Efficacy Trial In An Acute Liver Trauma Animal Model Of Novel Infusible Hemostatic Product: Red Blood Cell-Derived Microparticles (RMP)
Authors: Jy Wenche , Johansen Max E , Bidot Carlos , Pamukcu Rifat , Gomez-Marin Orlando , Ahn Eugene , Horstman Lawrence L , Ahn Yeon S 
Published: October 2019

Abstract

Background
Increasing evidence indicates that natural circulating red cell microparticles (RMP) contribute significantly to hemostasis. Accordingly, we produced RMP in vitro by high pressure extrusion of washed RBCs. We previously reported the hemostatic activity of our RMP product in vitro as well as its efficacy in arresting ear bleeding in rabbits, suggesting the potential of RMP as a hemostatic agent (Jy et al Thromb. Haemost., in press). Here we studied its hemostatic efficacy in a rabbit model of acute liver injury, as well as its short-term toxicity.

Methods
Male New Zealand White rabbits (mean weight 3.8kg) were randomly selected and assigned, one at a time, to an experimental (n = 9) or a control group (n = 10). All animals were sedated with 35mg/kg ketamine, 5mg/kg xylazine, and 0.01mg/kg glycopyrrolate 15 minutes before surgery. They were intubated and anesthetized with 2% isoflurane, and mechanically ventilated at approximately 20 breaths/min. The carotid artery and the jugular vein were canulated, and maintenance saline was administered at 20mL/hr. The abdomen was cleaned and shaved, and an incision was made from lower tip of the sternum to the bladder. A standardized injury of 9 incisions 30mm long by 4mm deep was inflicted to the liver, and two infusions of RMP (1.25x1011/kg) or saline were delivered via jugular catheter, first immediately post-incision, and again 10 minutes post-incision. Blood was collected in gauze pads at 10 min. intervals and weighed to calculate volume lost. Heart rate, blood pressure (BP), and temperature were continually monitored. The blood pressure (BP) of some of the animals fell very low (MAP<15) by 1 hr post-incision and resulted in death. Only those rabbits that survived 2 hr were used in analysis of blood loss and vital signs. Five of the 10 control rabbits survived 2 hr post-surgery (50%) compared to 5 of the 9 RMP-treated rabbits (56%). All surviving rabbits were euthanized with 0.2mL/kg Euthasol after 120 minutes.

Results
At early time points (30 – 60 min post-injury) when bleeding was most rapid, administration of RMP had no significant effect: mean (± SD) blood loss at 30 min was 46.1 ± 20.6 mL in the control vs. 26.6 ± 9.0 mL (p=0.07) in the RMP group. At 60 min it was 67.2 ± 24.3 mL vs. 53.4 ± 6.9 mL (p=0.23), respectively. Although at these early time points there were not significant differences, a trend of reduced blood loss was observed.
At the 90 min time point, blood loss was 78.6 ± 25.9 mL in the control group compared to 55.7 ± 5.6 mL in the RMP group (p<0.05), a relative difference of ≈ 30%. At 120 min, the corresponding values were 89.8 ± 29.3 mL and 57.7 ± 4.4 mL (p < 0.01), a relative difference of ≈ 36% .
Vitals were not significantly different between the control and RMP groups at any time point. Drop in BP tended to be greater in controls than in RMP rabbits. The difference in BP was marginally significant at 120 min. post-injury: systolic BP fell by 39.6 ±17.2 mmHg in controls vs. 18.6 ±15.0 mmHg in RMP-treated (p=0.07). In a short-term toxicity test, quadruple dose (5.0x1011/kg of RMP) was administered and effects observed for three hours: no obvious adverse effects on heart rate, BP, or temperature were observed.

Conclusion/Discussion
We previously reported dose-dependent reduction by RMP infusion in ear bleeding time of thrombocytopenic rabbits. Efficacy of infusions of RMP is evident in this severe hemorrhagic liver injury model after 30 - 60 min of acute injury when rapid bleeding was substantially slowing down. At 90 and 120 min post-injury, significantly less blood loss was observed in the RMP group compared to the controls, with relative differences of 30% and 36%. Studies are in progress to improve the model and to optimize dosages and scheduling of RMP administration for severe bleeding.

Disclosures:
No relevant conflicts of interest to declare.