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Novel Blood Products in Trauma Resuscitation: The Quest for a Dried Plasma Product
Shibani Pati MD PhDAssociate Professor
Blood Systems Research Institute andUniversity of California San Francisco- UCSF
San Francisco, CA
Disclosures:This work was funded by the DOD, BSRI and Entegrion Inc.
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•There have been a number of retrospective clinical studies (both civilian and military) suggesting that administration of higher FFP to platelet to RBC ratios, in massively transfused patients, is associated with a survival benefit.•These findings have formed the principles of DCR
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•Few of many
Background:
Borgman JOT Oct, 2007
Retrospective Seminal Military Study:
Plasma:RBC:PLT
Increased Balanced Ratios of FFP to RBCs improve outcomes in MT patients
across centers
Holcomb et al. Ann Surg 2008;248:445-458.
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Two trials that were designed to clarify the role and mechanismof FFP, RBC and platelet ratios in MT patients
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• 680 patients, 12 sites, pragmatic multicenter RCT• Tested 1:1:1 vs 1:1:2. Primary outcome mortality • There was no difference in overall deaths at 24 hours and 30 days.• However, deaths from exsanguination were less at 24 hours in the
1:1:1 group.• During the protocol period when 1:1:1 vs. 1:1:2 was actively being
administered, there were less deaths in the 1:1:1 group. • Interestingly, there was increased platelet and plasma usage in the
1:1:1 group in the first 24 hours with no increase in complications. Holcomb et al. JAMA, February 2015
These findings call for mechanistic studies into why balanced ratios with increased use of plasma and platelets provide a survival benefit
Trauma leads to disturbances in ICEImmunological functionCoagulationEndothelial function and integrity
The endothelium is the “platform” on which ICE disturbances occur. Which of these processes is the initiator of pathological disease is unclear.
We have chosen to explore the effects of blood products on vascular stability and the EOT of trauma.
Holcomb and Pati Hematology, 2013. doi: 10.1182/asheducation-2013.1.656.Jenkins et al. Shock. 2014 May;41 Suppl 1:3-12.
A new term has been coined: “Endotheliopathy of Trauma”- EOT
The Endotheliopathy of Trauma
Platelet Dysfunction
Tissue Hypoperfusion ATC Severe Injury
Hypothermia
Acidosis
HemodilutionTIC
DysfibrinogenemiaHyperfibrinolysis Endothelial Dysfunction
Inflammatory Response
Uncontrolled Hemorrhage
Damage Control Resuscitation (DCR)
• Primary Goals
– Hemorrhage control
– Avoid lethal triad
– Mitigate the Endotheliopathy of Trauma
• Risk factors for MT
– SBP < 110
– HR > 105
– HCT < 32
– pH < 7.25
• Transfusion Goal – 1:1:1 Plasma to Platelets to
RBCs
85% chance of MT
• Balanced ratios of components• Last blood product collected is first out• FWB only if components not available or
patient not responding • Platelets obtained in theater• Additional fibrinogen required• rFVIIa option if coagulopathy persists• TXA for patients requiring MT
Principles of Damage Control Resuscitation (DCR) in the Military
• We have formed the fundamental hypothesis:that if we prevent or repair endothelial dysfunction early after traumatic injury –we can improve outcomes.
• We hypothesize that plasma and platelets transfusion in traumarepairs endothelial injury and dysfunction.
• We further hypothesize that the effect of plasma and platelet transfusion leads to increased survival and improved secondary outcomes related to injury- ie decreased organ failure and long term disabilities
Hypothesis: Aside from hemostasis….
How do plasma and platelets work in DCR?
