To assess model performance, animals were classified as survivors and nonsurvivors at 6 h, and blood loss, fibrin-specific TEG, and endothelial activation by the concentration of Ang-2 in plasma were compared (Fig. 5). Intraperitoneal blood loss was significantly greater in nonsurvivors. Total unadjusted intraperitoneal blood loss (R = −0.75, P < 0.001) and adjusted blood loss for survival time (R = −0.88, P < 0.001) were also significantly and negatively associated with survival time in those animals receiving fluid resuscitation. At 60 min of prehospital resuscitation, fibrinogen concentration was decreased, and fibrin-specific clot formation was significantly impaired in nonsurvivors. Intraperitoneal blood loss was strongly and negatively correlated with both 60-min fibrinogen concentration (R = −0.85, P < 0.001) and fibrin-specific MA (R = −0.65, P = 0.005) in the fluid resuscitation groups. Angiopoietin 2 levels steadily increased over time in nonsurvivors during phase I, achieving an average 319% increase from baseline at 30 min and 780% increase from baseline at 105 min. Plasma Ang-2 levels were also increased in nonsurvivors compared with survivors by 30 min of prehospital resuscitation and remained elevated at 105 min of resuscitation, indicating early endothelial activation that was amplified in nonsurvivors.
Other swine models of hemodilution and bleeding from solid organ injury have produced similar results to ours. Fries et al. (10) demonstrated that high-dose fibrinogen concentrate (200 mg/dL) given after 65% hemodilution with gelatin could reduce blood loss from a liver injury and restore fibrin clot architecture. Similarly, Grottke (11) induced coagulopathy using HES and lactated Ringer’s solution to replace 80% of blood volume in a swine model. It was found that as little as 70 mg/kg of fibrinogen supplementation could prolong survival and reduce blood loss after blunt liver injury. These studies provide strong evidence for the critical role of fibrinogen to counteract coagulopathy and slow bleeding after injury. Our data also support a beneficial effect of fibrinogen supplementation to reduce life-threatening bleeding after injury. We add that the favorable effect of fibrinogen supplementation is preserved in the setting of high-pressure arterial bleeding.
Survivors demonstrated preserved fibrin-specific clot strength at 60 min after fluid resuscitation in this model. However, clot strength was similarly decreased in both FBG and HEX groups after fluid administration. Fibrinogen concentration was also preserved at baseline levels with sham resuscitation when both fibrinogen supplementation and fluid resuscitation were withheld. These results suggest that the primary effect of fibrinogen supplementation at the chosen dosage was to counterbalance the dilutional and anticoagulant effects of Hextend rather than a supranormal effect of fibrinogen on clot formation. Hextend has direct coagulopathic effects when given in similar dosage to ours (20 mL/kg) that are above and beyond hemodilution (22). The direct coagulopathic effect is due to the HES-induced interference with clot propagation and strength rather than impairment of thrombin generation and is recoverable with fibrinogen treatment (22, 30). We suspect that fibrinogen concentrate did preserve clot strength in this model. However, interanimal variation was too wide and mortality too early in the HEX group to definitively conclude a specific effect of fibrinogen concentrate because of the limited number of animals tested in each group (n = 7). Further studies powered to show differences in clot formation with fibrinogen concentrate are needed.
The goal of this model was to create favorable physiological conditions for severe hemorrhagic shock and endothelial dysfunction in the setting of free bleeding from a noncompressible internal wound that would be responsive to hemostatic interventions. Ideally, severe shock and endothelial dysfunction would be induced during phase I, yet animals would remain recoverable during phase II with surgical hemostasis and aggressive resuscitation. In keeping with these goals, we found that survival time in the resuscitated animals in this model was closely associated with the amount of internal blood loss, making it relevant to study the impact of hemostatic and fluid resuscitation strategies. The animals in the neg control group clearly died of hemorrhagic shock primarily from the initial catheter hemorrhage because intraperitoneal blood loss was very low, and lactate rose consistently until death. Nonsurvivors in the HEX group initially benefited from fluid resuscitation but continued to bleed and succumbed during phase I. Therefore, these animals represent a situation where fluid resuscitation promotes rebleeding and exsanguination. The two surviving animals in the HEX group likely survived because they demonstrated very little internal blood loss (mean, 10.2 [SD, 6.7] mL/kg). A single animal in the FBG group died quickly with a large amount of blood loss, whereas the remaining survivors in the FBG group bled very little, achieving a volume of adjusted intraperitoneal blood loss that was comparable to those receiving no fluid resuscitation. Overall, the survival benefit seen in the fibrinogen group may be attributed to decreased blood loss during fluid resuscitation. However, it is not clear if this effect can be attributed to an increase in fibrin-dependent clot strength.
Several limitations must be considered when interpreting the results of this study. First, this is a tightly controlled model of hemorrhage with clearly defined wound geometry and close monitoring of physiology. These conditions, along with obvious differences between humans and swine, do not allow for direct application of these results to the human trauma population. Next, the model does not fully reproduce native primary TIC where coagulopathy spontaneously develops after injury and hemorrhage without the influences of secondary coagulopathies. We suspect that the lack of additional tissue trauma precluded our ability to fully reproduce native TIC-like conditions in this model. We also are unable to separate protein colloidal and hemostatic effects of fibrinogen concentrate in this study. There may have been specific effects of fibrinogen as a colloidal protein that were independent of its hemostatic effects. We are planning future studies on this topic that will include a comparable nonhemostatic protein control such as albumin to investigate this possibility. We also noted an increase in PAP that coincided with fibrinogen infusion. The cause of this reaction and if it occurs similarly during fibrinogen infusion in humans remain unclear. Pigs are known to have similar adverse reactions to perfluorocarbon, particulate, and endotoxin infusions (36, 37). This response may be attributed to an acute inflammatory reaction causing pulmonary vascular constriction mediated by pulmonary macrophages because depletion of pulmonary macrophages in swine has also been shown to eliminate the increase in PAP associated with endotoxin infusion (38). There was also no simultaneous increase in systemic vascular resistance (data not shown), decrease in blood pressure, or decreased cardiac output to suggest that the PAP response was a product of systemic vasoconstriction similar to that seen with infusion of hemoglobin-based oxygen carriers (20). The response may also be specific to fibrinogen because isolated rabbit lungs have also demonstrated vasoconstriction and a sharp rise in PAP when perfused with soluble fibrin/fibrinogen-oligomers (39). This response was attenuated when pulmonary thromboxane generation was blocked. Although there were no adverse effects of the increased PAP on hemodynamics or outcomes in this study, the relevance to humans remains unclear, and further investigation is required. We also did not specifically investigate for pathological thrombosis of vessels or organs that might be associated with fibrinogen infusion. This is an important potential adverse effect of fibrinogen concentrate and requires further study using animal models specifically designed to assess thrombosis as a safety outcome.
The authors thank Nicole Bradbury, BS, for her special contributions, who was an integral member of our research team, who is no longer with us after unfortunately passing away unexpectedly; may she rest in peace.
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