Factor Xa aptamer

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Timeline

2009

An RNA aptamer (RNA11F7t), which was selected from a combinatorial library for its ability to bind factor Xa[1]

2018

They solved X-ray crystal structures of the aptamer bound to factor Xa (FXa)[3]

2022

The combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone[4]

Description

In 2009, Krishnaswamy, S. et al. presented the characterization of an RNA aptamer (RNA11f7t) that was developed by cyclical screening of a combinatorial library against factor Xa. They showed that the potent inhibition of thrombin formation catalyzed by prothrombinase unexpectedly resulted from the ability of RNA11f7t to bind factor Xa with high affinity and inhibit its interaction with factor Va on the membrane surface. In 2018, Sullenger, B. A. et al. determined the X-ray crystal structures of the aptamer bound to factor Xa (FXa). They demonstrated that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma[1,3].



SELEX

Krishnaswamy, S. et al. presented the characterization of an RNA aptamer (RNA11f7t) which had been developed by cyclical screening of a combinatorial library against factor Xa. They showed that the potent inhibition of thrombin formation, catalyzed by prothrombinase, unexpectedly resulted from the ability of RNA11f7t to bind factor Xa with high affinity and inhibit its interaction with factor Va on the membrane surface. After 11 rounds of selection, the products were digested with EcoR1 and BamH1 (New England Biolabs) and directionally cloned into pUC19 that had been linearized with the same enzymes. Individual clones were sequenced, and clonal RNA transcripts were analyzed in filter-binding assays. The lead molecule was systematically shortened to obtain a truncated version (RNA11F7t) that retained binding activity[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

FXa aptamer—11F7t, mFold predicted structure, 36 nts in length. Here we used ribodraw to complete the figure, through the 3D structure information. 11F7t was named by Krishnaswamy, S in the article[2,4].

5'-GAGAGCCCCAGCGAGAUAAUACUUGGCCCCGCUCUU-3'

drawing

3D visualisation

Sullenger, B. A. et al. able to crystallize and determine the structure of a ternary complex between GD-FXa_S195A, 11F7t, and rivaroxaban bound to the active site of the proteinase at 2.25 Å resolution. The PDB ID of this structure is 5VOF[3].

Additional available structures that have been solved and detailed information are accessible on Structures page.

(Clicking the "Settings/Controls info" to turn Spin off)      

drawing PDBe Molstar




Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 5VOF by X-ray crystallography. GD-FXaS195A (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with GD-FXaS195A.

drawing drawing


Ligand information

SELEX ligand

The dependence of the initial rate of thrombin formation on increasing concentrations of factor Va at different fixed concentrations of aptamer was analyzed by assuming mutually exclusive binding interactions in rapid equilibrium between factor Va versus Xa and aptamer. Analysis according to the cubic equation of the reference yielded fitted values for Kd for the membrane-dependent interaction between Xa and Va (Kd Xa, Va), the number of moles of Va bound per mole of Xa at saturation, Kd for aptamer binding to Xa (Kd RNA, Xa), the number of moles of aptamer bound per mole of Xa at saturation, the rate at zero Va, and the rate at infinite Va[1].

Name Ligand Affinity
Factor Xa aptamer Factor Xa 1.1 ± 0.2 nM

Structure ligand

Rivaroxaban is a factor Xa inhibitor used to treat deep vein thrombosis (DVT) and pulmonary embolism (PE). May also be used as thrombosis prophylaxis in specific situations.-----From Drugbank

PubChem CID: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

Drugbank: a comprehensive database with detailed information on drugs and drug targets.

Name PubChem CID Molecular Formula Molecular Weight CAS Solubility Drugbank ID
Rivaroxaban 9875401 C19H18ClN3O5S 435.9 g/mol 366789-02-8 50 mg/ml (in DMSO) DB06228
drawing drawing

Factor Xa is a vitamin K-dependent glycoprotein that converts prothrombin to thrombin in the presence of factor Va, calcium and phospholipid during blood clotting.-----From Uniprot

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
P00742 PIRSF001143 28.51 kDa
GGPYLQ ...... IVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK
1FAX 9606
drawing

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and selected the previous information with high similarity for presentation.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB Z-score RMSD Description
5VOF-H 48.5 0 Coagulation factor x
5VOE-H 46.4 0.3 Coagulation factor x
2Y5G-A 45.8 0.4 Activated factor xa heavy
2JKH-A 45.8 0.4 Activated factor xa heavy
2XC4-A 45.8 0.4 Activated factor xa heavy
3IIT-A 45.8 0.4 Activated factor xa heavy
2Y5F-A 45.8 0.4 Activated factor xa heavy
2Y5H-A 45.7 0.4 Activated factor xa heavy
2EI8-A 45.7 0.4 Coagulation factor x, heavy
3TK5-A 45.6 0.4 Factor x heavy chain


References

[1] An Anticoagulant RNA Aptamer That Inhibits Proteinase-Cofactor Interactions within Prothrombinase.
Buddai, S. K., Layzer, J. M., Lu, G., Rusconi, C. P., Sullenger, B. A., Monroe, D. M., & Krishnaswamy, S.
The Journal of biological chemistry, 285(8), 5212–5223. (2009)
[2] Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant.
Soule, E. E., Bompiani, K. M., Woodruff, R. S., & Sullenger, B. A.
Nucleic acid therapeutics, 26(1), 1–9. (2016)
[3] Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass.
Gunaratne, R., Kumar, S., Frederiksen, J. W., Stayrook, S., Lohrmann, J. L., Perry, K., Bompiani, K. M., Chabata, C. V., Thalji, N. K., Ho, M. D., Arepally, G., Camire, R. M., Krishnaswamy, S., & Sullenger, B. A.
Nature biotechnology, 36(7), 606–613. (2018)
[4] Combining Heparin and a FX/Xa Aptamer to Reduce Thrombin Generation in Cardiopulmonary Bypass and COVID-19.
Chabata, C. V., Frederiksen, J. W., Olson, L. B., Naqvi, I. A., Hall, S. E., Gunaratne, R., Kraft, B. D., Que, L. G., Chen, L., & Sullenger, B. A.
Nucleic acid therapeutics, 32(3), 139–150. (2022)