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683 Blood Coagulation and Platelet Function Are Impaired By the ARP2 p.R258G Variant in Mice

Program: Oral and Poster Abstracts
Type: Oral
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Targeting platelet reactivity in thrombosis
Hematology Disease Topics & Pathways:
Bleeding and Clotting, Research, Fundamental Science, Diseases, thrombotic disorders
Monday, December 11, 2023: 11:30 AM

Caitlin Schneider, B.S.1*, Marisa A Brake, PhD2, Amy Siebert-Mckenzie, PhD3*, Adrianna Jurek, BS1*, Audrey C Cleuren, PhD4,5, Linzi Hobbs, MD1, Alexander Johnston1*, Michael Powers, MS1*, Azaj Ahmed6*, Melissa Jensen6*, Javier Menendez, MS1*, Sanjana Dayal, PhD7, Anthony Washington, PhD1 and Randal Joseph Westrick, PhD1

1Oakland University, Rochester, MI
2Harvard University, Boston
3Versiti Blood Research Institute, Milwaukee, WI
4Oklahoma Medical Research Foundation, Oklahoma City, OK
5Life Sciences Institute, University of Michigan, Ann Arbor
6University of Iowa, Iowa City, IA
7University of Iowa Department of Internal Medicine, Iowa City

Introduction: Venous thromboembolism (VTE) displays significant heritability, yet the genes explaining this heritability are not completely characterized. We previously identified that tissue factor pathway inhibitor heterozygosity (Tfpi+/−) on the homozygous F5L background (F5L/L) produces a lethal perinatal thrombotic phenotype. Using a sensitized ENU mutagenesis screen, we discovered an autosomal dominant missense mutation in the Actr2 gene (p.R258G, Actr2G) that suppresses thrombosis and restores survival to F5L/L Tfpi+/− mice. Inheritance of the Actr2G mutation is associated with nearly complete suppression of the lethal F5L/L Tfpi+/- phenotype. Actr2 codes for the ARP2 protein, an essential component of the ARP2/3 complex, which mediates actin cytoskeletal rearrangements during platelet activation. We hypothesized that the Actr2G variant affects blood coagulation and platelet function to reduce thrombosis.

Methods: To investigate potential effects of the Actr2G mutation on the coagulation cascade, prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma-dependent thrombin generation assays (TGA) were used to evaluate clot formation. A mouse tail clip assay was used to measure bleeding time and quantify blood loss. Finally, platelets were assessed for functional defects using platelet spreading and aggregometry assays.

Results: Actr2+/G plasma had significantly prolonged PT and aPTT compared to wild type controls (N>17, p<0.03). There was also a significant reduction in Actr2+/G thrombin peak height compared to wildtypes (N=10; p=0.03). Despite the delayed time to clot formation, Actr2+/G mice did not exhibit a bleeding phenotype and showed no significant differences in blood loss from tail clip assays. Activated Actr2+/G platelets exhibited reduced surface area and adhesion (N=5; p<0.02), as well as significant defects in collagen induced whole blood aggregation (N=10; p<0.02).

Discussion: The results of the PT, aPTT and TGA indicate reduced thrombin generation potential, suggesting a defect in the common pathway of coagulation. This hypocoaguability may be due to a decrease in circulating procoagulant factors, or an increase in endogenous thrombin inhibitors. Both the significantly decreased aggregation and defective platelet spreading in the Actr2+/G mice indicate that Actr2+/G platelets are less responsive to agonists, a protective mechanism that contributes to overall thrombosis suppression.

Conclusion: In summary, our studies demonstrate the pleiotropic effects that the Actr2G mutation exerts in suppressing thrombosis. The combination of altered plasma coagulation and platelet reactivity likely contribute to suppression of the F5L/L Tfpi+/− phenotype. Defects in these actin-mediated processes precipitated by Actr2+/G are the likely root of the F5L/L Tfpi+/- phenotype rescue, demonstrating the power of this gene in maintaining hemostatic balance. However, the combination of these thrombosis suppressing phenotypes is not significant enough to prolong the bleeding time in mice with the Actr2G mutation. Thus, our results suggest that targeting actin proteins such as ARP2 for novel antithrombotic drug therapies may have clinical applicability with limited effects on bleeding.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH