Modulation of Vascular Stability in Traumatic Injury by Plasma
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1 Modulation of Vascular Stability in Traumatic Injury by Plasma Shibani Pati MD. PhD. Blood Systems Research Institute and University of California San Francisco Remote Damage Control Resuscitation Meeting Bergen, Norway 2012
2 Disclosures This work was funded in part by Entegrion Inc. Additional funding received from the DOD and BSRI.
3 Trauma and Injury Statistics Trauma remains the leading cause of death and disability in children and young adults. Hemorrhage is associated with 40% of early deaths. World wide there are 5 million deaths per year due to trauma Cost of $518 billion annually In the US 2005, a total of 173,753 injury-related deaths occurred in the equivalent to a 747 crashing into the ground every day The National Center for Injury Prevention and Control. Web-based Injury Statistics Query and Reporting System. US Department of Health and Human Services, CDC; Available at: Accessed May 22, 2009.
4 Years of Potential Life Lost (YPLL) Before Age 65 Years of Potential Life Lost (YPLL) <Age 65 Cause of Death YPLL Percent Injury 300, % Unintentional Injury 199, % Suicide 52, % Homicide 48, Cancer 137, % Heart Disease 107, % Perinatal Period 75, % Congenital Anomalies 43, % Cerebrovascular 21, % HIV 21, % Liver Disease 21, % All Others 220, % The National Center for Injury Prevention and Control. Web-based Injury Statistics Query and Reporting System. US Department of Health and Human Services, CDC; Available at: Accessed May 22, 2009.
5 Targeting the Endothelium in Traumatic Injury The Endothelial Organ 5 The endothelium is the platform on which a number of biological processes take place In both health and disease: coagulation, tissue oxygenation, inflammation. From Nachman and Rafi NEJM, 2008
6 How does trauma and resuscitation affect vascular endothelial function and stability?
7 Factors contributing to vascular endothelial Disruption of the endothelial glycocalyx Ischemia-Reperfusion Injury Aberrant coagulation-coagulopathy Reactive Oxygen Species Proteases (MMP-2, MMP-9) Inflammatory mediators Cytokines and Chemokines From Endothelial Biomedicine Yokoyama and Chaudry 2007
8 General Hypothesis: If we prevent or repair endothelial barrier function after traumatic injury we improve outcome and decrease organ injury. We have chosen to explore the effects of plasma based resuscitation on vascular stability in injury
9 Background: There have been a number of clinical studies (both civilian and military) suggesting that administration of higher FFP to RBC ratios in (massively transfused) MT patients is associated with a potential survival benefit. A number of papers all retrospective Borgman, Spinella et al. Trauma 2007 Kashuk et al. J Trauma 2008 Holcomb et al. Annals of Surgery 2008
10 Background: There have been a number of clinical studies (both civilian and military) suggesting that administration of higher FFP to RBC ratios in (massively transfused) MT patients is associated with a potential survival benefit. A number of papers all retrospective Borgman, Spinella et al. Trauma 2007 Kashuk et al. J Trauma 2008 Holcomb et al. Annals of Surgery 2008
11 Two clinical trials that will attempt to clarify the role and Multi mechanism center retrospective of FFP and blood study product ratios in MT patients 10
12 Early use of Plasma attributed to decreased 11 MOF lower, 20 vs 9% p < day survival higher 57 vs 38%, p < 0.01 Difference attributed to rapid implementation plasma and platelets, resulting in earlier transfusion of plasma and platelets J Trauma 2009
13 Early use of Plasma attributed to decreased 11 MOF lower, 20 vs 9% p < day survival higher 57 vs 38%, p < 0.01 Difference attributed to rapid implementation plasma and platelets, resulting in earlier transfusion of plasma and platelets J Trauma 2009
14 How does plasma work? The rationale for the use of FFP in MT patients has primarily focused upon replacing lost coagulation factors-establishing hemostasis, reversing Coagulopathy and the lethal triad. We think a bit differently. We hypothesize that many of the effects of plasma on mortality and organ injury in MT patients are due to stabilizing effects FFP on the vascular endothelium.
