Antiphospholipid Syndrome (APS) Is a Platelet Factor 4 (PF4)-Centric Immunothrombotic Disorder

Background: APS is a devastating immune-mediated disorder characterized by thrombosis and pregnancy complications in association with autoantibodies (aPL Abs) that bind β2-glycoprotein 1 (β2GP1), among other antigens. The physiological role of β2GP1 is uncertain, and the mechanism by which anti-β2GP1 aPL Abs lead to thrombosis is not fully elucidated. In 2010, it was shown that PF4, a platelet-specific chemokine released in large amounts following platelet activation, forms tetramers that bind to β2GP1 dimers. This PF4:β2GP1 interaction enhances recognition of β2GP1 by aPL Abs. Furthermore, aPL Abs induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin, that also bind PF4. PF4:NET complexes are key antigenic targets involved in the pathogenesis of heparin-induced thrombocytopenia (HIT) and in vaccine-induced thrombocytopenia (VITT).

Aim: We propose that PF4 bridges β2GP1 to NETs, leading to the formation of PF4:β2GP1:NET complexes that act as an important antigenic target in APS. To address this hypothesis, we studied the formation of PF4:β2GP1:NET complexes and asked whether they contribute to the prothrombotic state in in vitro assays and in a murine model of APS.

Methods: We studied plasma and isolated IgGs from four patients with “triple-positive” APS and thrombosis. Two distinct in vitro-binding studies were performed: i. Dynamic light-scattering (DLS) studies were done using β2GP1, PF4 and calf thymus (ct) DNA plus aPL IgGs in a Malvern Zetasizer Nano-ZS. In some experiments RTO, a monoclonal Ab that disrupts the PF4 tetramers, was added. ii. Isolated human neutrophils were adhered to fibronectin-coated Bioflux microfluidic channels, and then stimulated with phorbol myristyl acetate to induce NET release. NET-lined channels were then infused with β2GP1 and/or PF4. Functional studies to examine the importance of PF4:β2GP1:NETs in APS thrombosis were conducted in two systems: i. Microfluidic channels coated with fibronectin were lined with human umbilical endothelial cells (HUVECs) and then photochemically-injured with hematoporphyrin and blue-light exposure prior to adding whole blood from healthy donors to which β2GP1 and/or PF4 and/or aPL IgGs had been added. ii. A passive immunization model of isolated aPL IgGs were infused into double-transgenic human PF4/FcgRIIA-expressing mice or wildtype (WT) mice or mice lacking PF4 (mPF4^-/- mice). Rolling and adherence of neutrophils on cremaster venules were quantified before and after infusion of aPL IgGs. Platelet and neutrophil accumulation in laser injury-induced thrombi were measured.

Results: DLS studies showed that β2GP1 forms large complexes with PF4 plus ctDNA that binds aPL IgGs. Infused RTO decreased the formation of these large immune complexes in a dose-dependent fashion. In the NET microfluidic system, we observed that β2GP1 bound to exposed NETs only when PF4 was present. Additionally, just as in HIT and VITT, the ability of APS IgGs to lead to platelet-rich thrombus formation on the injured HUVECs was dependent on the inclusion of PF4. In a passive immunization murine model, we show that the induction of APS led to slowing of neutrophil rolling and neutrophil arrest on cremaster venules of mice in a PF4-dependent fashion, similar to that seen in HIT and VITT murine models. Moreover, infusion of aPL IgGs caused either hPF4/FcgRIIA or WT mice to develop occlusive platelet-rich thrombi at sites of laser injury in both arterioles and venules. Enhanced neutrophil accumulation developed in venules post-laser injury. Importantly, mice lacking PF4 were protected from enhanced arteriolar and venular thrombosis in the presence of APS IgGs. These findings were similar to those seen in prior studies of HIT- and VITT-induced prothrombotic mice models.

Conclusion: Our studies indicate that thrombosis in APS involves generation of PF4:NET complexes, as in HIT and VITT. In APS, β2GP1 is incorporated into these complexes forming PF4:β2GP1:NETs that are bound by anti- β2GP1 aPL IgG. In a passive murine immunization model of APS, these aPL IgGs increase neutrophil adhesiveness as well as platelet and neutrophil incorporation into laser-injury-induced thrombi in a PF4-dependent manner. Additional studies will be needed to relate these finding to clinical presentation and to ask whether strategies that block the formation of PF4:b2GP1:NET complexes would be an effective adjunctive therapy for APS.

McCrae:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ortel:Instrumentation Laboratory: Consultancy, Research Funding; Siemens: Research Funding; Stago: Research Funding; Up To Date: Honoraria. Rauova:NIH: Other: grant funding; Alexion: Other: grant funding; Astra Zeneca: Other: grant funding. Poncz:Alexion: Research Funding; Astra Zeneca: Research Funding.