Thrombin in the Activation of the Fluid Contact Phase in Patients with Hereditary Angioedema Carrying the F12 P.Thr309Lys Variant

Hereditary angioedema due to pathogenic FXII variants (HAE-FXII) is a rare dominant disease caused by increased activation of the plasma contact system. The most prevalent HAE-FXII variant, c.1032C > A p.Thr309Lys (FXII^309Lys), results in a smaller FXII protein with increased sensitivity to fluid-phase activation by poorly understood mechanisms. We aimed to investigate the functionality of the FXII^309Lys variant in 33 HAE-FXII patients, 25 healthy controls and 46 patients with congenital disorders of glycosylation (CDG). Activation of the plasma contact system was assessed by western blot and amidolytic assay in basal conditions or after treatment with either artificial or physiological activators. Recombinant wild-type and FXII^309Lys variants were expressed in S2 insect (Drosophila) cells. Amidolytic and fibrin generation assays were performed in fresh plasma samples. FXII^309Lys samples exhibited an increased electrophoretic mobility comparable with N-glycan-deficient FXII from CDG patients and asialo-FXII generated by neuraminidase treatment. They presented increased sensitivity to activation by dextran sulphate and silica which resulted in the generation of an aberrant 37-kDa heavy chain. We did not observe increased susceptibility of FXII^309Lys to proteolysis by exogenous or tPA-generated plasmin. However, both exogenous and endogenous thrombin cleaved the FXII^309Lys variant, releasing a 37-kDa fragment and resulting in enhanced proteolytic activation on the fluid phase. This model supports a sequential proteolytic activation process involving thrombin priming of FXII^309Lys, followed by kallikrein cleavage and generation of active βFXIIa. The present results and the observation that angioedema episodes in HAE-FXII patients occur predominantly during hypercoagulable situations suggest a key role for thrombin.