15 How does plasma work? The rationale for the use of FFP in MT patients has primarily focused upon replacing lost coagulation factors-establishing hemostasis, reversing Coagulopathy and the lethal triad. We think a bit differently. We hypothesize that many of the effects of plasma on mortality and organ injury in MT patients are due to stabilizing effects FFP on the vascular endothelium.
16 Another point of clinical relevance: The storage lesion of Plasma (FFP) AABB practice allows FFP to be thawed and kept up to 5 days at between 1-6 C (in the US) This practice is based upon the presence of adequate coagulation factor activity necessary for hemostasis. Advantages of storage are: -it can be present upon arrival in the in the ED -can be used as a primary resuscitative fluid -Decreases waste by 60-70% The storage lesion has been well described for RBCs but not FFP - Malone DL, Hess JR, Fingerhut A. Comparison of practices around the globe and suggestion for a massive transfusion protocol. J Trauma, Armand R, Hess JR. Treating coagulopathy in trauma patients. Transfus Med Rev 2003.
17 Another point of clinical relevance: The storage lesion of Plasma (FFP) AABB practice allows FFP to be thawed and kept up to 5 days at between 1-6 C (in the US) This practice is based upon the presence of adequate coagulation factor activity necessary for hemostasis. Advantages of storage are: -it can be present upon arrival in the in the ED -can be used as a primary resuscitative fluid -Decreases waste by 60-70% The storage lesion has been well described for RBCs but not FFP - Malone DL, Hess JR, Fingerhut A. Comparison of practices around the globe and suggestion for a massive transfusion protocol. J Trauma, Armand R, Hess JR. Treating coagulopathy in trauma patients. Transfus Med Rev 2003.
18 Effects of FFP and Storage on Coagulation
19 Changes in Coagulation Factors between Day 0 and Day 5 FFP Pati et al. JTrauma 2010 N= 10 donors
20 CAT Assay shows changes in Day 0 vs Day 5 FFP Platelet activated in Day 5 Pati et al. JTrauma 2010
21 Liver Injury Model of Hemorrhage shows Phillip A. Letourneau, Madonna McManus, Kendell Sowards, Weiwei Wang, Willa Wang, Nena Matijevic, Shibani Pati, Charles E. Wade, John B. Holcomb Journal of Trauma 2011
22 How does FFP affect EC function in injury? Molecular Cell Whole animal
23 In vitro: Endothelial Permeability Pati et al. J. Trauma, 2010
24 Critical Regulators of Endothelial Permeability
25 In vitro FFP reconstitutes EC Adherens Junctions A B FFP (h) 0 1/4 1/ VE-cad ZO-1 β-catenin E Norm HR HR + FFP HR + LR β-catenin GAPDH C Norm HR HR + FFP HR + LR VE-Cadherin D 1.6 Relative VE-cad Level * Norm HR # * HR + FFP # HR + LR
26 FFP but not LR repairs EC Adherens Junctions after hypoxic injury Close up with Confocal microscopy VE-Cad β-catenin t=20 minutes DAPI Merge Control Hypoxia Pati et al - unpublished Hypoxia+FFP Hypoxia LR
27 In vitro Day 5 FFP Alters PEC signaling and decreases Adherens Junction Stability Pati et al - unpublished Net Implication Increased Leak with stored FFP
28 Gaps between Endothelial Cells Diminish with FFP Treatment AFM Microscopy Average Gap Size: Control- no gaps Hypoxia µm Hypoxia/Plasma µm Control Hypoxia Hypoxia/FFP
29 In vivo :Hemorrhagic Shock Mouse Model Lung Edema is inhibited by FFP Lung Edema * * Kozar-Pati submitted for publication N= 10 animals/ treatment group
