Platelet Dysfunction after Trauma: Characterization and the study of function. Mitchell Jay Cohen MD FACS Associate Professor In Residence Associate Trauma Medical Director Director of Trauma Research University of California San Francisco

Platelets: A primary mediator?

San  Francisco  General  Hospital  

•  San  Francisco’s  only  trauma  center  •  Cares  for  all  pa7ents  with  trauma7c  injury  in  San  Francisco,  regardless  of  ability  to  pay  

•  Serves  100,000  pa7ents  per  year  •  Cares  for  4,900  injured  pa7ents  per  year  

SFGH  Surgical  Research  Lab  

Hypoxia

FXa binds to FVa on the cell surface

The complex between TF and FVIIa activates FIX and FX

Tissue factor (TF) is exposed and binds to FVIIa or FVII which is subsequently converted to FVIIa

Trauma, and Hypoxia induce the initiation of coagulation

Initiation phase

The FXa/FVa complex converts small amounts of prothrombin into thrombin

The small amount of thrombin generated activates FVIII, FV, FXI and platelets locally.

Activated platelets bind FVa, FVIIIa and FIXa

FXIa converts FIX to FIXa

Amplification phase

The FVIIIa/FIXa complex activates FX on the surfaces of activated platelets

Fibrin

The “thrombin burst” leads to the formation of a stable fibrin clot.

FXa in association with FVa converts large amounts of prothrombin into thrombin creating a “thrombin burst”.

Propagation phase

Coagulopathy in the wild...

Maladaptive response to trauma. Early coagulopathy, later hypercoagulable state and loss

of cytoprotectivity.

J Trauma, 2007 65%

34%

19%

0

10

20

30

40

50

60

70

(Low) 1:8 (Medium) 1:2.5 (High) 1:1.4

Mo

rtal

ity

P < 0.05

Don’t forget platelets.

Sugges7ons  platelets  are  important  aGer  injury.  

•  Correla7on  of  number  to  outcome.  •  Benefit  for  empiric  platelet  resuscita7on.  •  Func7onal  data.  

Platelet Number and Mortality

Brown L, Cohen M J Trauma 2011

Benefits of platelet transfusions.

¡  Recent trends in hemostatic resuscitation ratios have revolutionized care of the critically injured

¡ Appropriate FFP:RBC ratios are commonly a focus of therapy and research ¡  Intuitive mechanism

¡  Highly evolved research techniques

¡  Higher platelet:RBC ratios show a similar survival advantage ¡  Less clear explanation

¡  Difficult to study

¡  Rare in clinical practice

Holcomb et al., 2008. Ann Surg 248(3):447.

Ann Surg 2008

PROMMTT  showed  benefit  from  early  platelet  transfusion  despite  poor  and    slow  platelet  use.  

Platelet function in trauma ¡  Platelet function is impaired after trauma ¡  Flow cytometry for activation markers

¡  Platelet microparticle analysis

¡  Light aggregometry

Jacoby et al., 2001. J Trauma 51(4):639.

Platelet function assays ¡  Platelet count

¡  Bleeding time

¡  Single platelet counting

¡  Flow cytometry

¡ Aggregometry

Background: Platelet function assays ¡  Resting platelets are non-thrombogenic

¡ Activation leads to aggregation and adhesion

¡  Light aggregometry ¡  Measures occlusion of an aperture leading to

decreased light transmission by platelet aggregates

¡  Impedance aggregometry ¡  Measures increase in continuous electrical

impedance between charged wires as platelets adhere to charged surface

Maurer-Spurej & Devine, 2001. Lab Invest 81:1517.

¡  Prospective collection of citrated whole blood from critically-injured trauma patients on arrival and throughout ICU stay

¡  Point-of-care measurement of platelet activation using Multiplate® multiple electrode aggregometry

¡ Matching to parallel standard laboratory values

¡  Link with resuscitation and outcomes data

Impedance aggregometry

Impedance aggregometry

Impedance aggregometry

Platelet agonists

Ther Adv Cardiac Dis, 2008. London: SAGE.