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Multiple Units of Blood components are transfused into MT patients– We need to know how these
products work and optimize them
Transfused intoone patient on a Friday Nightat OHSU
Our Working Biological Model
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Pati et al. JOT 2010
Hypothesis:Plasma and Plateletsare Reparativeof the EOT
VE-Cadherin
0 1/4 1/2 1 2 4 6FFP (h)
GAPDH
-catenin
ZO-1
In vitro: FFP inhibits EC permeability, repairs EC Adherens Junctions and inhibits EC inflammation
-catenin
FFP inhibits EC permeability
FFP increases VE-Cadherin Expression
FFP repairs Adherens Junctions
FFP inhibits EC-Leukocyte BindingControl
FFP-0
Pati et al. JOT 2010 and Wataha et al. Transfusion 2012
VE-Cadherin
In Vivo: Hemorrhagic Shock Mouse ModelLung Injury is inhibited by FFP
Lung Edema is inhibited by FFP
**
Lung Injury is inhibited by FFP Lung Inflammation is inhibited by FFP (MPO)
Peng, Pati… Kozar Shock 2013
Model
Lung Vascular Permeability in HS is Inhibited by FFP
Shock Shock + FFP
-0
-10,000
Pix
el in
ten
sity
Potter…Pati, 2015 Journal of Trauma Acute Care Surgery
Per
mea
bilit
y
FFP Inhibits Pulmonary Vascular Permeability induced by Hemorrhagic
Shock in Mice
SHAM SHOCK FFP
-0
-10,000
Pix
el in
tens
ity
More Leakage
Less Leakage
Sham
Shock FFP LR
0
1
2
3
4
5
Nor
ma
lize
dP
erm
ea
blity
*
N=5 mice/group
NS
Potter….Pati, Journal of Trauma Acute Care Surgery, 2015
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Novel and New Blood Products on the Horizon
• Fresh Whole Blood
• Frozen RBCs
• Frozen Cryopreserved Platelets (CPP)
• Cold 4C Stored Platelets
• Dried Platelets: Thrombosomes
• Plasma: FFP, Thawed plasma, Liquid plasma, dried plasma (Lyophilized, Spray Dried)
• Obtained from whole blood and frozen at -20 oC within 8 hours of collection
• Stored up to 1 year at -20 oC• Freeze/thaw results in diminished factor
function• Approximately 30 minutes to thaw• After thaw it can be stored up to 5 days at 1-6 oC
Fresh Frozen Plasma
• Once thawed, FFP can be maintained at 1 –6 oC for up to 5 days
• Minimal degradation of factor function (<10%)• Mitigates logistical difficulties of maintaining a
balanced resuscitation
Thawed FFP
Data Holcomb Lab
• Similar to FFP and used interchangeably • Frozen by 24 hours • Additional testing (anti - HLA antibodies, TRALI)• Further fractionation of plasma• Not approved to create thawed plasma
FP24
• Never frozen• Approved for 26 days storage at 1-6 oC• Minimal factor degradation• Immediately available
Liquid Plasma
0
40
80
120
160
200
LQP‐0 LQP‐5 LQP‐10 LQP‐20 LQP‐26
FII
FV
FVII
VIII
FIX
FX
FXI
FXII
FXIII
vWF ag
% Activity
Increase in FVII, XII and vWF in some plasma units: cold promoted activation>88% of the initial activities retained
41%
51%
Data Holcomb Lab
Back to the Future- Dried Plasma
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-Logistically superior -Storage for long periods of time (2 year)-Rapid reconstitution (minutes)-Can be concentrated 2X or 3X-Pathogen Reduced (SD/PRT)-Universal Donor Possible-Minimal factor activity loss (some loss of V and VII and SDP-vWF)-Can be used in remote areas and far forward care is possible-US products are in early FDA trials
Dried Plasma
Commercially Available Dried Plasma
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GermanLyoPlas
FrenchFLyP
South AfricanBioplasma
German LyoPlasSingle donor (Blood type compatibility)Stored up to 15 months200,000 TFNs – 0.023% major complications similar to FFPSD treatedFew minute reconstitution
