Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors

Verhoef, Daniël; Visscher, Koen; Vosmeer, C. Ruben; Cheung, Ka Lei; Reitsma, Pieter H.; Geerke, Daan P.; Bos, Mettine H.A.

doi:10.1038/s41467-017-00647-9

Cited by 33 publications

(40 citation statements)

References 62 publications

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“…In exploration of RIV rescuing agents, Verhoef et al. () found that the F174A mutant of FXa was resistant to the RIV‐like inhibitor, Apixaban, while retaining the catalytic activity, and suggested local modifications on S4 pocket may reduce the binding affinity of the direct inhibitor to FXa. However, Verhoef's computational simulations mainly focused on the mobility of the 99 loop in wild‐type and P. textilis isoform FXa, and did not elaborate the molecular mechanism of the F174A mutant in detail.…”

Section: Introductionmentioning

confidence: 99%

An unexpected dynamic binding mode between coagulation factor X and Rivaroxaban reveals importance of flexibility in drug binding

et al. 2019

Chem Biol Drug Des

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Rivaroxaban (RIV) is a direct oral anticoagulant (DOAC) targeting activated coagulation factor X (FXa). An earlier study reported the F174A mutant of FXa resistant to a RIV‐like inhibitor, Apixaban. In current study, the detailed molecular mechanism of the resistance has been explored by molecular dynamics simulations on the impaired interactions between RIV and FXa in the damaged S4 pocket of F174A mutant. Besides, an unexpected relative stable binding mode of S1′S1 was revealed, which required dynamic motions of Gln192 and Gln61 to allow the morpholinone moiety of RIV to shift into the S1′ pocket and form strong interactions. These dynamic motions of RIV and critical residues might be important in drug design for direct inhibitors of coagulation factors.

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Section: Introductionmentioning

confidence: 99%

An unexpected dynamic binding mode between coagulation factor X and Rivaroxaban reveals importance of flexibility in drug binding

et al. 2019

Chem Biol Drug Des

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“…The HEK cells were transfected with pcDNA‐FIX using the calcium phosphate coprecipitation method . To generate cell lines stably expressing FIX, individual clones were expanded and selected in media containing 450 μg/mL geneticin essentially as described previously for recombinant FX expression . In following, the stable HEK‐FIX cells were transiently transfected with pcDNA‐VKORC1.…”

Section: Methodsmentioning

confidence: 99%

“…15 To generate cell lines stably expressing FIX, individual clones were expanded and selected in media containing 450 μg/mL geneticin essentially as described previously for recombinant FX expression. 16 In following, the stable HEK-FIX cells were transiently transfected with pcDNA-VKORC1. Transfection with FIX and VKORC1 was confirmed by performing RT-PCR to demonstrate transcription of the transgene.…”

Section: Cell Culture and Transfection Of Recombinant Human Factor IXmentioning

confidence: 99%

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1

Pakdaman

Vatandoost

Bos

2019

Biotechnology Progress

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Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ‐carboxylation might be of interest. The expression and functional activity of vitamin K‐dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ‐carboxylation that is mediated by the enzymes γ‐carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX‐producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT‐PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme‐linked immunosorbent assay and coagulation test. In addition, to quantify γ‐carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX‐producing HEK cells, resulting in a 3.2‐fold higher expression of functional FIX, which displayed a 1.4‐fold enhanced specific activity. Moreover, a 3.9‐fold enhanced recovery of fully γ‐carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ‐carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

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“…65 Both venom proteins comprise exceptional structural and functional features, 66 with venom FXa carrying specific modifications of the substrate-binding aromatic S4 subpocket within its active site that disrupt high-affinity engagement of the direct FXa-inhibiting oral anticoagulants. 67 Human FX variants comprising a similarly modified S4 subsite resulting either from a single point mutation at position Phe174 (chymotrypsinogen numbering) or insertion of a unique snake venom FXa 99-loop demonstrated a 10-to 100-fold loss of FXa inhibitor-sensitivity. 67 As these FX variants are able to restore hemostasis in plasma inhibited by the FXa inhibitors, they have the potential to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.…”

Section: Risk Stratification Of Patients With Acute Pulmonary Embolismentioning

confidence: 99%

“…67 Human FX variants comprising a similarly modified S4 subsite resulting either from a single point mutation at position Phe174 (chymotrypsinogen numbering) or insertion of a unique snake venom FXa 99-loop demonstrated a 10-to 100-fold loss of FXa inhibitor-sensitivity. 67 As these FX variants are able to restore hemostasis in plasma inhibited by the FXa inhibitors, they have the potential to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation. The gut microbiota is an environmental factor that impacts vascular physiology, locally in the intestine, 68 but also through remote signalling.…”

Section: Risk Stratification Of Patients With Acute Pulmonary Embolismentioning

confidence: 99%

Illustrated State‐of‐the‐Art Capsules of the ISTH 2019 Congress in Melbourne, Australia

Ward

Andrews

2019

Research and Practice in Thrombosis and Haemostasis

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The 27th Congress of the International Society of Thrombosis and Haemostasis (ISTH) is an international conference held July 6‐10, 2019, in Melbourne, the capital of the state of Victoria, Australia. The ISTH congress has previously been held every other year, with the Scientific and Standardization Committee (SSC) meeting held annually, until 2019 when it became one combined annual meeting of the ISTH and SSC. The conference covers clinical and basic aspects of hemostasis and thrombosis, and this year includes 5 Plenary lectures and >50 State of Art (SOA) lectures, presented by internationally recognized speakers, as well as numerous oral session and poster presentations selected from submitted abstracts, including many early career and reach the world support recipients. This SOA review article in RPTH contains concise Illustrated Review Articles or ‘Capsules’ consisting of short text, three references and a figure, with topics including stroke, cancer‐associated thrombosis, hemophilia, coagulation, the interface between infection and inflammation, and in the experimental and discovery areas, megakaryocyte biology and platelet production, structure‐function of key receptors and coagulation factors, and emerging new roles for thrombotic/hemostatic factors. Together, these articles highlight novel findings which will advance knowledge and with the potential to change clinical practice and improve outcomes. It is hoped that conference attendees and followers will enjoy utilizing the images for ongoing education and during the conference for live tweeting during sessions, to assist in the broadcasting and promotion of the science to those unable to attend, or who have chosen to attend a concurrent session. Use #IllustratedReview and #ISTH2019 on social media.

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Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors

Cited by 33 publications

References 62 publications

An unexpected dynamic binding mode between coagulation factor X and Rivaroxaban reveals importance of flexibility in drug binding

An unexpected dynamic binding mode between coagulation factor X and Rivaroxaban reveals importance of flexibility in drug binding

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1

Illustrated State‐of‐the‐Art Capsules of the ISTH 2019 Congress in Melbourne, Australia

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