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 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 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing 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 pretreatment 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-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01).

Conclusion: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function.

Level of evidence: Prognostic and Epidemiological; Level III.

Figure 1.

Ex vivo plasma group comparisons including thrombin and control groups.

Figure 2.

Area under the curve for ex vivo plasma group comparisons including thrombin and control groups.

Figure 3.

Ex vivo plasma with and without 30-min pre-incubation with 3K3A-aPC. TP: Trauma plasma, or ex vivo plasma

Figure 4.

Area under the curve for ex vivo plasma with and without 30-min pre-incubation with 3K3A-aPC. TP: Trauma plasma, or ex vivo plasma [ISS >15, BE <−6].

Figure 5.

Rac1/RhoA Ratio of treatment groups as compared to untreated control, where the ratio of the aPC + TP is statistically significantly greater than all other groups.