Protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw

Abstract

Background: Clinical studies have shown that resuscitation with fresh frozen plasma (FFP) is associated with improved outcome after severe hemorrhagic shock (HS). We hypothesized that in addition to its effects on hemostasis, FFP has protective and stabilizing effects on the endothelium that translate into diminished endothelial cell (EC) permeability and improved resuscitation in vivo after HS. We further hypothesized that the beneficial effects of FFP would diminish over 5 days of routine storage at 4 degrees C.

Methods: EC permeability was induced by hypoxia and assessed by the passage of 70-kDa Dextran between monolayers. Thrombin generation time and coagulation factor levels or activity were assessed in FFP. An in vivo rat model of HS and resuscitation was used to determine the effects of FFP on hemodynamic stability.

Results: Thawed FFP inhibits EC permeability in vitro by 10.2-fold. Protective effects diminish (to 2.5-fold) by day 5. Thrombin generation time is increased in plasma that has been stored between days 0 and 5. In vivo data show that day 0 FFP is superior to day 5 FFP in maintaining mean arterial pressure in rats undergoing HS with resuscitation.

Conclusion: Both in vitro and in vivo studies show that FFP has beneficial effects on endothelial permeability, vascular stability, and resuscitation in rats after HS. The benefits are independent of hemostasis and diminish between days 0 and 5 of storage.

Figure 1

Day 0 FFP inhibits PECs permeability in vitro, which diminishes after 5 days of routine storage at 4°C. PECs were seeded in transwell chambers (0.4-μM pore size) and allowed to adhere for 48 hours. PECs were cultured with different concentrations (100%, 30%, and 5%) of FFP in media. FFP was stored and aged at 4°C between days 0, 5, and 10. Permeability was induced by 3% hypoxia in hypoxia incubators for 18 hours. Permeability was determined by the passage of 70-kDa fluorescein isothiocyanate-conjugated dextran to the bottom chamber over 1 hour. The 30-minute time point is presented here. All decreases in permeability were analyzed according to the mean and standard deviation of three experiments, and they were statistically significant (p < 0.05).

Figure 2

Representative curve of thrombin-generating capacity of FFP between days 0 and 5 of storage. As a global measure of the ability to form a clot, we studied the effect of storage on the capacity of the FFP to generate thrombin using a CAT Assay. The plot shows representative thrombin generation time curves from a single-donor FFP on days 0 and 5.

Figure 3

Effects of FFP at days 0 and 5 on MAP in rats subjected to hemorrhagic shock. Rats were hemorrhaged over 10 minutes and resuscitated with a fixed volume of plasma after 60 minutes. One hour after HS, FFP was administered: day 0 (group 1, n = 5) and day 5 (group 2, n = 5). FFP was infused at a volume equal to the blood lost. Data indicate that FFP infused at day 0, but not at day 5, restores blood pressure to baseline in hemorrhaged rats. These studies suggest that resuscitation with day 0 FFP is superior to day 5 FFP in maintaining hemodynamic stability. B, baseline.

Figure 4

Working biological model of the mechanism of action of FFP. This figure depicts our working biological model of the mechanism of action of FFP. HS leads to a deviation of the vasculature from homeostasis. HS induces hypoxia, endothelial cell tight junction breakdown, inflammation, and leukocyte diapedesis. FFP repairs and “normalizes” the vascular endothelium by restoring tight junctions, building the glycocalyx, and inhibiting inflammation and edema, all detrimental processes that are caused by iatrogenic injury with fluids such as lactated ringer's solution.