TY - JOUR

T1 - The functional role of the autolysis loop in the regulation of factor X upon hemostatic response

AU - Bonde, Amalie Carnbring

AU - Lund, Jacob

AU - Hansen, Jens Jacob

AU - Winther, Jakob Rahr

AU - Nielsen, Per Franklin

AU - Zahn, Stefan

AU - Tiainen, Peter

AU - Olsen, Ole Hvilsted

AU - Petersen, Helle Heibroch

AU - Rose Bjelke, Jais

N1 - This article is protected by copyright. All rights reserved.

PY - 2022

Y1 - 2022

N2 - 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). 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. 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 participate in all aspects of FX regulation. In plasma-based 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.

AB - 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). 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. 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 participate in all aspects of FX regulation. In plasma-based 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.

U2 - 10.1111/jth.15624

DO - 10.1111/jth.15624

M3 - Journal article

C2 - 34927362

VL - 20

SP - 589

EP - 599

JO - Journal of Thrombosis and Haemostasis

JF - Journal of Thrombosis and Haemostasis

SN - 1538-7933

IS - 3

ER -