Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation

Abstract

Background Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. Methods We used electric cell-substrate impedance sensing (ECIS) to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 μg/mL solution of 3K3A-aPC at 180, 120, 60, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via ECIS for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey’s multiple comparison’s test. Results Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pre-treatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60, 120, and 180-minute pre-treatment groups was also higher than TP alone (mean diff. 16.30 95% CI [7.93, 24.67], 19.43 95% CI [11.06, 27.80], and 18.65 95% CI [10.28, 27.02], all p < .0001 respectively. Rac1/RhoA activity was higher in the aPC pre-treatment group when compared to all other groups (p < 0.01). Conclusions Pre-treatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anti-coagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thrombo-inflammation for injured patients by minimizing aPC’s role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. Level of Evidence Level III, Prognostic/Epidemiological