30 In vivo FFP restores pulmonary endothelial integrity and decreases Acute Lung Injury * Lung Injury * A Sham HS HS + FFP HS + LR VE-cad β-catenin Occludin ZO-1 Actin Kozar-Pati submitted for publication
31 Endothelial Glycocalyx is reconstituted by FFP Electron Microscopy The glycocalyx is a ubiquitious barrier that protects the underlying endothelium PLOS One 2011
32 Syndecan-1 is restored by FFP in Pulmonary Alveolar Cells Kozar et al. PLOS1 2011
33 Syndecan-1 is restored by FFP in Pulmonary Alveolar Cells Kozar et al. PLOS1 2011
34 What about Dried Plasma? Lyophilized Plasma Use began during WWII Lyophilized plasma with ascorbic acid decreases inflammation in hemorrhagic shock. Hamilton GJ, Van PY, Differding JA, Kremenevskiy IV, Spoerke NJ, Sambasivan C, Watters JM, Schreiber MA. J Trauma Aug;71 (2):292-7; discussion Hemostatic and pharmacologic resuscitation: results of a long-term survival study in a swine polytrauma model. Alam HB, Hamwi KB, Duggan M, Fikry K, Lu J, Fukudome EY, Chong W, Bramos A, Kim K, Velmahos G. J Trauma Mar;70(3):
35 Why do we need a dried plasma product? Current FFP Preparation/Delivery Time: mins Blood type specific and limited supply of Universal Type AB Plasma (4% population) Potential Waste & Breakage: 4-37% Shelf life: 1 year frozen, 5 day unfrozen Storage Requirements: Freezers/ refrigerators Access limited to large trauma and acute care facilities Universal Reconstitution/prep-time: < 5 mins = immediate transfusion treatment Universal no ABO typing required No Waste reconstitute when needed Shelf life: 2-3 years Storage Requirements: TBD (Room Temperature +) Expanded access: Military Field Use, Emergency use, Aeromed Flight Services, National Disaster Stockpile Programs
36 The questions are: Does dried plasma (SDP or LyP) function like Day 0 FFP? Is dried plasma superior to Day 5 FFP? Can it treat the Lethal Triad? HYPOTHERMI A ACIDOSIS COAGULOPATHY Current Resuscitation 1:1:1 ratio of FFP/Plt /RBC Saline Artificial colloids Future of Resuscitation? 1:1:1 ratio Dried Human Plasma Easily reconstituted Easy to transport
37 Dried Plasma Products 2 products that are in development are being tested 1) Spray Dried Solvent Detergent Treated Plasma (SDP) Source: Entegrion 2) Lyophilized Plasma (LyP) Source: Hemcon Our studies have focused around effects on vascular endothelial function and inflammation
38 How does SDP compare to FFP in its effects on Endothelial Permeability? Groups compared are: 1) Control untreated 2) Hextend 3) LR 4) SD-FFP (solvent detergent treated pre-spray drying) 5) SDP
39 SDP and FFP are equivalently protective on EC Pati et al submitted
40 Time dependent changes reveal equivalence of minutes
41 EC Permeability Coefficients Reveal Equivalence 5% 10% 30% Pati et al submitted [P= J/A C] J= Flux, A area, C- Concentration,
42 The protective effects of SDP and FFP on EC TER/ permeability last up to 4 days post-treatment ECIS- Endothelial Cell Impedance System Inhibition of EC Permeability SDP and FFP Controls Treatment Added Thrombin Added
43 FFP and SDP are less protective 3kD 10kD No Donor Variability Pati et al submitted
44 Albumin is not the critical component in FFP All groups were subjected to antibody column depletion of albumin EC Permeability Pati et al submitted
