Trauma-targeted delivery of tranexamic acid improves hemostasis and survival in rat liver hemorrhage model

Abstract

Background: Trauma-associated hemorrhage and coagulopathy remain leading causes of mortality. Such coagulopathy often leads to a hyperfibrinolytic phenotype where hemostatic clots become unstable because of upregulated tissue plasminogen activator (tPA) activity. Tranexamic acid (TXA), a synthetic inhibitor of tPA, has emerged as a promising drug to mitigate fibrinolysis. TXA is US Food and Drug Administration-approved for treating heavy menstrual and postpartum bleeding, and has shown promise in trauma treatment. However, emerging reports also implicate TXA for off-target systemic coagulopathy, thromboembolic complications, and neuropathy.

Objective: We hypothesized that targeted delivery of TXA to traumatic injury site can enable its clot-stabilizing action site-selectively, to improve hemostasis and survival while avoiding off-target effects. To test this, we used liposomes as a model delivery vehicle, decorated their surface with a fibrinogen-mimetic peptide for anchorage to active platelets within trauma-associated clots, and encapsulated TXA within them.

Methods: The TXA-loaded trauma-targeted nanovesicles (T-tNVs) were evaluated in vitro in rat blood, and then in vivo in a liver trauma model in rats. TXA-loaded control (untargeted) nanovesicles (TNVs), free TXA, or saline were studied as comparison groups.

Results: Our studies show that in vitro, the T-tNVs could resist lysis in tPA-spiked rat blood. In vivo, T-tNVs maintained systemic safety, significantly reduced blood loss and improved survival in the rat liver hemorrhage model. Postmortem evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma-targeted activity of the T-tNVs.

Conclusion: Overall, the studies establish the potential of targeted TXA delivery for safe injury site-selective enhancement and stabilization of hemostatic clots to improve survival in trauma.

Keywords: fibrinolysis; hemorrhage; rat model; targeted delivery; tranexamic acid; trauma.

FIGURE 1

(A) Cellular and molecular components of hemostasis involving platelets, coagulation factors, thrombin (FIIa) generation, fibrinogen (Fg) conversion to fibrin, fibrin crosslinking of by FXIIIa and fibrin degradation by plasmin (formed by activation of plasminogen (Plg) by tPA); in trauma-associated hyperfibrinolysis, the fibrin degradation processes are upregulated while inhibitors for these processes (e.g., PAI-1) are downregulated; tranexamic acid (TXA) is a potent inhibitor of tPA, Plg, and plasmin, and thus has antifibrinolytic activity that can maintain clot stability. (B) Schematic of targeted delivery of TXA at the trauma injury site from T-tNVs anchored to clot-associated active platelets, for site-specific antifibrinolytic action

FIGURE 2

Representative fluorescence confocal images showing red fluorescent FMP-decorated nanovesicles (designated in the article as trauma-targeted nanovesicle or tNV) can bind specifically with activated platelets (blue) within crosslinked fibrin-rich (green) clots formed from platelet-rich plasma (PRP) in presence of thrombin

FIGURE 3

(A) Schematic of TXA-loaded trauma-targeted nanovesicle (T-tNV) preparation. (B) Reaction schematic for NHS-fluorescein labeling of TXA (Fluor-TXA). (C) Representative DLS characterization of multiple batches of T-tNVs show an approximate diameter of ~170 nm. (D) Encapsulation efficiency (EE) of TXA in T-tNVs for multiple batches show an approximate encapsulation of ~ 60%. (E) Diffusive release profile of Fluor-TXA from T-tNVs at 37°C over 12 hours (data shown for first 2 hours at 10-minute intervals and for the next 10 hours at 2-hour intervals)

FIGURE 4

(A) ROTEM profiles of rat whole blood spiked with tPA and treated with saline (no TXA), free TXA, empty (no TXA) platelet-targeted nanovesicle (tNV) or TXA loaded untargeted nanovesicles (T-NV). [A1] rat whole blood (WB). [A2] WB spiked with 0.75 mg/mL tPA showing rapid onset of lysis. [A3] Treatment with tNVs could not rescue the clot from lysis. [A4] Treatment with free TXA rendered resistance to lysis. [A5] Treatment with T-NVs rendered resistance to lysis similar to free TXA treatment. (B) ROTEM parameters from these studies, demonstrating that at 30-minute time point only free TXA and T-NV were able to completely resist lysis. (C) Percent (%) lysis of tPA-spiked rat blood at 60 minutes, further demonstrating that compared to treatment with saline or empty tNV, treatment with free TXA or T-NVs reduce clot lysis at statistically significant levels

FIGURE 5

In vivo safety evaluation with tNVs as well as T-tNVs (n = 3 rats per group) show that intravenous administration of these nanovesicles themselves (tNVs) or the corresponding TXA-loaded formulation (T-tNVs), at dose of 8.8 mg/kg does not drastically affect vitals over a 24-hour period (A and B). Representative heart rate and SpO₂ traces over the first 60-minute period are shown in C and D. A total of six animals (three per group) were used for these studies

FIGURE 6

(A) Schematic of rat liver injury hemorrhage model setup (details described in Methods). (B) Postinjury vitals of animals administered intravenously with saline, free TXA, T-NV, or T-tNV show that hemorrhagic injury causes higher heart rate while SpO₂ stays consistent due to efficient use of ventilation. (C) Blood loss analysis postinjury upon administration of the various treatment groups shows that rats treated with T-tNVs undergo statistically significantly reduced blood loss (P < 0.05) compared with rats treated with saline or free TXA; furthermore, T-tNV-treated rats exhibit substantially reduced variance in blood loss data compared with T-NV-treated rats. (D) One-hour survival analysis postinjury and treatment administration shows that T-tNV-treated rats exhibited significantly higher survival from all other treatment groups (P < 0.01 compared with saline-treated and free TXA-treated groups, and P < 0.05 compared with T-NV-treated group, by log-rank test). (E) At 72 hours postinjury and treatment, T-tNV-treated rats continued to show improved survival compared with T-NV-treated rats; none of the saline-treated and free TXA-treated rats survived 72 hours. Number of animals: n = 6 per treatment group

FIGURE 7

Representative images from histopathologic analysis (hemoxylin and eosin staining) of excised tissue sections from rats subjected to liver injury and administered with various treatments, show that T-NVs or T-tNVs do not have any off-target effects on any of the clearance organs; in contrast, free TXA-treated rats show prominent pulmonary congestion, specifically the alveoli filled with floccular pink material, infiltration of polymorphonuclear neutrophils, and red blood cell congestion indicative of edema and inflammation, suggesting pulmonary embolism

FIGURE 8

Representative confocal microscopy images of immunostained tissue sections from injured liver site of rats administered with the various treatment groups show that rats treated with saline, free TXA and T-NVs form suboptimal clot structure at the liver injury site, while rats treated with T-tNVs exhibit substantial colocalization of T-tNVs with platelets in the hemostatic clot and the clot structure for T-tNV-treated rats appeared more robust