Variants from Patients with Hemophilia Impair Proper Carboxylation of Factor IX
ISTH Academy. Popp N. Jul 10, 2019; 273845; OC 76.4 Topic: FVIII/IX
Nicholas Popp
Nicholas Popp
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OC 76.4

Variants from Patients with Hemophilia Impair Proper Carboxylation of Factor IX

N. Popp1,2, K. Lannert3, J. Johnsen3,4, D. Fowler1,5
1University of Washington School of Medicine, Department of Genome Sciences, Seattle, United States, 2University of Washington School of Medicine, Medical Scientist Training Program, Seattle, United States, 3Bloodworks Northwest, Seattle, United States, 4University of Washington School of Medicine, Department of Medicine, Division of Hematology, Seattle, United States, 5University of Washington School of Medicine, Department of Bioengineering, Seattle, United States

Main Topic: Coagulation and Anticoagulation
Category: FVIII/IX

Background: γ-carboxylation of the GLA domain of Factor IX (FIX) is required for its activation and participation in the coagulation cascade. Previous work has shown that A37T, which lies in the carboxylation recognition motif, leads to warfarin sensitivity. Furthermore, many variants within the GLA domain have been identified in patients with hemophilia B, but the mechanism of dysfunction remains unknown. The presumed processive nature of the carboxylation process leads us to hypothesize that variants within and around the GLA domain may directly interfere with γ-carboxylation and thus lead to disease.
Aims: To determine whether and how variants from patients with hemophilia B impact carboxylation.
Methods: A cell-based reporter consisting of a Tet-inducible promoter, prothrombin signaling peptide, the FIX GLA domain, and a transmembrane domain was genomically integrated into HEK293T cells using Bxb1 recombinase. After integration, cells expressing variant or wildtype FIX GLA domain at their cell surface were incubated with 50nM vitamin K +/- 100 µM warfarin, stained with a APC-conjugated FIX carboxylation-specific antibody, and then analyzed using flow cytometry in triplicate. For variant comparison, the median of each fluorescence measurement was taken and scaled such that warfarin-treated cells were zero and wildtype cells were one.
Results: Cells expressing GLA domain variants from patients with hemophilia B showed complete or near complete loss of carboxylation. Variant E79A, isolated from a patient with moderate hemophilia, showed the highest γ-carboxylation of any variant, approximately 25% of wildtype. Another variant from a patient with severe hemophilia at the same site, E79K was indistinguishable from warfarin-treated FIX.
Conclusions: We employed a reporter assay for FIX variants to test their effect on the degree of carboxylation, and by proxy, pro-coagulant activity. Variants from patients with hemophilia B show a quantitative loss in the degree of carboxylation that appears to correlate with disease severity, indicating a potential loss of function mechanism.

[Scaled median fluorescence of Factor IX variants]

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