Anti-Phospholipid Syndrome (APS) Antibody (Ab)-Induced Thrombosis Can be Blocked By Platelet Factor 4 (PF4)-Directed Abs: A Novel Therapeutic Approach for APS?

Background: APS is an autoimmune thromboinflammatory disease characterized by thrombocytopenia, thrombosis, and/or complications in pregnancy. APS occurs in association with one or more antiphospholipid (aPL) Abs/IgGs whose major target is b2-glycoprotein I (b2GPI), a protein previously shown to bind to human (h) PF4, a platelet-specific chemokine with a high affinity for polyanions including DNA in neutrophil extracellular traps (NETs) and von Willebrand factor (vWF). No further studies have been conducted to characterize the role of hPF4:β2GPI complexes in APS-related thrombosis. We hypothesized that b2GPI binds to hhPF4 adhered to NETs or vWF to form potent, prothrombotic antigenic complexes.

Aim: We now aim to determine whether PF4:b2GPI:NETs or vWF are central to APS-mediated thrombosis and evaluate whether hPF4-blocking antibodies can prevent these events.

Methods: The following anti-hPF4 monoclonal (mo) Abs were studied: (1) the murine heparin-induced thrombocytopenia (HIT)-like anti-hPF4 KKO, (2) IgG4 humanized KKO (G4KKO), (3) humanized vaccine-induced immune thrombotic thrombocytopenia (VITT)-like anti-hPF4 Ab 1E12, (4) deglycosylated 1E12 (DG1E12), and (5) a murine PF4-tetramer disaggregating moAb RTO. These Abs were infused through Bioflux microfluidic channels coated with NETs, released from healthy donor neutrophils stimulated with phorbol 12-myristate 13-acetate and then treated with β2GPI (20 µg/ml), hPF4 (6.5 µg/ml) and APS IgGs (100 µg/ml). A polyclonal anti-β2GPI Ab (a gift from Dr. McCrae) was used to visualize β2GPI binding to NETs.

In a separate set of studies, human umbilical vein endothelial cells (HUVECs) were grown to confluence in Bioflux channels and then subjected to a hematoporphyrin (HP)-induced photochemical injury that stimulates vWF release. Whole blood from healthy donors was supplemented with additional β2GPI (20 µg/ml), PF4 (25 µg/ml) and purified IgGs (100 µg/ml) from four distinct patients with “triple-positive” APS with associated thrombi and was then flowed through the HUVEC-lined channels with or without the various anti-hPF4 moAbs. Some studies included DNase1 (100 U/mL, Sigma-Aldrich) or ADAMTS13 (0.7 µg/ml, R&D Systems).

Results: In studies with NET-lined microfluidic channels, we did not detect binding of b2GPI to unmodified NETs, but found b2GPI adhered well to PF4-coated NETs. β2GPI binding to PF4-coated NETs was completely inhibited by VITT-like moAb 1E12, but unaffected by HIT-like Ab KKO, which binds to a different antigenic site on PF4. In the experiments in which whole blood was infused through HUVEC-lined channels following HP injury, platelet thrombosis and complement deposition were significantly higher when all three proteins - PF4, β2GPI and APS IgG - were present. Inclusion of either DNaseI or ADAMTS13 partially blocked thrombosis. Inclusion of the HIT-like G4KKO, the VITT-like DG1E12 and RTO showed marked inhibition of platelet- and complement-rich thrombi in the presence of APS IgG.

Conclusions: Our studies support the importance of PF4:b2GPI:NET and vWF complexes in APS in in vitro model systems. b2GPI requires the presence of PF4 to be anchored to NETs. This interaction can be blocked by inclusion of the VITT-site-binding moAb 12E12, but not to the HIT-site-binding moAb KKO, suggesting that the hhPF4:b2GPI:NET complex involves the VITT site. The fact that both ADAMTS13 as well as DNase1 can reduce APS Ab-induced thrombosis in our HUVEC-lined microfluidic system, suggests that PF4:b2GPI forms complex on both NETs and VWF. Anti-hPF4 Abs that block either the HIT or VITT sites on hPF4 or disrupt PF4 tetramerization can effectively prevent thrombi on both NETs and vWF. These studies provide new mechanistic insights into a central role for PF4 thrombosis in the presence of anti-b2GPI⁺ APS Abs and identify potential new non-anticoagulant therapeutics that may be useful in this challenging prothrombotic setting.

McCrae:Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: 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; Stago: Research Funding; Siemens: Research Funding; Up To Date: Honoraria. Gruel:Stago: Honoraria, Research Funding. Rollin:Stago: Honoraria, Research Funding. Poncz:Alexion: Research Funding; Astra Zeneca: Research Funding.