Activated Protein C Drives the Hyperfibrinolysis of Acute Traumatic Coagulopathy

Abstract

Background: Major trauma is a leading cause of morbidity and mortality worldwide with hemorrhage accounting for 40% of deaths. Acute traumatic coagulopathy exacerbates bleeding, but controversy remains over the degree to which inhibition of procoagulant pathways (anticoagulation), fibrinogen loss, and fibrinolysis drive the pathologic process. Through a combination of experimental study in a murine model of trauma hemorrhage and human observation, the authors' objective was to determine the predominant pathophysiology of acute traumatic coagulopathy.

Methods: First, a prospective cohort study of 300 trauma patients admitted to a single level 1 trauma center with blood samples collected on arrival was performed. Second, a murine model of acute traumatic coagulopathy with suppressed protein C activation via genetic mutation of thrombomodulin was used. In both studies, analysis for coagulation screen, activated protein C levels, and rotational thromboelastometry (ROTEM) was performed.

Results: In patients with acute traumatic coagulopathy, the authors have demonstrated elevated activated protein C levels with profound fibrinolytic activity and early depletion of fibrinogen. Procoagulant pathways were only minimally inhibited with preservation of capacity to generate thrombin. Compared to factors V and VIII, proteases that do not undergo activated protein C-mediated cleavage were reduced but maintained within normal levels. In transgenic mice with reduced capacity to activate protein C, both fibrinolysis and fibrinogen depletion were significantly attenuated. Other recognized drivers of coagulopathy were associated with less significant perturbations of coagulation.

Conclusions: Activated protein C-associated fibrinolysis and fibrinogenolysis, rather than inhibition of procoagulant pathways, predominate in acute traumatic coagulopathy. In combination, these findings suggest a central role for the protein C pathway in acute traumatic coagulopathy and provide new translational opportunities for management of major trauma hemorrhage.

Figure 1. HUMAN STUDY – activated Protein C (aPC) is associated with concentration dependent functional changes in coagulation but preservation of thrombin generation

ROTEM is EXTEM unless specified. ANOVA with Dunnett’s post hoc correction. (A) Elevated aPC in trauma patients are associated with diminished A5 *p<0.05 vs. normal aPC (≤3 ng/ml). (B) Maximum Clot Firmness (MCF) falls in the presence of high aPC in trauma patients *p<0.05 vs. normal aPC (≤3 ng/ml). (C) Factor V (FV) falls in association with increasing aPC in trauma patients *p<0.05 vs. normal (aPC ≤3 ng/ml). (D) Inverse relationship between von Willebran Factor (vWF): Factor VIII (FVIII) ratio and elevated aPC indicative of selective inhibition of FVIII activity in humans following severe injury *p<0.05 vs. normal (aPC ≤3 ng/ml). (E) Endogenous Thrombin Potential (ETP) remains unchanged in trauma patients regardless of Factor V level. (F) Only minimal (non-significant) prolongation of Clotting Time (CT) is observed with high levels of aPC in trauma patients.

Figure 2. HUMAN STUDY

Fibrinogen loss and fibrinolysis are only observed in association with elevated activated Protein C (aPC) levels: One-way ANOVA with Dunnett’s post hoc correction or two-way ANOVA between subjects (A, D, F) with Bonferroni post-tests. (A) At high aPC levels of Prothrombin Fragment 1+2 (PF1+2) generation (thrombin production), there is a trend toward reduced fibrinogen in the presence of elevated aPC (p=0.08) (B) Fibrinogen is inversely related to rising aPC in trauma patients *p<0.05 vs. aPC ≤3ng/ml. (C) In trauma patients FIBTEM Maximum Clot Firmness (MCF) falls in association in rising aPC *p<0.05 vs. aPC ≤3ng/ml. (D) High levels of aPC (>9ng/ml) have a significant interaction (p<0.001) on tissue Plasminogen Activator (tPA) associated Plasmin-Antiplasmin (PAP) generation in trauma. *p<0.05, PAP ug/L: aPC ≤3 ng/ml vs. aPC >9ng/ml. (E) High aPC (aPC>9 ng/ml) was associated with lower Plasminogen Activator Inhibitor -1 (PAI-1) in the presence of shock (Base Deficit >4mmol/l) compared to aPC ≤3 ng/ml but this did not achieve significance (p=0.10). (F) Increased thrombin generation (PF 1+2) is associated with elevated D-dimers (fibrinolysis) with significantly greater fibrinolytic activity in the presence of high levels of aPC (interaction of aPC >9ng/ml, p<0.05) *p<0.05, PF 1+2 ng/ml: aPC ≤3 ng/ml vs. aPC>9ng/ml.

Figure 3. MURINE STUDIES – activated Protein C (aPC) rise after trauma hemorrhage is attenuated in Factor V Leiden (FVL) animals but preservation of Factor V function does not fully prevent ATC

(A) Trauma Hemorrhage (TH) in Wild Type (WT) mice is associated with a significant rise in aPC (WT Time 0 [T0]: 1.5ng/ml vs WT Time 60 mins [T60]: 10.3 ng/ml, *p<0.05) but attenuated in thrombomodulin knock-in (TMKI) animals. (B) Attenuation of Acute Traumatic Coagulopathy with normal Maximum Clot Firmness (MCF) in transgenic mice subject to TH *p<0.05 vs. WT T0. (C) Clotting Time (CT) is prolonged after TH in WT but not TMKI mice *p<0.05 WT T0 vs. WT T60 although baseline CT were significantly different between WT and TMKI at T0 (18 sec vs 22 sec, p<0.05). (D) CT is prolonged in both WT and FVL mice subjected to TH *p<0.05, T0 vs. T60 (WT and FVL).

Figure 4. MURINE STUDIES – activated Protein C (aPC) is central to fibrinolysis in Acute Traumatic Coagulopathy

(A) Attenuation of fibrinolytic activity in thrombomodulin knock-in (TMKI) mice subjected to Trauma Hemorrhage (TH) *p<0.05 (WT: Time 0 [T0] vs. WT: Time 60 mins [T60]) (WT: T60 vs TMKI: T60). (B) In WT mice after TH, fibrinogen fell significantly but TMKI animals were protected from this fibrinogen depletion *p<0.05 (WT: T0 vs. WT: T60) and (WT: T60 vs TMKI: T60). (C) Improved median survival times after TH in TMKI mice vs. WT *p<0.05.