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TRIP: Thrombotic Risk ProgressionTRIP: Thrombotic Risk Progression
Paul A. Gurbel, MDDirector, Sinai Center for Thrombosis Research
Sinai Hospital of Baltimore Associate Professor of Medicine
Johns Hopkins University School of Medicine Baltimore, MD
Transition to an Unstable Coronary Syndrome is Marked Transition to an Unstable Coronary Syndrome is Marked by Hypercoagulability, Platelet Activation, Heightened by Hypercoagulability, Platelet Activation, Heightened
Platelet Reactivity, and Inflammation: Platelet Reactivity, and Inflammation: Results of the Results of the TThrombotic hrombotic RIRIsk sk PProgression (TRIP) Studyrogression (TRIP) Study
Udaya S. Tantry, Kevin P. Bliden, Rolf P. Kreutz, Joseph DiChiara, Paul A. Gurbel,
Sinai Center for Thrombosis Research,
Sinai Hospital, Baltimore, MD, USA
MI, Stroke, CV Death
Pathobiology of Coronary ThrombosisPathobiology of Coronary Thrombosis
Atherosclerosis
Atherothrombosis
Plaque Rupture
Inflammation (local and systemic)
Platelet ReactivityHypercoagulability
Vulnerable Blood
Vulnerable Patient
• Coronary atherothrombosis is a progressive disease influenced by inflammation,
hypercoagulability and heightened platelet function, ultimately leading to catastrophic
events after plaque rupture.
• Independent studies have linked ischemic events to:
A) Hypercoagulability (increased fibrinogen and vWF; and high platelet-fibrin clot
strength) (1,2).
B) Ex vivo measurements of platelet activation and high platelet reactivity (3).
C) Elevated inflammation markers, especially C-reactive protein (CRP) (4,5).
• To date, no single study prospectively evaluated all of these markers of
pathophysiological processes together in patients with various stages of acute
coronary syndrome.
1. Feinbloom D, Arterioscler Thromb Vasc Biol. 2005;25:2043-2053. 2. Gurbel PA, et al. J Am Coll Cardiol. 2005;46:1820-6.
3. Gurbel PA, et al. Rev Cardiovasc Med. 2006;7 Suppl 4:S20-8. 4. Willerson JT, et al. Circulation 2004;109:II2-II10.
5. Granger DN, et al. Hypertension. 2004;43:924-931.
Background
• To simultaneously study hypercoagulability, platelet
function, and inflammation at various clinical stages of
CAD.• To determine whether changes in these markers indicate a
transition in clinical disease state.
OBJECTIVE
• All patients treated with at least 81mg daily aspirin for 1 wk before enrollment.
*All patients with MI and 30% of patients with UA treated with heparin prior
to blood draw.
Patients(n = 188)
Patients Undergoing Stenting (n=117)
(n = 71) Asymptomatic CAD (AS)
• CAD documented by prior angiography >6 months prior to enrollment.
(n = 84) Stable Angina (SA)
• Undergoing PCI for at least 1 lesion.• All lesions had 75% luminal diameter stenosis.
(n = 26) Unstable Angina (UA)
• Typical symptoms associated with ECG changes, requiring emergent hospitalization and a 75% luminal diameter stenosis.
(n = 7) Acute MI (MI)
• Typical symptoms and elevation of cardiac markers and a 75% luminal diameter stenosis.
Methods- Blood Sampling/Processing
• Symptomatic patients - indwelling femoral vessel sheath in cath. lab.
• Asymptomatic patients- venipuncture.
• Samples transferred to vacutainer tubes containing 40 USP lithium heparin
for thrombelastography (TEG) and 3.2% citrate for flow cytometry and
multianalyte profiling.
• Flow cytometry and TEG - conducted immediately.
• Multianalyte profiling plasma samples stored at -700C until analysis.
Thrombelastography
• 1mL heparinized blood transferred to a vial containing kaolin (hydrated aluminum
silicate), an intrinsic pathway activator.
• 500μL activated blood transferred to a heparinase vial to neutralize heparin.
• 360μL neutralized blood was immediately added to a heparinase-coated cup in the
TEG.
Whole Blood Flow Cytometry
• GPIIb/IIIa receptors determined by multicolor analysis before and after stimulating
with 5M ADP:
- Fluorescein isothiocyanate-conjugated PAC-1 antibody
(recognizes active GPIIb/IIIa)
- R-phycoerythrin conjugated CD41a antibody (recognizes total GPIIb/IIIa)
• Total and activated GPIIb/IIIa receptor levels expressed as mean fluorescence
intensity (MFI).
MultiAnalyte Profiling (MAP)• Fluorokine® MultiAnalyte Profiling (MAP) using a Luminex® 100™ analyzer,
(Rules Based Medicine, Inc., Austin, TX).
• Plasma samples incubated with fluorokine colored microspheres coated with a
specific antibody directed against an analyte then washed and incubated with
biotinylated antibodies specific for the analyte and phycoerythrin.
• Markers: CRP, fibrinogen, IL-1, IL-3,4,5,7,8,10,and 18; MCP-1, MIP-1, MIP-1,
RANTES, TNF- , TNF- , VCAM-1, VEGF, and vWF.
• As microsphere passes through
detection chamber, a red laser
excites internal dyes allowing the
classification of the microsphere
specific for each analyte.
