PB0189 - Low normal levels of factor V enhance thrombin generation in hemophilia A

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

DM

Dougald Monroe

Professor
University of North Carolina Blood Research Center, Chapel Hill, NC, USA.
Chapel Hill, North Carolina, United States
CB

Christine Baird

Professional Research Assistant
University of Colorado Denver
Aurora, Colorado, United States
JP

Julie Peterson

Research Scientists
Versiti
Milwaukee, Wisconsin, United States
Aaron Fogelson, PhD

Professor
University of Utah
Salt Lake City, Utah, United States
Karin Leiderman, PhD

Associate Professor
Colorado School of Mines
Golden, Colorado, United States
SS

Suzanne Sindi

Professor
Department of Applied Mathematics, University of California Merced
Merced, California, United States
MS

Michael T. Stobb

Assistant Professor
Coe College
Cedar Rapids, Iowa, United States
AM

Alan E. Mast

Sr Investigator
Versiti Blood Research Institute
Milwaukee, Wisconsin, United States
MM

Marilyn J. Manco-Johnson

Professor of Pediatrics
University of Colorado Hemophilia & Thrombosis Center
Aurora, Colorado, United States

Presenting Author(s)

Keith B. Neeves, PhD

Professor
University of Colorado Anschutz Medical Campus
Aurora, Colorado, United States

Background: We previously reported that a mathematical model of coagulation predicted low normal levels (50%-75%) of factor V (FV) enhance thrombin generation in hemophilia A (Link, JTH, 18, 2020). The study suggested that FV and FVIII compete for cleavage by FXa during the initiation of coagulation.

Aims: To test the hypothesis that substrate competition for FXa is responsible for enhanced thrombin generation in hemophilia A plasma with low amounts of FV.

Methods: 1) A purified system of FV, FVIII, FXa and phospholipids examined the biochemical feasibility of the substrate competition mechanism, wherein the generation of FVIIIa was the output. 2) Tissue factor (TF) initiated thrombin generation was measured in synthetic plasma containing the zymogens and co-factors of the extrinsic pathway and anticoagulants (ATIII, +/-TFPI) at varying levels of FV (25%-150%). 3) A mixture study of FVIII-deficient (5%) human plasma with normal FV and immunodepleted of FV was performed using calibrated automated thrombography (CAT). 4) CAT initiated with 1 or 5 pM TF was used to measure thrombin generation in 75 severe to mild hemophilia A plasmas. Prothrombin, FV, FVIII, total TFPI, and TFPIα levels were measured in these plasma samples and Pearson correlation coefficients were calculated against thrombin generation metrics.

Results: In the purified system, there was an inverse relationship between FV concentration and the amount of FVIIIa generated, indicating competition for FXa between the two substrates. In both synthetic and human plasma, there was an inverse relationship between FV concentration and thrombin generation metrics (Fig. 1). There was negative correlation between FV concentrations and these thrombin generation metrics in hemophilia A plasmas (p < 0.01).

Conclusion(s): Four different biochemical systems support the hypothesis that low normal levels (50%-75%) of FV enhance thrombin generation in hemophilia A plasma. These experiments support the substrate competition mechanism for FXa predicted by our mathematical model.