The functional role of the autolysis loop in the regulation of factor X upon hemostatic response

Abstract

Background:The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity.Objectives: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX. Methods:We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma. Results:The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, 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). K i for the inhibition by full-length TFPI of these FXa variants was increased by 7-to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2-to 15-fold compared with wild-type. 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 FXa variants. Conclusion:The autolysis loop participates in all aspects of FX regulation. In plasmabased assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced.

Site‐specific functional roles of the Factor X activation peptide in the intrinsic tenase‐mediated Factor X activation

“…Prior to the first purification step, cell supernatants were supplemented with 10 m m CaCl 2 and 5 m m benzamidine, and pH was adjusted to 6.0. In the first step, FX variants were purified by immunoaffinity chromatography using a Ca 2+ ‐dependent anti‐FX Gla domain‐specific antibody [15] coupled to Sepharose 4 FF resin packed in a Tricorn 10/50 column. Bound protein was eluted in a buffer containing 60 m m EDTA and subsequently purified further by anion‐exchange chromatography to isolate the fully γ‐carboxylated Gla domain.…”

“…Recombinant FX protein variants were expressed in HEK-293 cells and purified from cell culture supernatants by a two-step integrated chromatographic approach as previously described [15]. Furin processing, protein purity and the degree of homogeneity were evaluated by reducing SDS/PAGE and SE-HPLC.…”

The Interaction of Factor Xa and IXa with Non-Activated Antithrombin in Michaelis Complex: Insights from Enhanced-Sampling Molecular Dynamics Simulations

“…Bonde et al have studied the role of the autolysis loop of factor X, a region with a critical role in the AT interaction as well as other functions of the factor, by introducing artificial glycosylation sites using mutagenesis. The authors found that adding such glycans to the loop affects many aspects of factor Xa's biochemistry, including activation, procoagulant activity and inhibition by antithrombin [15].…”