The functional role of the autolysis loop in the regulation of factor X upon hemostatic response
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The functional role of the autolysis loop in the regulation of factor X upon hemostatic response. / Bonde, Amalie Carnbring; Lund, Jacob; Hansen, Jens Jacob; Winther, Jakob Rahr; Nielsen, Per Franklin; Zahn, Stefan; Tiainen, Peter; Olsen, Ole Hvilsted; Petersen, Helle Heibroch; Rose Bjelke, Jais.
I: Journal of Thrombosis and Haemostasis, Bind 20, Nr. 3, 2022, s. 589-599.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 288188048