173 - Tropomyosin 1 Promotes Platelet Adhesion and Clot Contraction Separate From Its Roles in Developmental Hematopoiesis

Publication Number: 2167.173

Victor Tsao, Childrens Hospital of Philadelphia, Plano, TX, United States; Po-Lun Kung, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Alina D. Peshkova, University of Pennsylvania, PHILADELPHIA, PA, United States; Kim Ha, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Gennadiy Fonar, Mayo Clinic Alix School of Medicine, ROCHESTER, MN, United States; Nkemdilim A. Okoli, Childrens Hospital of Philadelphia, Boston, MA, United States; Brian M. Dulmovits, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Rong Qiu, Children's Hospital of Philadelphia, Philadelphia, PA, United States; Rolf Dale P. Bates, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Janelle Yeboah, Childrens Hospital of Philadelphia, Baltimore, MD, United States; Carson Shalaby, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; Tyler R. Truex, Eastern University, Ephrata, PA, United States; Vladimir Muzykantov, University of Pennsylvania, Philadelphia, PA, United States; Jacob W. Myerson, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Christopher Thom, CHOP/UPenn, Philadelphia, PA, United States

Background: Neonatal thrombosis incidence has risen almost 3-fold in the past 20 years and carries significant morbidity. In general, thrombotic mechanisms are incompletely understood. We hypothesized that novel genetic determinants of thrombosis may be hidden in human genetics data sets. Genome-wide association studies (GWAS) have linked the Tropomyosin 1 (Tpm1) gene locus to quantitative platelet trait variation. Tpm1 encodes an actin-binding protein that stabilizes actin filaments and regulates cell adhesion and contractility, though its role in platelets was unknown. We hypothesized that Tpm1 mediates platelet adhesion and hemostasis.

Objective: To determine how Tpm1 regulates platelet adhesion, clot contraction, and thrombosis, and to identify its potential role as a genetic determinant of thrombotic risk.

Design/Methods: Using murine in vivo models, we created conditional Tpm1 knockout (Tpm1KO) models using Cdh5Cre or VavCre to ablate Tpm1 in embryonic hemogenic endothelium or nascent hematopoietic stem and progenitor cells (HSPCs).

Results: Both models ablated Tpm1 in adult peripheral blood by qPCR and produced healthy Tpm1KO mice in normal Mendelian ratios. Cdh5Cre Tpm1fl/fl mice increased formation of definitive hemogenic endothelial cells in embryos (immediate precursors). However, postnatal HSPCs and adult blood counts remained unchanged. This suggested separate roles for Tpm1 in embryonic and adult blood systems. Genetic colocalization analysis confirmed a platelet trait-specific GWAS locus at the Tpm1 locus: alleles that decrease TPM1 expression increase platelet counts.

We hypothesized that Tpm1 deficiency might diminish adhesion based on actin perturbations. Static adhesion assays showed reduced adhesion to fibronectin and fibrinogen and led to longer in vivo platelet half-life in healthy adult mice by almost 50%. Platelet-fibrin binding mediates blood clot contraction, which prevents vascular occlusion during thrombosis. Tpm1KO delayed clot contraction in assays of whole blood and platelet rich plasma.

Perturbed clot contraction can worsen vascular occlusion. In ferric chloride–induced thrombosis, Tpm1KO caused faster occlusion and larger clots within 5–10 minutes, versus >30 minutes in littermate controls.

Conclusion(s): Our findings reveal a new role for Tpm1 and cytoskeletal regulation in platelet adhesion and clot contraction. Loss of Tpm1 perturbs clot contraction, worsening vascular occlusion and thrombosis. This mechanism represents a new target for modifying thrombotic risk, and will spur comparison studies to ascertain the relevance of these thrombotic mechanisms in adults and neonates.