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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session E

S33 - Effects of anticoagulants on neutrophil extracellular trap-mediated clotting in horses

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Dysfunctions in hemostasis, both hyper- and hypocoagulability, can lead to life-threatening complications in critically ill veterinary and human patients. Inflammatory conditions disrupt the balance between blood clot formation and breakdown, often resulting in pro-thrombotic conditions. Systemic inflammation can drive hemostatic dysregulation by activating innate immune cells, particularly neutrophils. Neutrophils both enhance and promote platelet activation through the release of neutrophil extracellular traps (NETs). Concurrent cytokine release from neutrophils suppresses fibrinolysis and clot breakdown, further promoting thrombosis. This project investigates the effect of clinically relevant anti-thrombotic therapeutics with different mechanistic approaches on NET-mediated ex vivo thrombosis in adult horses. To mimic the inflammatory states found in sepsis, and to induce NET release, blood is stimulated with phorbol 12-myristate 13-acetate (PMA). We hypothesize that NET-mediated clots are resistant to fibrinolysis and that antithrombotics will be more effective in unstimulated blood compared to PMA-stimulated blood.

Materials and

Methods: : To test this hypothesis, blood was collected from 8 healthy adult horses using sodium citrate as an anticoagulant. Half of the collected whole blood was then stimulated with phorbol 12-myristate 13-acetate (PMA) to induce NET release while the other half was left unstimulated and treated with an equal volume of media. Following a 4-hour stimulation period, plasma containing platelets and white blood cells was treated with anticoagulants (enoxaparin, antithrombin-III), antithrombotics (tissue plasminogen activator, clopidogrel, aspirin) or a vehicle control as a negative control. Multiple experiments were performed to assess both prophylactic and therapeutic functions of the pharmacological agents in both stimulated and unstimulated clots. Clot absorbance assays and confocal microscopy of plasma based clots were performed to assess prophylactic antithrombotic use, while microfluidic devices were used to assess anticoagulant treatment in equine fibrinogen based clots.

Results, Conclusions, and Discussions:: Our study provides a stronger understanding of coagulation management in adult horses to further improve clinical treatment of coagulopathies in critically ill equine patients, an area with limited evidence-based guidance. Clot absorbance assays revealed that tissue plasminogen activator (tPA) produced the most substantial effect on both stimulated and unstimulated clots. This can be seen in Figure 2, as both stimulated and unstimulated tPA had a significant decrease in absorbance in comparison to the other treatment groups. This suggests that utilization of tPA in a clinical setting is more effective for prophylactic use. Stimulation significantly increased the rate of clot polymerization and decreased the rate of degradation across all treatment groups. Clots polymerized in the presence of antithrombin-III (ATIII) exhibited significantly lower maximum turbidity compared to untreated clots. This finding suggests that ATIII was able to minimize blood clot formation. In confocal analysis, treatment type, cellular presence, and stimulation did not significantly affect the percent change in fibrin fiber density, with a p value of >0.05. Microfluidic analysis showed that stimulation increased cellular attachment. Notably, a large horse-to-horse variability was observed, highlighting the need for a larger sample size to better evaluate the findings and increase the power of this study. Horses serve as a valuable translational model for human medicine due to their similarities in blood composition, robust immune response, and high incidence of systemic inflammation and sepsis across diverse clinical conditions emphasizing the importance of this study. The investigators look to expand its findings to foals to evaluate age-related variations in coagulation and antithrombotic treatment.

Acknowledgements and/or References (Optional): : This project was supported by funding from the American Heart Association. ​

Thank you to Shannon Chiera for assistance in sample collection and processing.