Background: Coagulation factor IXa is the proteinase component of the intrinsic Xase complex which catalyzes the activation of factor X. The RNA aptamer DTRI-177 was selected as a subnanomolar binder of the zymogen, factor IX and the proteinase factor IXa. This aptamer serves as a potent anticoagulant by inhibiting factor X activation but with a minor effect on active site function of IXa. Although the mechanism of inhibition by DTRI-177 remains unresolved, evidence suggests it may allosterically modulate IXa function and also possibly interfere with the IXa/VIIIa interaction within intrinsic Xase.

Aims: To resolve the X-ray structure of the IXa – aptamer complex and obtain insights into the mechanism of inhibition.

Methods: Crystals were obtained for Gla-domainless factor IXa _S195A in the absence or presence of aptamer. X-ray diffraction data were collected at 1.9 A resolution for des-Gla-IXa _Ser195Ala and at 2.8 A for the complex with aptamer.

Results: The aptamer binds to the proteinase domain of IXa without occluding the active site (Figure 1). Predominant interactions involve residues (highlighted in violet) with a buried area of 790 A². The extensive and complementary interaction surface explains the high affinity and specificity of the interaction between DTRI-177 and IXa. It includes residues in the 165-helix previously implicated in the interaction with VIIIa and key residues previously shown to interact with heparin: K₁₂₆, N₁₂₉, K₁₃₂, R₁₆₅, R₂₃₃ (Figure 1 inset). Accordingly, DTRI-177 was able to compete with the binding of either IX or IXa to heparin Sepharose. A comparison with the structure of apo-IXa also revealed structural perturbations surrounding the catalytic residues induced by the aptamer.
X-ray structure of anticoagulant RNA aptamer DTRI-177 in complex with factor IXa shown in a canonical view.

Conclusions: The surface on IXa occupied by the aptamer reveals an important site for regulation of the intrinsic Xase function.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/crystal-structure-of-factor-ixa-with-an-anticoagulant-aptamer-bound-to-an-allosteric-site/