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Substitution of the Factor IX Activation Sites for Characterization of the Factor IX Activation Pathway and Partially Activated Intermediates

1Leiden University Medical Center / Hakim Sabzevari University, Leiden, Zuid-Holland, Netherlands, 2Leiden University Medical Center, leiden, Zuid-Holland, Netherlands, 3Div. Thrombosis and Hemostasis, Einthoven Laboratory, for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands, Leiden, Zuid-Holland, Netherlands, 4Leiden University Medical Center, Leiden, Zuid-Holland, Netherlands, 5Leiden University Medical Center, Leiden, The Netherlands, Leiden, Zuid-Holland, Netherlands, 6Leiden University Medical Center

Abstract Number: OC 74.1

Meeting: ISTH 2022 Congress

Theme: Coagulation and Natural Anticoagulants » FVIII/IX

Background: Conversion of the zymogen factor (F)IX to its activated serine protease state FIXa-beta proceeds through sequential cleavage at Arg145 and Arg180 by the extrinsic tissue factor-FVIIa complex or by intrinsic FXIa. Proteolysis at Arg145 results in the activation intermediate FIX-alpha that displays limited amidolytic activity, while single cleavage at Arg180 generates partially active FIXa-alpha. As modifications in FIX aimed at improving its activity may affect the activation pathway and activity of intermediate species, detailed knowledge on these key processes is essential.

Aims: Generate FIX variants comprising substitutions at the Arg145 and Arg180 activation sites to establish a framework for detailed characterization of FIX activation species and variants.

Methods: Recombinant variants FIX-R145Q, FIX-R180Q, and FIX-R145Q-R180Q were generated and purified to homogeneity. The variants were functionally assessed for both extrinsic and intrinsic activation pathways.

Results: Under conditions in which wild-type (WT-) FIX was efficiently converted to FIXa-beta by both the extrinsic and intrinsic activators, FIX-R180Q was mostly proteolyzed to FIX-alpha while FIX-R145Q was only partially converted to FIXa-alpha. In case of the latter, prolonged incubations with supraphysiological FXIa concentration led to additional protein products suggestive of aspecific proteolysis of the FIX heavy chain. These findings confirm that cleavage at Arg145 site is most efficient, which is required for Arg180 to become available for subsequent proteolysis. No proteolysis was observed when incubating FIX-R145Q-R180Q with the extrinsic or intrinsic activators. Extrinsically or intrinsically triggered thrombin generation assessments in which FIX-deficient plasma was supplemented with FIX variant demonstrated 50% activity for FIX-R145Q relative to WT-FIX, while no thrombin generation was observed for the other variants, in line with earlier observations.

Conclusion(s): These FIX variants allow for analysis of the FIX activation pathways. We next aim to obtain detailed insight into the contribution of activation intermediates to FIX function, which is essential for the knowledge-based design of therapeutic FIX variants.

To cite this abstract in AMA style:

Vatandoost J, Cheng S, Cheung K, Shifai N, Mayorga M, Strijbis V, Bos M. Substitution of the Factor IX Activation Sites for Characterization of the Factor IX Activation Pathway and Partially Activated Intermediates [abstract]. https://abstracts.isth.org/abstract/substitution-of-the-factor-ix-activation-sites-for-characterization-of-the-factor-ix-activation-pathway-and-partially-activated-intermediates/. Accessed October 1, 2023.

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