French MilitaryUp to 11 donors/unit (Universal AB)Stored up to 24 monthsABO UniversalAvailable to US Special Forces on IRB protocolTransfusions with no major adverse eventsIntercept Treated6 minute reconstitution
Pooled up to 1500 donorsSD treatedABO UniversalStored below 25 C1000s transfused<10 min reconstitution
Pusateri et al Transfusion 2016
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Investigating Dried Plasma: Proving Non-Inferiority
Currently in the United States there are 3 main companies Involved in the development of a dried plasma product
1) Entegrion- SDP2) Velico- LP- Device for Blood Banks3) Vascular Solutions/Teleflex- LP
Also Terumo (Device for Blood Banks) and Cerus
Comparison of Spray Dried Plasma (SDP) to FFP in vitro
% Decr
ease in
Transw
ell Pe
rmeab
ility
0
10
20
30
40
50
60
10% Permeability w/ 3 kDa FITC- dextran, t= 45 minutes
0
10
20
30
40
50
60
70
80
10% Permeability w/ 10 kDa FITC-
dextran, t= 45 minutes
Control LR Hextend FFP-0 SDP Control LR Hextend FFP-0 SDP
SDP and FFP Equivalently inhibit Paracellular Pulmonary Endothelial Permeability to 3 and 10kD Molecules
NSNS
* *
Wataha et al. Transfusion 2012
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SDP and FFP Equivalently inhibit Leukocyte Pulmonary Endothelial Cell Binding
% De
creas
e in E
ndoth
elial
Leuk
ocyte
Bind
ing
5% Concentration
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
30% Concentration
Control LR HextendFFP-0
SDPFFPO FFPE Control LR Hextend SDPFFPO FFPE
* NS NS
*
Wataha et al. Transfusion 2012
ECIS Assay
• Endothelial Cell Impedance Assay (Applied Biophysics)
http://biophysics.com/products.php
Increased Resistance = Decreased Permeability
SDP and FFP increase TEER in a dose dependent fashion but not LR
0.0 0.5 1.0 1.5 2.0 2.50.9
1.0
1.1
1.2
1.3
1.4
1.5
Time (hrs)
Norm
aliz
ed
Res
ista
nce
LR5%
10%30%
0.0 0.5 1.0 1.5 2.0 2.50.9
1.0
1.1
1.2
1.3
1.4
1.5
FFP
Time (hrs)
Norm
aliz
ed
Res
ista
nce 5%
10%
30%
0.0 0.5 1.0 1.5 2.0 2.50.9
1.0
1.1
1.2
1.3
1.4
1.5
SDP
Time (hrs)
Norm
aliz
ed R
esis
tan
ce 5%
10%30%
A B
C
Dec
reas
ed P
erm
eabi
lity
0.0 0.5 1.0 1.5 2.0 2.50.9
1.0
1.1
1.2
1.3
1.4
Time (hrs)
Nor
maliz
ed R
esis
tance
Control
10% LR
10% SDP
10% FFP
LRFFP
SDP0.0
0.1
0.2
0.3
0.4
% in
cre
ase
of
Norm
aliz
ed
Res
ista
nce
5%
10%
30%
10%
LR
10%
SDP
10%
FFP
0.0
0.1
0.2
0.3
0.4
% in
crea
se o
f Norm
aliz
ed R
esis
tance
A
B
C*p<0.05
SDP and FFP increase TEERs but not LR
* **
FFP and SDP Equivalently Reconstitute Endothelial Integrity-Adherens Junctions
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How does FFP and SDP regulate Gene Expression in Pulmonary Vascular Endothelial Cells?
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SDP and FFP similarly modulateEndothelial Gene Expression
HUVECs
MediaRemoveMedia
10% Treatment in Media
1 hourin Serum
freeMedia
No Serum
RemoveMedia
AddTreatment
Media
10% Treatmentin Media
Incubate16 hours
10% Treatment in Media
Harvestcells
AmplifyRNA
(MirVana Kit)
Run rtPCRArray(Qiagen)
LR
Con
trol
SD
P
FF
PN
THBS1
CA V1SERPINE1
FN1CTNNA1
MMP1
GJ A1HYAL 2
ACTN1IT GB1
VWFANXA5
MMP2
CDH5IT GA5
SOD1TFPI
RHOB
CSF2IL 1B
PF4TIMP3
PDGFRA
GJ A5FGF1
CCL5CLDN4
CX3CL1
COL18A1FL T1
IT GB3PECAM1
TNFRSF1 0C
TIMP1PGF
BAXTEK
CASP3IT GA V
CTNNB1
KDRTNFSF1 0
SDC3GPC1
SDC2
KITCCL2
SDC4PLA T
NOS3
AD AM1 7CASP6
PLA UPLG
TYMP
SELPLGSELE
VCAM1SELL
SDC1PTGI S
TNF AIP3
CASP1ANGPT 1
GJ A4IL 6
PLA2G4C
ICAM1THBD
SNCAI PVEGF A
F AS
AC ERIPK1
OCLN
− 1 0 1
Row Z−Score
Color Key
Heat Map
C LR SDP FFP
FFP and SDP up regulate ACE and GJA4 in primary endothelial cells after 8 hours
FFP vs Control SDP vs Control
