Increase in activated protein C mediates acute traumatic coagulopathy in mice

Abstract

In severely injured and hypoperfused trauma patients, endogenous acute coagulopathy (EAC) is associated with an increased morbidity and mortality. Recent human data correlate this coagulopathy with activation of the protein C pathway. To examine the mechanistic role of protein C in the development of EAC, we used a mouse model of trauma and hemorrhagic shock, characterized by the combination of tissue injury and severe metabolic acidosis. Mice were subjected to one of four treatment groups: 1) C, control; 2) T, trauma (laparotomy); 3) H, hemorrhage (MAP, 35 mmHg x 60 min); 4) TH, trauma + hemorrhage. After 60 min, blood was drawn for analysis. Compared with C mice, the TH mice had a significantly elevated activated partial thromboplastin time (23.3 vs. 34.5 s) and significantly increased levels of activated protein C (aPC; 2.30 vs. 13.58 ng/mL). In contrast, T and H mice did not develop an elevated activated partial thromboplastin time or increased aPC. Selective inhibition of the anticoagulant property of aPC prevented the coagulopathy seen in response to trauma/hemorrhage (23.5 vs. 38.6 s [inhibitory vs. control monoclonal antibody]) with no impact on survival during the shock period. However, complete blockade of both the anticoagulant and cytoprotective functions of aPC caused 100% mortality within 45 min of shock, with histopathology evidence of pulmonary thrombosis and perivascular hemorrhage. These results indicate that our unique mouse model of T/H shock mimics our previous observations in trauma patients and demonstrates that EAC is mediated by the activation of the protein C pathway. In addition, the cytoprotective effect of protein C activation seems to be necessary for survival of the initial shock injury.

Figures

Fig. 1. Hemodynamic and respiratory responses to hemorrhagic shock

A–B, Changes in MAP (A) and respiratory rate before and during hemorrhagic shock (B). Control mice underwent catheter placement only. Trauma mice received 2-cm midline laparotomy and closure, followed by catheter placement. Hemorrhage mice underwent catheter placement, followed by hemorrhagic shock (blood withdrawal(s) necessary to maintain MAP = 35 ± 5 mmHg for 60 min). Trauma + hemorrhage mice received 2-cm midline laparotomy and closure, catheter placement, and hemorrhagic shock. Data are expressed as mean ± SD (n = 10 per group).

Fig. 2. Systemic markers of hypoperfusion in mice after TH shock

A–D, Mice underwent either TH, H, T, or C (no trauma, no shock). Blood was drawn after 60 min of hemorrhagic shock via IVC puncture. Data expressed as mean ± SD (n = 5 per group). A, *P < 0.02 (C vs. H, T vs. H); **P < 0.02 (C vs. TH, T vs. TH). B, *P < 0.01 (C vs. T); **P < 0.01 (C vs. H, T vs. H); ***P < 0.01 (C vs. TH, T vs. TH). C, *P < 0.01 (C vs. H, T vs. H); **P < 0.01 (C vs. TH, T vs. TH).

Fig. 3. Trauma + hemorrhagic shock is associated with increases in aPTT (in seconds) and aPC (in nanograms per milliliter) in mice

A and B, Mice underwent either TH, H, T, or C (no trauma, no shock). Activated partial thromboplastin time (in seconds) and aPC (ng/mL) levels were measured after 60 min of hemorrhagic shock. Data were expressed as mean ± SD. C (n = 6), T (n = 6), H (n = 10), TH (n = 10). A, *P = 0.02 (C vs. TH, T vs. TH, H vs. TH). B, **P < 0.02 (C vs. TH, T vs. TH, H vs. TH).

Fig. 4. Inhibition of the anticoagulant function of PC prevents the development of acute traumatic coagulopathy in mice

Mice were pretreated with a mAb that inhibits the anticoagulant function of PC (mAb 1591) or with an isotype control mAb (mAb 1761). After 10 min, the mice then underwent TH. Activated partial thromboplastin time values were measured after 60 min of hemorrhagic shock. Data were expressed as mean ± SD. C, 1761 (n = 5); TH, 1761 (n = 9); C, 1591 (n = 5); TH, 1591 (n = 10). *P < 0.05 (C vs. TH mAb 1761). *P < 0.05 (TH [1761] vs. TH [1591]).

Fig. 5. Complete inhibition of PC before TH leads to 100% mortality within 45 min of shock

Mice were pretreated with one of three monoclonal antibodies: mAb 1609 inhibits both the cytoprotective and anticoagulant functions of PC, mAb 1591 inhibits only the anticoagulant function of PC, and mAb 1761 is an isotype control monoclonal antibody. After 10 min, the mice then underwent TH. n = 14 mice per group. All C mice (no trauma, no shock) in all treatment groups (mAbs 1761, 1591, and 1609) survived (curves are not shown).

Fig. 6. Complete inhibition of PC before TH leads to pulmonary artery thrombosis, perivascular hemorrhage, and alveolar hemorrhage in mice

A–C, Mice were pretreated with one of three monoclonal antibodies: A, mAb 1761, an isotype control monoclonal antibody; B, mAb 1591, inhibits only the anticoagulant function of PC; C, mAb 1609, inhibits both the cytoprotective and anticoagulant functions of PC. After 10 min, the mice then underwent TH. Lungs were harvested after 60 min of hemorrhagic shock or at the time of death. Lungs were fixed, sectioned, and hematoxylin and eosin stained. Images obtained at 2003 magnification. All C mice (no trauma, no shock) in all treatment groups (mAbs 1761, 1591, and 1609) had no pulmonary pathology (sections not shown).