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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