Mechanisms Of Vasoprotection By Resuscitation With Thawed Fresh Frozen Plasma In Hemorrhagic Shock

Chapter One:The mechanisms and function of thawed Fresh Frozen Plasma Induces Nitric Oxide ProductionHemorrhagic shock (HS) is the leading cause of death in civilian and military trauma. Fresh frozen plasma (FFP) has been used for HS in trauma centers with survival benefit compared to traditional resuscitation fluids such as lactated Ringer (LR). Because nitric oxide (NO) is a key mediator of normal endothelial function, we hypothesized that part of the beneficial effects of FFP is due to its ability to induce NO production and vasodilation during HS. In a standardized rat HS-resuscitation model, we found that FFP resulted a much higher and earlier NO production in the sera of FFP-resuscitated rats than HS alone rats as determined by a NO/Nitrite/Nitrate assay. On the contrary, LR suppressed HS-induced NO levels at all the time points examined. Acetylcholine induced a significant increase in vasodilation in FFP-resuscitated HS rats compared to HS alone rats (49±4 mmHg vs 37±4 mmHg). In vitro, FFP induced NO production in the conditioned media of FFP-treated human pulmonary microvascular endothelial cells (HPMECs) and dermal lymphatic endothelial cells (HDLECs). Phospho-kinase array and Western blotting revealed that FFP resulted in activation of AMPKα1, Akt1 and eNOS in both HPMECs and HDLECs. eNOS activation was confirmed in the lung tissues of FFP-resuscitated HS rats. Pretreatment with Compound C (AMPK inhibitor), LY294002 (PI3K/Akt inhibitor) or L-NAME (NOS inhibitor) inhibited AMPKα1/Akt1/eNOS cell signaling resulted in FFP-induced NO production and eNOS activation in HPMECs. Together, this study reveals a novel mechanism by which FFP exerts its vasoprotective effect.Chapter Two:Effect of thawed FFP on Endothelial Cell Migration and its MechanismsHemorrhagic shock (HS) is a leading cause of death after trauma. Recent clinical studies have shown that resuscitation with earlier and increased amounts of fresh frozen plasma (FFP) is associated with improved outcomes after severe HS. Many trauma centers are starting to use thawed plasma, an approved product that can be stored for up to 5 days at 4℃. FFP has been shown to contain significant levels of transforming growth factor-β(TGF-β) which is involved in endothelial cell migration. We hypothesized that FFP promotes endothelial migration, and the storage of FFP may alter its growth factor levels and signaling, thereby leading to decreased efficacy. Using migration as a functional endpoint, we examined the changes of TGF-βsignaling in response to Day 0 versus Day 5 FFP. Human pulmonary microvascular endothelial cells (HPMECs) and human dermal lymphatic endothelial cells (HDLECs) were treated with Day 0 and Day 5 FFPs and were subjected to migration assays and immunoblotting. We found that (1) Day 0 FFP induced endothelial cell migration under both normoxia and hypoxia conditions and this effect diminished significantly in Day 5 FFP; (2) TGF-β1 protein level increased significantly in Day 5 FFP compared to Day 0 FFP; (3) Inhibition of TGF-βtypeⅠreceptor ALK5 enhanced cell migration induced by both Day 0 and Day 5 FFPs; and (4) FFP induced a significant increase in phospho-Smad2/3, which was inhibited by ALK5 inhibitors. Compared to Day 0 FFP, Day 5 FFP induced greater phospho-Smad2/3 in both cell types. Our data suggest that TGF-β/ALK5/Smad2/3 signaling is involved in inhibition of FFP-induced cell migration, and standard storage of FFP may decrease the FFP efficacy on cell migration through increased TGF-P 1 level and TGF-β1/ALK5/Smad2/3 signaling.