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Presentation Details
| The Mexican American Restricted Arg500Leu F2 Variant Prothrombin-RGV Encodes the Zymogen of an Antithrombin-Sensitive Hypocoagulable Thrombin that Likely Protects Against Venous Thromboembolism (VTE) Henry J.Mead1, Vincent P.Diego2, 3, Marcio A.Almeida2, 3, Luis Pena Marquez2, Afshin Ameri4, Juan M.Peralta2, 3, Laura Almasy11, Harald H.Goring2, 3, Michael Recht5, 6, Joanne E.Curran2, 3, Kevin R.Viel3, Miguel A.Escobar7, 8, Tammuella Chrisentery-Singleton9, 10, Sarah Williams-Blangero2, 3, John Blangero2, 3, Tom E.Howard2, 3. 1Haplogenics, Brownsville, TX, USA.2South Texas Diabetes and Obesity Institute and Department of Human Genetics, College of Sciences, Brownsville, TX, USA.3Histonis, Portsmouth, NH, USA.4Department of Pediatrics, Division of Hematology and Oncology, Georgia Health Sciences University, Augusta, GA, USA.5Yale Center for bleeding and Clotting disorders, New Haven, CT, USA.6, Yale University School of Medicine, New Haven, CT, USA.7Division of Hematology and Oncology, Department of Medicine, University of Texas Health Science Center, Houston, TX, USA.8Gulf States Hemophilia and Thrombophilia Center, Houston, TX, USA.9American Thrombosis & Hemostasis Network, Hickory, NC, USA.10Ochsner Clinic Foundation, New Orleans, LA, USA.11Department of Biomedical and Health Informatics, Lifespan Brain Institute, Philadelphia, PA, USA |
Abstract
BaACKGROUND: Inherited-thrombophilias underlie most idiopathic-VTE but often go undiagnosed due to limitations of current coagulation-screening-assays. In a recent association-scan of hemostasis traits and genotypes of exome-wide nonsynonymous (ns)-SNPs measured in 790 subjects of the South Texas Family Study (STFS), we identified “Prothrombin Rio Grande Valley (Prothrombin-RGV)”, a Mesoamerican-origin R500L-protein-altering F2 ns-SNP whose minor-allele showed statistically-significant dose-dependent prolongations of both PT- and aPTT-clot-time-tests and decreased Factor II-coagulant-activity (FII:C). Upon prothrombinase-cleavage/-activation of prothrombin to thrombin, R500 becomes R168, which, due to being in the front rim of its active-site cleft, contributes to efficient fibrinogen cleavage via electrostatically-stabilized substrate binding through S3-subsite-interactions. Based on established aspects of prothrombin structure/function (Figure-1), we predict the modest hypocoagulability measured for Prothrombin-RGV results from an induced structural alteration in Thrombin-RGV that impairs fibrinogen binding/cleavage (Hypothesis-1). OBJECTIVES: Based on new insights concerning antithrombin-mediated thrombin-inhibition and prothrombinase-catalyzed prothrombin-activation, our goal was to use the thrombin-generation (TG)-assay (which evaluates coagulation-parameters not assessable with clot-time-tests) to investigate two other plausible consequences of the R500L-structural change including: impeding antithrombin-binding/-inactivation of thrombin, similar to Prothrombin-Yukuhashi, R553L, a nearby identical subtitution (Figure-1) recently found to be a rare potent thrombophilia despite showing similarly prolonged PT- and aPTT-clot-times, and decreased FII:C levels (Hypothesis-2); and perturbing the open-/closed-conformational-equilibrium of prothrombin in plasma, which, by shifting >20% of FII to its open (more slowly prothrombinase-cleaved/-activated) form, would delay formation of thrombin and cause mild hypocoagulability, analogous to Prothrombin-Perijá, Gly548Ala, a rare dysprothrombinemia identified in patients with excessive-bleeding after trauma/surgery, that reduces the prothrombin-activation rate by destabilizing its closed form (Hypothesis-3). METHODS: Frozen platelet-poor citrated-plasma from 25 STFS subjects (four R500/R500, 20 L500/R500, and one L500/L500) were quick-thawed (37°C) and assayed in duplicate for thrombin-generation (TG) on the calibrated-automated-thrombogram (CAT)-instrument (Thermo-Fisher) by adding 80mL/well to the appropriate microbottom-plate wells with 20mL PPP-reagent providing 1pM recombinant-human-Tissue-Factor + 4pM phospholipids, final assay concentration, after TG-activation by adding 20mL FluCa-reagent containing the thrombin-specific fluorogenic-substrate and CaCl2. For each subject, we used Thrombinoscope-software-v5.0, to record/measure TG based on a thrombin calibrator, and T-tests, to statistically compare the means of three TG-assay-parameters [Lagtime-for-TG (LTG); Endogenous-Thrombin-Potential (ETP); and Start-of-Tail-for-TG (STTG)] in heterozygotes versus wildtype-homozygotes. We assessed all SNVs in, or within ~0.5Mbp upstream or downstream of, the F2 for LD with prothrombin-RGV using r2 and, of the 11 found (r2>0.2), seven exhibited strong LD (r2>0.8) (Figure-2). RESULTS/DISCUSSION: Though mean ETP for R500/L500-heterozyotes (1,793.14_nM·min) was greater than for wildtypes (1,130.70_nM·min), the difference was not significant, p=0.154. Moreover, the mean STTG for heterozygotes (32.01_minutes) was borderline-significantly shorter than for wildtypes (37.67_minutes), p=0.053, opposite to that expected if thrombin-RGV was antithrombin-inactivation-resistant. Finally, since mean LTG for heterozygotes (10.08_minutes) was not significantly longer than wildtypes (9.58_minutes), p=0.804, Prothrombin-RGV does not perturb the FII-conformational-equilibrium toward its open (more slowly prothrombinase-cleaved/-activated) form. CONCLUSIONS: Satistical-analyses of our TGA-results support Hypothesis-1, that prothrombinase-cleavage/-activation of Prothrombin-RGV yields an antithrombin-inactivation-sensitive hypocoagulable-thrombin-variant, and reject alternative Hypotheses-2/-3. Though this ns-SNP (F2_c.G1628T) wasn’t genotyped in the multi-ethnic Million-Veteran-Program, we are testing the hypothesis it protects against VTE via marginal-genotype-association-analyses of flanking SNPs in strong LD.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.