Prolonging coagulant activity of factor Xa under hemophilic conditions by site-specific N-glycosylation of the surface-exposed autolysis loop
The regulation of Factor X (FX) is critical to maintain hemostasis. To gain insights to the regulation of the active and zymogen form of coagulation FX, we probed specific molecular interactions by introducing novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. Introduction of N-glycans in the autolysis loop of these FX variants decreased Factor VIIa (FVIIa)-mediated activation ~3-fold and prothrombin activation 2- to 10-fold presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor Factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, while ATIII inhibition in the presence of the heparin-analogue Fondaparinux was modestly increased by 2- to 15-fold compared to wild type. To probe the in vitro hemostatic effect of the FX variants, the thrombin generation potential in FX-depleted plasma was evaluated. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FX variants with N-glycosylation at positions 148-150. These results indicate that residues of the surface-exposed autolysis loop and residues close by participate in FX activation, proteolytic activity and inhibition of FXa by TFPI and ATIII. In plasma-based assays, a modest decrease in FX-activation rate appeared to compensate for the collective reduction in inhibitor interactions.