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At the level of EC gene expression – few significant differences were found between FFP and SDP
In Vivo GOLD StandardHemorrhagic Shock and Trauma model
in C57BL6 mice
Instrumentation &Acclimation
Post shock
Time (min)0-60 60 90 120-30 30
Shock
Blood
dra
w
Blood
gas
Mea
sure
d
Resus
citat
ion
150 180
Free
ambu
latio
n
Blood
gas
mea
sure
d
lung
s ha
rves
ted
Cannu
las R
emov
ed
90 Minute Shock Model
210
Fixed Pressure Acute Model of Hemorrhagic Shock and LaparotomyChesebro BB, Rahn P, Carles M, Esmon CT, Xu J, Brohi K, Frith D, Pittet JF, Cohen MJ.Increase in activated protein c mediates acute traumatic coagulopathy in mice. Shock. 2009;32:659-665
FFP and SDP elevate MAPs after HS equally
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n=7 mice
MAPs continuous readings
SDP and FFP equally correct the Negative Base Excess in HS
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Presh
ock
Sham HSFFP
SDP LR-20
-15
-10
-5
0
5
Base excess
Bas
e E
xces
s (m
mo
l/L) FFP and SDP were equivalent in
All other laboratory parameters tested • Electrolyte chemistries• Blood gases• Metabolic parameters
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Sham
Shock FFP
SDP LR
0
1
2
3
4
5
Norm
aliz
ed
Pe
rme
abl
ity
A **
n.s.
SHAM SHOCK LRFFP SDP
-0
-10,000
Pix
el in
tens
ity
More Leakage
Less Leakage
B
FFP and SDP equivalently inhibit pulmonary vascular permeability induced by hemorrhagic
shock in mice
VE-Cadherin and Adherens Junctions are Preserved in by FFP and SDP but not LR
VE-Cadherin is the single most critical molecular/cellularregulator of vascular permeability
50 um
Pericytes are equally preserved in FFP and SDP Lungs
45
NG2 (Green)
50 um
CD68 positive monocytes and macrophage infiltrates are decreased by SDP and FFP resuscitation but not LR
Red- CD68, Blue- MPO
CD68
50 um
Quantitative Results demonstrating equivalently decreased lung inflammation and increased endothelial integrity in FFP and SDP mice
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How does FFP and SDP regulate Vascular Endothelial Gene Product (cytokines, chemokines
and growth factors) Production and Secretion?
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Regulation of Cytokine, Chemokine and Growth Factor Production in ECs by FFP and SDP
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Endothelial Cells
Hypoxia 1%
FFP, LR, SDP
Harvest Conditioned MediaLuminex Panels Run50 cytokine, chemokine, and growth factors
sEGF
Control
FFP LRSDP
0
1000
2000
3000
4000
5000
Co
nc
en
tra
tio
n (p
g/m
L)
GCSF
Control
FFP LRSDP
0
2
4
6
8
10
Co
nc
en
tra
tion
(pg
/mL
)
sHER-2
Control
FFP LRSDP
1
10
100
1000
10000
100000
Co
nc
en
tra
tion
(p
g/m
L)
HGF
Control
FFP LRSDP
0
50
100
150
200
Co
nc
en
tra
tion
(pg
/mL
)
Cytokine, Chemokine and Growth Factor Differences
SDP equivalently alter expression of many Endothelial growth factors,chemokines and cytokines
Control
FFP LRSDP
0
50
100
150
200
Co
nc
en
tra
tion
(pg/
mL
)
VEGF-C
Control
FFP LRSDP
0
100
200
300
400
Co
nce
ntr
atio
n (p
g/m
L)
VEGF-A
Control
FFP LRSDP
0
100
200
300
400
Co
nc
en
tra
tion
(pg
/mL
)
VEGF-D
Vascular Permeability: SDP and FFP decrease expression of permeability factors VEGF-A and VEGF-D
Control
FFP LRSDP
0
500
1000
1500
2000
Co
nc
en
tra
tion
(pg
/mL
)
Angiopoietin -2sTIE-2
Control
FFP LRSDP
0
500
1000
1500
2000
Co
nc
entr
atio
n (p
g/m
L)
Vascular Permeability: SDP and FFP increase expression of anti-permeability factor Tie-2 which
inhibits Ang-1/Ang-2 signaling
Angiopoietin-1 and Angiopoeitin-2 bind to Tie-2 and regulate vascular stability.Ang-1 and Ang-2 bind to sTie-2R. Net: Inhibits permeability.