Patient demographics ¡  101 critically-injured patients

¡  376 individual samples

¡ Normal mean platelet count

¡ No admission thrombocytopenia

Age 41.3 ± 19.3 BMI 26.2 ± 5.4

Blunt injury 69.0% ISS 23.9 ± 14.8

Base deficit -5.3 ± 6.0 GCS 9 (5-15)

Temperature 35.8 ± 0.8 Prehospital IVF 250 (50-1000)

Hematocrit 39.6 ± 5.4 Platelet count 274.4 ± 85.4

INR 1.1 (1.1-1.3) PTT 27.5 (25.4-31.5) pH 7.28 ± 0.17

Base deficit -5.3 ± 6.0

Platelet function on admission

Admission values (N=78) Normal range

ADP 44.6 ± 20.4 38-85 TRAP 86.6 ± 27.0 69-117 AA 44.3 ± 28.3 40-91

Collagen 44.7 ± 19.7 43-90

¡ Mean admission platelet function within normal range

¡  Using manufacturer normal ranges: ¡  45.5% had admission platelet hypofunction to at least one

measured agonist

¡  91.1% had platelet hypofunction at some time during ICU stay

Platelet function vs. platelet count

010

020

030

040

050

0

Plat

elet

cou

nt

0 20 40 60 80 100 120 140ADP response (AUC)

AADP

010

020

030

040

050

0

Plat

elet

cou

nt

0 20 40 60 80 100 120 140ADP response (AUC)

BTRAP

010

020

030

040

050

0

Plat

elet

cou

nt

0 20 40 60 80 100 120 140ADP response (AUC)

CAA

010

020

030

040

050

0

Plat

elet

cou

nt

0 20 40 60 80 100 120 140ADP response (AUC)

DCollagen

R2=0.417 R2=0.514

R2=0.504 R2=0.427

Platelet function over time

010

020

030

040

050

0Pl

t * 1

0^3/

uL

0 24 48 72 96 120Hours

EPlatelet count

2030

4050

AUC

0 24 48 72 96 120Hours

AADP

5060

7080

90AU

C

0 24 48 72 96 120Hours

BTRAP

2030

4050

60AU

C

0 24 48 72 96 120Hours

CAA

3040

5060

AUC

0 24 48 72 96 120Hours

DCollagen

Multivariate predictors of platelet hypofunction

OR P 95% confidence interval Age 1.041 0.032 (1.003 – 1.081)

Base deficit 0.871 0.026 (0.772 – 0.983) GCS 0.833 0.010 (0.726 - 0.957)

Platelet count 0.994 0.150 (0.987 – 1.002)

Platelet hypofunction: Outcomes

Platelet hypofunction Normal function P-value (N=39) (N=52)

Hospital LOS 6 (2-27) 10 (6.5-20) 0.090 ICU LOS 3.5 (1-14) 3 (2-14) 0.436

Vent-free days 12 (0-26) 26 (7.5-27) 0.040 24h mortality 20.0% 2.1% 0.009

Platelet hypofunction associations with mortality

0.2

5.5

.75

1

Surv

ival

0 200 400 600 800

Hours enrolled

ADP response >=38ADP response < 38

AADP

0.2

5.5

.75

1

Surv

ival

0 200 400 600 800

Hours enrolled

AA response >=40AA response < 40

CAA

0.2

5.5

.75

1

Surv

ival

0 200 400 600 800

Hours enrolled

TRAP response >=69TRAP response < 69

BTRAP

0.2

5.5

.75

1

Surv

ival

0 200 400 600 800

Hours enrolled

Collagen response < 43Collagen response >=43

DCollagen

*

*

0.2

5.5

.75

1

Surv

ival

0 200 400 600 800

Hours enrolled

Platelet count >=220Platelet count < 220

EPlatelet count

Platelet hypofunction predicts mortality

Univariate P Multivariate P ADP 0.958 0.007 0.974 0.109 TRAP 0.976 0.023 0.981 0.047 AA 0.969 0.004 0.964 0.018

