Pam3CSK4 As a Second Hit in NLRP3-Dependent Activation of Monocytes Primed With Antiphospholipid Antibodies

Antiphospholipid antibodies (aPL) are drivers of inflammation and thrombosis in antiphospholipid syndrome (APS). However, the molecular mechanisms underlying infection-induced exacerbation of APS remain incompletely understood. To identify novel pathways contributing to the pro-inflammatory and pro-coagulant activation of monocytes in APS, we screened fourteen Toll-like receptor (TLR) ligands for their ability to induce tissue factor (TF) expression in a monocyte-like cell line (THP-1). Among these, Pam3CSK4, a synthetic TLR1/2 ligand, emerged as a potent inducer of TF in the presence of aPL. Subsequent experiments revealed that Pam3CSK4 synergized with aPL to amplify both TF expression and IL-1β secretion. This synergistic effect was mediated via NF-κB signaling and required activation of the NLRP3 inflammasome. aPL priming promoted ATP release, providing the second signal for NLRP3 activation. Inhibition of NLRP3 markedly reduced Pam3CSK4-induced TF expression, suggesting a critical role for inflammasome signaling in monocyte procoagulant responses. These in vitro findings were confirmed in a mouse model of APS, supporting the pathogenic relevance of the TLR1/2-NLRP3 axis in vivo. Taken together, our results validate the "second hit" paradigm in APS and suggest a novel TLR1/2-NLRP3 axis as a key mediator of infection-driven inflammatory and thrombotic responses in aPL-primed monocytes.