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