as RNA and DNA trigger Toll-like receptors and are important in mediating host defense but also in triggering and maintaining autoimmunity.⁸  It is obvious that charged endogenous molecules and antibodies directed against these molecules are involved in these defense mechanisms. In fact, most routine laboratories use lupus-anticoagulant insensitive partial thromboplastin time (PTT) reagents to avoid the frequent detection of prolonged PTTs in children with recent infections. (Prolonged PTTs are often caused by antiphospholipid antibodies.) Potentially, PF4 too could have a role in bacterial host defense.⁹  It is an intriguing hypothesis that β2-GPI and PF4 play a role in balancing a system of self- and nonself-recognition. Understanding the underlying mechanisms may provide important insights into the complex interactions of host defense and mechanisms of autoimmunity.