Introduction:
Endothelial damage and extravasation are deleterious consequences of hemorrhagic shock (HS), associated with inflammation, loss of the endothelial glycocalyx barrier and hyperpermeability. Fresh frozen plasma (FFP) has been shown to preserve the glycocalyx and mitigate endothelial dysfunction following HS, although the mediators of this phenomenon remain unknown. Antithrombin III (ATIII) is a plasma protein with potent anticoagulant and anti-inflammatory activity. ATIII levels are decreased following trauma and particularly in those suffering from HS. Previous work in other disease models has shown that ATIII plays an important role in preserving the endothelial glycocalyx. We hypothesized that following HS, the vascular protective effects of FFP therapy are mediated through ATIII.
Methods and Results:
In vitro:
Confluent endothelial cells were pre-treated with FFP (n=12), ATIII deficient FFP (AT-Def FFP, n=12) or Vehicle (n=12) followed by treatment with TNF-α. The electrical impedance was monitored in real time over 24 H to detect changes in cell-to-cell junctions. We found that FFP treatment prevented the TNF-α -induced endothelial disruption showing a 1.6 and 1.5-fold improvement at 12 and 24 H respectively (vs. Vehicle, p<0.0001). AT-Def FFP showed some protection at 12 H (1.2-fold vs. Vehicle, p=0.05), although it was not maintained at 24 H (0.86-fold vs. Vehicle, p=0.34).
In vivo:
Under anesthesia
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30 male mice were subjected to a well-described fixed pressure exsanguination model. HS was achieved by sustaining a MAP of 35 mmHg for 90 min followed by resuscitation with either FFP (n=10) or AT-Def FFP (n=10). The expression of the glycocalyx marker, syndecan-1, in lung tissue was measured by immunofluorescence. We found that syndecan-1 expression increased in the FFP treatment group (1.6±0.4, p=0.001) vs. Control (1.0±0.26, n=10). However, these effects were absent in the AT-Def FFP treated group (1.3 ±0.3, p=0.13).
Conclusions:
These results suggest that ATIII mediates both the protective and reparative effects of FFP. Further studies are needed to elucidate the endothelial intracellular and extracellular signaling pathways mediating the ATIII-induced endothelial barrier preservation.
Clinical guidelines for cryosupernatant transfusions