Conclusions on Dried Plasma:
1) Dried plasma and FFP have equivalent capability to achieve hemostasis
2) Dried plasma and FFP equally inhibit endothelial permeability and increase trans-endothelial resistances (TEER).
3) Dried plasma and FFP similarly influence EC gene expression and EC cytokines, chemokines and factors.
4) In vivo, in hemorrhagic shock, Dried plasma and FFP similarly regulate MAPs, Base deficit correction, pulmonary vascular permeability, lung inflammation and lung endothelial integrity.
53 54
Is there Donor Variability in FFP?
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Donor Variability in Plasma Thrombin Generation by CAT Assay
Spinella et al JOT 2015
Donor Variability in FFP from 8 donors from Blood Centers of the Pacific, San Francisco
VEGF induced TEER drop
Effects of Plasma on Endothelial Permeability
Donor Variability in FFP from donors from Blood Centers of the Pacific, San Francisco
SDP
FFP(used in study)
Dec
reas
ed P
erm
eabi
lity
Maximal Protection
FFPs that are share a line are NOT significantly different from each other For example, 29,28, and 37 (connected by line B are not different, but 36 and37 are different. (8 wells per FFP batch)
Donor Variability in FFP found in GroupsD
ecre
ased
Per
mea
bilit
y
Donor Variability in FFP: Differential Capacity to Restore MAPs after Hemorrhage
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-30 0 30 600
30
60
90
90 100 110 120
FFP33 vs FFP30
Time (Min)
MA
P (
mm
Hg) FFP33
FFP30
*HS
FFP 30FFP 33
Red B-cateninGreen- VE-CadherinYellow- Merge
Donor Variability: Age and Gender Effects on Outcomes
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Exposure of aged mice to young blood latein life is capable of rejuvenating cognitive function
Parabiosis Model
Exposure of aged mice to young blood is capable of rejuvenating skeletal muscle
Papers of InterestScience May 2014Nature Med May 2014
Questions to consider:1) Does the age and gender of thedonor blood affect outcomes in recipients?2) What are the soluble factors that modulate outcomes?3) Can one determine the effect of age/gender of donor bloodRetrospectively on trauma outcomes?4) A prospective observational or randomized controlled trialStudying age and gender specific blood components may beworthy of future investigations?
Was Bram Stoker Right?
Over 1000 proteins in plasmaMany are biologically activeMany have unknown functionsSuppression of dysfunctional inflammationDecreased endothelial permeabilityRebuilding the glycocalyxDecreased Organ Failure
Potential Benefits of Plasma (Aside from hemostasis)
Blood Systems Research Institute SF:
Michael Busch MD PhD- Director
Phillip Norris MD
Roberta Bruhn PhD
Sheila Keating PhD
Xutao Deng PhD
My Lab (Tyler Menge, Kathryn Wataha, Daniel Potter, Stuart Gibb)
Yuhai Zhao-UT
My Lab
Daniel Potter PhD
Gail Baimukanova MD PhD
Stuart Gibb PhD
Nathan Bucay
BSI-Arizona
Dr. Peter Tomasulo MD
Dr. Frank Nizzi DO
Acknowledgements
UT HoustonDr. Rosemary KozarKozar Lab (Dr.Xue and Dr.Peng)John Holcomb and Charles WadeTien Ko and LabBiostats at MDA (Li Shen and Jing Wang)
WUSTL- Phillip SpinellaUCSF- Mitchell CohenByron Miyazawa
OHSU Martin Schreiber
Entegrion Joseph DacortaMichael Galliger