TRIP: Thrombotic Risk Progression TRIP: Thrombotic Risk Progression Paul A. Gurbel, MD Director,...

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


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


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


Stable Angina

Unstable Angina


p=0.48

Time to Platelet-Fibrin Clot Formation(Indicator of Thrombin Generation Time)


Stable Angina

Unstable Angina


P<0.001

Prothrombotic Markers

vWF

(ug

/mL

)

0

10

20

30

40

50

60

p=0.34

p=0.17p=0.67


Stable Angina

Unstable Angina


Fibrinogen

0

1

2

3

4

5

6

(m

g/m

L)

p=0.22 p=0.26

p=0.15


Stable Angina

Unstable Angina


Inflammation Markers(u

g/m

L)

0

5

10

15

20

25

p=0.006

p=0.2p=0.2

C-Reactive Protein


Stable Angina

Unstable Angina


0

5

10

15

20

25

(pg

/mL

)

p<0.001p=0.11

p=0.13

Interleukin- 8


Stable Angina

Unstable Angina


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)


Stable Angina

Unstable Angina


0

7

14

21

28

35

(pg

/mL

)

p<0.001

p=0.52 p=0.48


Stable Angina

Unstable Angina


Macrophage Inflammatory Protein-1 Alpha

Biomarker ProfileAsymptomatic

-CADStableAngina

UnstableAngina


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

Question Question

&&

AnswerAnswer

TRIP: Thrombotic Risk Progression TRIP: Thrombotic Risk Progression Paul A. Gurbel, MD Director,...

Documents

Transcript of TRIP: Thrombotic Risk Progression TRIP: Thrombotic Risk Progression Paul A. Gurbel, MD Director,...