Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Abstract

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains

“…The results presented so far are in disagreement with recent work by Heestermans et al 13 showing that removal of amino acids Gln 317 to Ser 339 from the FXII PRR (supplemental Figure 3) interferes with autoactivation induced by poly-P, dextran sulfate, or kaolin. We prepared FXII with a deletion of the entire PRR between residues Thr 278 and Leu 338 (FXII-ΔPRR; Figure 4A ).…”

“…In reviewing the method used to create deletion mutants in the study by Heestermans et al, 13 we noted that introducing EcoR1 restriction endonuclease sites into the FXII cDNA to remove the sequence encoding Gln 317 to Ser 339 may have changed the adjacent residue Cys 340 at the C terminus of the PRR. Cys 340 forms a disulfide bond with Cys 467 that connects the heavy and light chains after FXII conversion to FXIIa.…”

“… 6 , 9-11 There are conflicting data regarding the mechanism by which FXII binds to surfaces. Work by Clark et al 12 suggested that a surface-binding element resides in or near the EGF1 domain, while a recent study by Heestermans et al 13 localized surface binding to the PRR. Both studies used FXII molecules containing deletions of specific domains or parts of domains, which could produce deleterious effects on overall protein structure.…”

Commentary on “Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa”: Small molecule factor XIa inhibitor asundexian allows for safer anticoagulation

“…Classical activated partial thromboplastin time (APTT)‐based diagnostic assays have little if any sensitivity for measuring FXI levels in this required therapeutic range and it is challenging to precisely monitor FXI levels in patients. However, just recently a coagulation assay that can precisely measure FXIa activity in a broad range of FXI has emerged 9 …”

“…However, just recently a coagulation assay that can precisely measure FXIa activity in a broad range of FXI has emerged. 9 Targeting FXI with antisense oligonucleotides (ASO) and an array of function interfering antibodies that block FXIa, FXIIa-mediated FXI activation, or deplete FXI from the circulation have been demonstrated to protect from arterial and venous thrombosis in rodents, primates, and patients, 10,11 respectively. the potent antithrombotic activity of FXI-neutralizing agents therapy-associated bleeding has remained very low indicating that interference with FXI might be superior to currently used anticoagulation strategies.…”