45 SDP and FFP reconstitute EC Adherens Junctions Merged Pati et al submitted
46 How does SDP compare to FFP in its effects on endothelial cell inflammatory processes?
47 FFP and SDP treated ECs suppress TNFα dependent inflammatory cell binding FFP Pati et al submitted
48 Endothelial Adhesion Molecule Expression is not altered by FFP or SDP treatment..other mechanisms? Leukocyte Adhesion Endothelial Adhesion Molecule Expression Pati et al submitted VCAM-1 ICAM-1 E-Selectin
49 How does dried plasma compare to Day 5 FFP in its effects on EC permeability?
50 Dried (Lyp) plasma is superior to Day 5 FFP in regulation of EC permeability Pati et al submitted
51 How does dry plasma compare to FFP in vivo?
52 Lyophilized Plasma Inhibits ALI in Mouse Model of HS * * Group Sham HS no resuscitation HS + Hextend HS + NS HS + LR HS + FFP0 HS + FFP5 HS + lyophilized plasma N= 10 animal/group Pati et al submitted. % survival at 3 days * *
53 Working Biological Model?????? Improved outcome in patients
54 Pre-clinical Data Conclusions: FFP inhibits endothelial permeability both in vitro and in vivo. FFP inhibits endothelial junction breakdown in vitro and in vivo FFP in HS and Trauma models decreases leukocyte-ec interactions and acute lung injury FFP restores shock induced depletion of the EC glycocalyx Aged (Day 5) FFP has a diminished capacity to restore endothelial stability and junctions in vitro. In vivo FFP 5 shows a detrimental effect on survival. Data are in progress with the pulmonary effects. The effects of FFP in these defined pre-clinical measures of outcome can be recapitulated by dried plasma products: SDP and LyP.
55 Summary Our studies and those of others suggests that resuscitation paradigms and mechanisms of action of blood products and fluids in trauma are worthy of further investigation since current research shows that effects are multiple and not necessarily due solely to repletion of circulating blood volume or hemostasis, but possibly due to other effects.
56 Summary Our studies and those of others suggests that resuscitation paradigms and mechanisms of action of blood products and fluids in trauma are worthy of further investigation since current research shows that effects are multiple and not necessarily due solely to repletion of circulating blood volume or hemostasis, but possibly due to other effects.
57 Future Directions: What are the soluble factors X in FFP that mediate these effects on Endothelial Function? We have embarked upon a proteomics based approach to attempt to identify the soluble factor(s) X in FFP and their mechanism of action Not one factor We can capitalize on the differences between Day 0 and Day 5 FFP to id active proteins.
58 Future Directions: How does plasma change whole genome expression in the vascular endothelium? Control FFP Affymetryx full genome arrays of treated ECs Figure 11. Heat Map array analysis of Affymetryx data of LP vs FFP-0 showing that differences do exist at the gene expression levels. Blue genes indicate downregulation by treatment and Red/Yellow genes indicate upregulation.
59 Balancing Scales of Endothelial Function in Injury Homeostasis Lung Injury
60 Balancing Scales of Endothelial Function in Injury Injury (Traumatic or Infectious) TNFa MMPs IL-1 ROS Lung Injury
61 Balancing Scales of Endothelial Function in Injury KGF TIMP-3 Ang-1 IL-10 Novel Therapeutics MSCs, FFP, soluble factors TNFa VEGF IL-1 ROS REPAIR Lung Injury
62 Balancing Scales of Endothelial Function in Injury Endothelial Normalization
63 What is clear is that: Stability and integrity of the vascular endothelium is critical to good health. Trauma
64 Funding Sources This work was funded by 1) Department of Defense 2) Entegrion Inc.
65 Acknowledements Blood Systems Research Institute SF: Michael Busch- Director Phillip Norris My Lab (Tyler Menge, Kathryn Wataha, Daniel Potter, Stuart Gibb) Yuhai Zhao-UT UT Houston Director- CeTIR- John Holcomb Kozar Lab (Dr.Xue and Dr.Peng) Biostats at MDA (Li Shen and Jing Wang) WUSTL- Phillip Spinella UCSF- Mitchell Cohen
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