Collagen 0.944 0.001 0.954 0.026

¡ Adjusted for age, GCS, base deficit, and platelet count

Platelet hypofunction predicts mortality

0.00

0.25

0.50

0.75

1.00

Sens

itivity

0.00 0.25 0.50 0.75 1.00

1 - SpecificityArea under ROC curve = 0.3381

AADP

0.00

0.25

0.50

0.75

1.00

Sens

itivity

0.00 0.25 0.50 0.75 1.00

1 - SpecificityArea under ROC curve = 0.2313

CAA

0.00

0.25

0.50

0.75

1.00

Sens

itivity

0.00 0.25 0.50 0.75 1.00

1 - SpecificityArea under ROC curve = 0.4213

BTRAP

0.00

0.25

0.50

0.75

1.00

Sens

itivity

0.00 0.25 0.50 0.75 1.00

1 - SpecificityArea under ROC curve = 0.2831

DCollagen

0.00

0.25

0.50

0.75

1.00

Sens

itivity

0.00 0.25 0.50 0.75 1.00

1 - SpecificityArea under ROC curve = 0.3665

EPlatelet count

AUC=0.769* AUC=0.717*

AUC=0.662 AUC=0.579

AUC=0.634

Summary ¡  Platelet dysfunction exists after traumatic injury, and is

not identifiable by standard laboratory measures. ¡  45% of critically injured patients have platelet

hypofunction on arrival despite normal platelet counts

¡  Older age, lower base deficit, and lower GCS are independent risk factors for platelet hypofunction

¡  Platelet dysfunction after trauma correlates with poor outcomes. ¡  Mortality in patients with platelet hypofunction is nearly 10-

fold higher than those with normal platelet function

¡  Poor platelet response to AA and collagen on admission are independent predictors of mortality

Summary ¡ Clinically significant platelet dysfunction exists and

carries a grave prognosis, but remains invisible to treating clinicians

¡  Impedance aggregometry reliably identifies this platelet dysfunction in injured patients

¡  Rapid, point-of-care platelet function testing will lead to better targeted therapy and better outcomes after trauma

That’s  great  but  what  should  we  give?  

Storage  Lesion  of  Apheresis  Platelets?  

Cold  platelets?  

•  Current  blood-­‐banking  procedures  call  for  apheresis  derived  platelets  to  be  stored  at  22°C  for  5  days.  

•  The  only  measurements  controlled  for  platelets  are  pH  and  sterility.  

•  These  procedures  are  mainly  derived  from  research  done  in  the  1970’s,  focusing  on  hemosta7c  func7onality  and  circula7on  survival.  

•  Consequently,  storage  at  4°C  had  been  eliminated  due  to  elevated  clearance  aGer  transfusion.  

Platelets  and/or  whole  blood?  

¡ WB variants (11 of each) ¡ Room temperature WB ¡ Cooled WB ¡ Modified room temperature WB ¡ Modified cooled WB

¡ RWB (23 of each) ¡ 1:1:1 (RBC:FFP:PLTS) ¡ 2:1:1 (RBC:FFP:PLTS)

Generating whole blood and reconstituted whole blood.

Non-modified WB variants

Platelet-modified WB variants

÷6

ROOM TEMP WB

COOLED WB

MODIFIED COOLED

WB

MODIFIED ROOM TEMP

WB

RBC

1 2

1 1

1 1

1:1:1 RWB

2:1:1 RWB

Laboratory methods

¡ Rotational thromboelastometry (ROTEM)

¡ Standard coagulation measures

¡ CBC

¡ Fibrinogen, factors II, V, VII, VIII, IX, X, ATIII, and protein C

¡ Statistical analysis: ¡ Student’s t test/ANOVA (normal)

¡ Wilcoxon rank sum/Kruskal Wallis (skewed) ¡ Fisher’s exact test for proportions (proportions)

1:1:1 2:1:1

Comparisons of resuscitation products.

¡ Comparison of 1:1:1 to 2:1:1 RWB variants

¡ Comparison of 4 types of whole blood

¡  cooled, room temp, modified cooled, modified room temp

¡ Comparison of modified WB to 1:1:1

760.57  

952.43  

CFT(s)  

56.27  

38.57  

45.83  

50.35  

48.65  

30.43  

38.82  

43.22  

Alpha(°)   a10(mm)   a20(mm)   MCF(mm)  

EXTEM

*

*

* * *

145.91  

199.61  

CT(s)  

1:1:1   2:1:1  

1:1:1 2:1:1

Comparisons of resuscitation products.