(Identification).• A green laser excites
phycoerythrin fluorescence
associated with binding of the
biotinylated antibody (Quantification).
Patient DemographicsAS (n=71) SA (n=84) UA (n=26) MI (n=7)
Age (yrs) 6610 6711 7111 5912Male (%) 68 67 50 67BMI 30.57 29.45 29.36 29.46Risk Factors/Past Medical Hx (%)Current Smoking 10 17 19 57Former Smoking 31 36 27 29Family History of Coronary Artery Disease 34 55 31 43Hypertension 65 71 77 57Hyperlipidemia 85 76 85 57Diabetes Mellitus 27 34 69 43Prior Myocardial Infarction 21 26 42 43Prior Coronary Artery Bypass Grafting 32 18 31 0Prior Percutaneous Transluminal CoronaryAngioplasty
28 37 38 14
Peripheral Vascular Disease 9 6 12 14Stroke 7 11 4 0Medical Therapy (%)Lipid TreatmentStatinother
84822
69636
81738
867214
ACE-Inhibitors 58 56 27 71Beta Blockers 63 83 85 83Laboratory ValuesWhite Blood Cell Count (*106/mm3) 6.52 7.83 7.32 6.91Platelets (*109/mm3) 23472 23167 25685 24786Creatinine (mg/dL) 1.20.7 1.10.4 1.30.5 1.40.8
0
8
16
24
32
40
48
56
AsymptomaticPatients
Stable Angina Unstable AnginaAc
tiva
ted
GP
IIb/II
Ia-U
nst
imu
late
d (
MF
I)
p=0.051
p<0.0001
Platelet Activation
Platelet Reactivity
0
50
100
150
200
250
300
AD
P-S
tim
ula
ted
GP
IIb/II
Ia (
MF
I)
p=0.002
p=0.14
AsymptomaticPatients
Stable Angina Unstable Angina
Pla
sm
ino
gen
Ac
tiva
tor
Inh
ibit
or
Ty
pe
1
(ng
/mL
)
Fibrinolysis Inhibitor
0
45
90
135
180
p=0.01p=0.28
p=0.02
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
Prothrombotic Markers
60
62
64
66
68
70
72
(mm
) p=0.002
p=0.053
0
2
4
6
8
(min
)
p=0.98 p=0.11
Platelet-Fibrin Clot Strength
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
p=0.48
Time to Platelet-Fibrin Clot Formation(Indicator of Thrombin Generation Time)
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
P<0.001
Prothrombotic Markers
vWF
(ug
/mL
)
0
10
20
30
40
50
60
p=0.34
p=0.17p=0.67
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
Fibrinogen
0
1
2
3
4
5
6
(m
g/m
L)
p=0.22 p=0.26
p=0.15
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
Inflammation Markers(u
g/m
L)
0
5
10
15
20
25
p=0.006
p=0.2p=0.2
C-Reactive Protein
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
0
5
10
15
20
25
(pg
/mL
)
p<0.001p=0.11
p=0.13
Interleukin- 8
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
Inflammation Markers (
ng
/mL
)
0
3
6
9
12
15
p<0.001
p=0.8 p=0.64
Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
0
7
14
21
28
35
(pg
/mL
)
p<0.001
p=0.52 p=0.48
AsymptomaticPatients
Stable Angina
Unstable Angina
MyocardialInfarction
Macrophage Inflammatory Protein-1 Alpha
Biomarker ProfileAsymptomatic
-CADStableAngina
UnstableAngina
MyocardialInfarction
Interleukin-1 (ng/mL) 0.40.01 0.30.01 0.30.01 0.4 0.1
Interleukin-3 (ng/mL) 0.70.1 0.70.1 0.80.1 1.4 0.4
Interleukin-4 (pg/mL) 13 0.9 333.0* 300.01 29 9
Interleukin-5 (pg/mL) 18 1.5 131.3* 13.22.2 13 3.4
Interleukin-7 (pg/mL) 107 7 806** 778 87 14
Interleukin-10 (pg/mL) 9.2 0.6 131** 132 144
Interleukin-18 (pg/mL) 206 9 19810 18216 21341
Monocyte ChemoreactantProtein (pg/mL) 163 6 1689 16412 17640
Macrophage InflammatoryProtein- (pg/mL)
91 5 20237** 13714 12014
Vascular Cell AdhesionMolecule –1 (ng/mL) 446 18 49519 47834 39425
Vascular EpitheliumGrowth Factor (pg/mL) 263 9 24013 30928 23529
* P<0.05, **p<0.01; p values are between A-CAD vs. SA
• Our data suggest that: A) A distinct pathophysiological state of heightened platelet reactivity to ADP, platelet activation, hypercoagulability, and inflammation marks the development of symptomatic cardiovascular disease from chronic stable
disease. B) Ex vivo measurements indicating high tensile platelet-fibrin clot strength highlight the patient vulnerable to thrombosis.
• Our data support the concept that reactive and activated platelets influence
inflammation creating a viscious cycle leading to a prothrombotic state culminating in unstable coronary disease.
• Further studies are required to investigate the primary mechanisms activating the prothrombotic state that destabilizes the disease.
Conclusions
Reactive Platelets
Inflammation
? ?Prothrombotic State(Hypercoagulability)
Unstable Coronary Disease
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