PB1121 - Human missense variants in F3 impair the initiation of blood coagulation

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Shabbir A. Ansari, MSc PhD

Research Fellow in Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School
Boston, Massachusetts, United States

Co-Author(s)

JF

James T. Flaumenhaft

Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
MB

Marisa A. Brake, PhD

Postdoctoral Fellow
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School
Boston, Massachusetts, United States
NP

Natasha Panwar

Research Assistant
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
NP

Nishtha Pathak

Research Assistant II
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
JL

Jacob G. Ludington

Research Fellow in Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School
Boston, Massachusetts, United States
Ernest Turro

Associate Professor
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai
New York, New York, United States
Sol Schulman, MD, PhD

Assistant Professor of Medicine
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School
Boston, Massachusetts, United States

Background: F3 encodes tissue factor (TF), which binds factor VII (FVII/FVIIa) to initiate blood coagulation, but the extent to which human F3 variants impact blood coagulation is unknown.

Aims: Determine the impact of human F3 missense mutations on TF-triggered coagulation activation.

Methods: The five most common and five rare F3 missense substitutions were introduced by site-directed mutagenesis. Full-length TF was expressed in CHO-K1 cells and localization, surface expression, FX activation, and plasma clotting time were determined. Soluble TF was purified from E. coli and kinetic parameters of FX activation and FVIIa amidolytic activity were determined. Genotype-based recall within the BioMe Biobank enabled evaluation of coagulation activation by ELISA for selected rare variants and age- and sex-matched controls.

Results: None of the human F3 missense variants with minor allele frequency (MAF)>1x10^-3 impacted TF localization or TF-dependent coagulation activation, even when FVII/FVIIa was limiting. In contrast, two rare human missense variants, Asp90Asn (MAF=4.0x10^-6) and Gly196Arg (MAF=2.4x10^-5), had < 20% (p=0.002) and < 50% (p=0.018) specific activity for FX activation on the cell surface. Purified soluble Asp90Asn demonstrated 130-fold (0.002+/-0.001 nM^-1min^-1) and Gly196Arg 32.5-fold (0.008+/-0.0007 nM^-1min^-1) reduced Kcat/Km as compared to wild-type (0.26+/-0.03 nM^-1min^-1), effects attributable to impaired catalytic function rather than substrate affinity. Asp90Asn eliminated FVIIa amidolytic activity suggesting impaired FVIIa binding, consistent with the orientation of Asp90 at the TF-FVIIa interface. Asp90Asn failed to clot plasma and Gly196Arg exhibited prolonged plasma clotting time (p < 0.001). An individual heterozygous for Gly196Arg had normal PT, aPTT, and FVII antigen but reduced FVIIa-antithrombin complex (96 pM) compared to matched (286+/-135 pM, n=4) and population (194+/-94.5 pM, n=36) controls lacking F3 variation.

Conclusion(s): No common but selected rare human missense substitutions in F3 impair the TF-dependent activation of blood coagulation. These variants are not captured by routine clinical assays but may contribute to human bleeding or thrombotic risk.