¡ Comparison of 1:1:1 to 2:1:1 RWB variants

¡ Comparison of 4 types of whole blood

¡  cooled, room temp, modified cooled, modified room temp

¡ Comparison of modified WB to 1:1:1

    ROOM TEMP   COOLED  MODIFIED, COOLED  

MODIFIED, ROOM TEMP      

    n=11   SD   n=11   SD   n=11   SD   N=7   SD   p-value  WBC (x10^3/µL)   4.40   1.31   4.51   1.28   4.15   1.32   4.01   1.51   0.7594  Hgb (g/dL)   11.24   1.47   11.52   1.26   10.39   1.22   10.90   1.27   0.2698  Hct (%)   37.22   3.80   37.26   3.13   33.77   3.34   35.56   3.55   0.0973  Plts (x10^9/L)   202.10   67.72   191.90   66.97   299.40   74.19   307.14   78.80   0.0031  

CBC

202.10 191.90

299.40 307.14

Plts

ROOM TEMP

COOLED

MODIFIED, COOLED

MODIFIED, ROOM TEMP *

    ROOM TEMP   COOLED  MODIFIED, COOLED  

MODIFIED, ROOM TEMP      

    n=11   SD   n=11   SD   n=11   SD   n=7   SD   p-value  INR   1.29   0.20   1.27   0.20   1.27   0.15   1.32   0.14   0.8897  PT   15.77   1.92   15.61   1.94   15.70   1.46   16.09   1.35   0.9498  PTT   53.89   18.45   52.55   16.79   48.84   11.73   56.36   16.71   0.7353  Factor II   69.10   7.75   73.73   7.99   72.73   10.30   70.71   9.07   0.4136  

Factor V   60.00   14.48   56.70   15.34   49.18   19.66   52.14   12.58   0.4510  

Factor VII   77.22   16.82   77.67   14.46   76.56   16.19   72.83   8.61   0.4960  

Factor VIII   59.40   34.51   56.73   27.76   55.91   26.19   59.29   24.86   0.9173  Factor IX   86.18   17.56   89.73   19.07   82.00   17.04   87.86   12.69   0.7644  Factor X   74.00   12.37   74.82   14.38   73.55   13.79   72.14   13.16   0.9812  ATIII   73.73   18.28   77.82   18.98   77.18   19.16   77.29   18.90   0.6770  Protein C   83.90   11.28   85.50   11.71   85.50   12.96   84.71   10.72   0.9869  Fibrinogen   225.82   56.04   227.73   55.60   225.91   43.33   213.43   39.66   0.9392  

Factors

49.90

35.09

42.82

48.91 48.50

34.09

43.18

49.00

56.27

44.64

55.27

61.73

57.57

45.57

54.86

60.29

Alpha(°) a10(mm) a20(mm) MCF(mm)

EXTEM

220.00

622.72

184.00

762.36

200.27 209.27 207.14 203.86

CT(s) CFT(s)

ROOM TEMP

COOLED

MODIFIED, COOLED

MODIFIED, ROOM TEMP

*

Results

¡ Comparison of 1:1:1 to 2:1:1 RWB variants

¡ Comparison of 4 types of whole blood

¡ Comparison of modified WB to 1:1:1

    MODIFIED WB   1:1:1          N=18   SD   N=23   SD   p-value  

WBC (x10^3/µL)  4.09   1.36   0.01   0.03   0.0000  

Hgb (g/dL)   10.60   1.23   9.05   1.07   0.0003  Hct (%)   34.51   3.43   28.93   3.46   0.0000  

Plts (x10^9/L)  302.59   73.75   129.62   22.23   0.0000  

CBC

4.09

0.01

WBC

10.60  

9.05  

Hgb  

Modified  WB  

1:1:1  

34.51  

28.93  

Hct  

302.59  

129.62  

Plts  

*

* * *

1:1:1

    MODIFIED WB   1:1:1          N=18   SD   N=23   SD   p-value  

INR   1.32   0.14   1.31   0.18   0.8232  PTT   56.35   16.71   41.76   4.68   0.0117  PT   16.09   1.35   15.98   1.71   0.7858  

Factor II   71.94   9.61   65.00   19.81   0.0848  

Factor V   50.33   16.89   52.55   22.70   0.7332  

Factor VII   75.07   13.41   72.00   26.74   0.6876  

Factor VIII   57.22   24.99   76.35   32.17   0.0201  

Factor IX   84.28   15.37   86.22   25.39   0.6453  

Factor X   73.00   13.17   66.52   16.12   0.1648  ATIII   77.22   18.49   72.65   41.19   0.0697  

Protein C   85.18   11.73   80.10   22.25   0.2787  Fibrinogen   221.06   41.22   241.65   67.16   0.2345  

Factors 1:1:1

56.78  

45.00  

55.11  

61.17  

56.27  

38.57  

45.83  

50.35  

Alpha(°)   a10(mm)   a20(mm)   MCF(mm)  

*  *  

EXTEM modified vs. 1:1:1 RWB

207.17  

760.57  

CFT(s)  

Modified  WB  

1:1:1  

Conclusions

¡ First characterization study

¡ 1:1:1 > 2:1:1

¡ modified WB > non-modified

¡ modified WB > 1:1:1

1:1:1

2:1:1

Non-modified

Modified

INR

PTT

EXTEM

CT

EXTEM

CFT

EXTEM

ALPH

A

EXTEM

a10

EXTEM

MC

F

So  now  that  we  know  there  is  platelet  dysfunc7on  aGer  trauma    

how  do  we  study  it?  

