Novel PFAS Alternatives 6:2 Cl-PFESA and HFPO-TA as Plasma FXII Activators: Experimental Evidence and Computational Mechanistic Insights

Abstract Per- and polyfluoroalkyl substances (PFAS) can persist in human blood and may perturb hematological homeostasis by interacting with coagulation factor XII (FXII), the initiator of the contact system. This study combined in vitro, ex vivo, and in vivo experiments with computational modeling to evaluate the effects of legacy PFAS (PFOS and PFOA) and their alternatives (6:2 Cl-PFESA, HFPO-DA, HFPO-TA, and HFPO-TeA) on FXII and downstream contact activation. In vitro and ex vivo screening showed that 6:2 Cl-PFESA had a greater capacity to activate FXII than PFOS, whereas HFPO-TA induced more pronounced FXII cleavage and a larger, albeit modest, increase in activated FXII (FXIIa) activity relative to PFOA. Both alternatives also triggered plasma prekallikrein activation. Intravenous exposure to 6:2 Cl-PFESA significantly increased plasma FXIIa activity in mice, suggesting that this PFAS alternative may perturb the contact system in vivo. Structure-activity analysis showed that, unlike linear PFAS, FXII activation induced by HFPO alternatives did not follow a simple carbon chain-length dependence. Molecular dynamics simulations and virtual alanine scanning analysis using FXII crystal and AlphaFold structures suggested a domain-selective mechanism, with the carboxylic PFAS favoring the fibronectin type-II domain (Fib-II) and the sulfonic PFAS favoring both the Fib-II and the proline-rich (PR) domain. These findings uncover new activators of the contact system, informing the potential hematological effects of PFAS alternatives, and provide computational support for a possible domain-selective mechanism for PFAS–FXII interactions.