Puta7ve  Func7on  

•  Platelets  must  circulate  without  adhesion  and  ac7va7on  while  in  constant  contact  with  an  undamaged  endothelium,  ac7vate  and  s7ck  at  the  site  of  injury  form  an  adhesive  plug  un7l  no  longer  needed  and  dissipate  without  further  downstream  or  systemic  damage.  

Steps  to  Platelet  Ac7on  

•  Ini7al  capture,  ac7va7on  and  adhesion  to  damaged  vessel  wall.  

•  Recruitment  of  addi7onal  platelets  to  the  forming  plug  via  genera7on  of  soluble  platelet  agonists  and  αIIbβ3  platelet-­‐platelet  interac7ons  

•  Stabiliza7on  of  platelet  plug  

Steps  to  Platelet  Func7on  

•  Ini7al  capture  •  Binding  of  GPIb–IX–V  complex  on  platelet  surface  to  VWF  

and  binding  of  αIIbβ3  to  VWF.  

•  Ac7va7on  – Collagen  GPVI  promotes  engagement  of  integrin  α2β1  to  form  platelet  monolayer  

– Ac7va7on  also  through  local  release  of  agonists  to  G  protein  coupled  receptors  on  platelet  surface  inclding  TxA2  and  ADP.  

– This  forms  a  local  enviornment  for  thrombin  ac7va7on  and  a  dense  hemosta7c  plug.  

Steps  to  Platelet  Func7on  

•  Ac7va7on  – Collagen  GPVI  promotes  engagement  of  integrin  α2β1  to  form  platelet  monolayer  

– Ac7va7on  also  through  local  release  of  agonists  to  G  protein  coupled  receptors  on  platelet  surface  inclding  TxA2  and  ADP.  

– This  forms  a  local  environment  for  thrombin  ac7va7on  and  a  dense  hemosta7c  plug.  

– αIIbβ3  binds  fibrinogen  and  plasma  protein  – This  is  different  in  different  types  of  injury  and  different  vessels!  

Intracellular  Signaling  

•  PLC  ac7va7on  produces  IP3  to  raise  CA++    leading  to  αIIbβ3    

Poten7al  Mechanisms  

Actual  platelet  dysfunc7on  via  binding,  ac7va7on,  platelet-­‐platelet  interac7ons,  G  protein  receptor  signaling  (including  but  not  limited  to  G-­‐G  interac7ons,  RGS  proteins),  adhesion  ligand  receptors  (numerous),  the  local  environment,  changing  flow  and  shear,  and  the  remainder  of  the  coagulant  milieu.  

How  should  this  be  studied?  

Stalker  Blood  2013  

Take  home  points…  

•  There  is  platelet  dysfunc7on  aGer  trauma.  •  Platelet  transfusions  early  show  a  large  mortality  benefit.  

•  Platelet  transfusions  late  are  associated  with  organ  failure  (ARDS  etc)  

•  There  is  considerable  variability  in  platelet  func7on.  

•  It  is  hard  to  define  func7on  and  even  more  difficult  to  know  which  mechanisms  to  study.  

Acknowledgements

•  Alan Hubbard •  Adam Arkin •  Amor Menzes

•  Ivan Diaz •  Anna Decker

 NIH  GM-­‐085689  DOD  W911NF-­‐10-­‐1-­‐0384   NIH TACTIC NL 13025

¡  The  Cohen  Lab  ¡  Ryan  Vilardi  ¡  David  Dong  ¡  Mary  Nelson  ¡  Brin  Reddick  ¡  Xiaoming  Yao    

 

 ¡  Lucy  Kornblith  ¡  Ben  Howard  ¡  Byron  Miyazawa  

¡  Man  Kutcher  

 

mcohen@sfghsurg.ucsf.edu