Cardiovascular Pathologies and Cardiovascular Pathologies and Thrombosis in Devices Thrombosis in Devices ––

Combining Clinical and Biomedical Combining Clinical and Biomedical Engineering ApproachesEngineering Approaches

Danny Bluestein, Ph.D.Danny Bluestein, Ph.D.

Abdominal Aortic Aneurysm (AAA)

Abdominal Aortic Aneurysm (AAA)Pathologic remodelingGradual weakening of the abdominal aorta, Intraluminal thrombus depositionMay be fatal if ruptured

Reliable predictor for AAA rupture is not available

Currently AAA size is the main criterion used to justify risky surgical intervention

FSI simulations Patient specific AAA geometries reconstructed from medical imagesIncludes the AAA wall and intraluminal thrombus (ILT). Anisotropic wall properties based on anisotropic material modelADINA 8.4

Patient specific Patient specific AAAAAA

CTCT--images reconstruction images reconstruction with segmentation with segmentation

softwaresoftware

CFD simulationsCFD simulationsBlood flow induced Blood flow induced wall shear stress wall shear stress

distributiondistribution

CFD simulationsCFD simulationsBlood flow patterns Blood flow patterns within the aneurysmwithin the aneurysm

FSI simulationsFSI simulationsVon Mises stresses Von Mises stresses

developing in the AAA developing in the AAA wallwall

Potential Potential rupture rupture

pointpoint

Patient Specific Diagnostics Patient Specific Diagnostics of Abdominal of Abdominal Aortic Aneurysm (AAA)Aortic Aneurysm (AAA)

“The Dancing Aneurysms”

Group portrait of the usual

suspects

AAA Geometries with ILT- fusiform vs. saccular(patients images and reconstructed geometry)

IntralumenalThrombus (ILT)

AAA Lumen

Orthotropic Material Models( ) ( ) ( ){ }( )

( ){ }

21 1 2 1 1 2 1

isotropic part

212

4,62

anisotropic

4 6

3 3 exp 3 1

2exp 12

( ) ( ) , ( ) ( )

ii

ij a i b j ij a j b i

W C I C I D D I

k k Jk

J C n n J C n n

=

= − + − + − −

⎡ ⎤+ −⎣ ⎦

= =

∑

1 4 4 4 4 4 4 4 4 4 2 4 4 4 4 4 4 4 4 43

1 4 4 4 44 2 4 4 4 4 43

G. A. Holzapfel, T. C. Gasser, R. W. Ogden, .A New Constitutive Framework for Arterial Wall Mechanics and a Comparative Study of Material Models., Journal of Elasticity, 61: 1-48, 2000.

J. P. V. Geest, M. S. Sacks, and D. A. Vorp, "The effects of aneurysm on the biaxial mechanical behavior of human abdominal aorta," Journal of Biomechanics, vol. 39, pp. 1324-1334, 2006

Use a nonlinear regression to fit experimental data with orthotropic model

Numerically pull a 2 cm2

specimen replicating experimental results to validate orthotropic model

Strain Energy (Isotropic Vs. Anisotropic)

J. P. V. Geest, M. S. Sacks, and D. A. Vorp, "The effects of aneurysm on the biaxial mechanical behavior of human abdominal aorta," Journal of Biomechanics, vol. 39, pp. 1324-1334, 2006

•The peak and average stresses for anisotropic material are larger than the isotropic model.

• Indicating that the isotropic model underestimates stresses for a patient specific geometry

•Underestimating the risk of rupture.

AAA – OrthotropicFully coupled fluid structure interaction simulation

Isotropic Mooney-Rivlin 2x ExaggerationOrthotropic Material

Isotropic Anisotropic

Stresses on the wall for patient 2

Patient 3

Patient 7 Patient 9 Patient 11

ILT

BloodDomain

Calcification

Cross section in calcification area and zoomed region

calcification

calcification

Comparison of the stresses for the same region with (left) and without (right) calcification

calcification

Cross section in calcification area and zoomed regionCross section in calcification area and zoomed region

calcification

calcification

Comparison of the stresses for the same region with (left) and without (right) calcification

calcification

Libby P. J Intern Med. 2000;247:349-358.

PHASE I: Initiation PHASE II: ProgressionPHASE I: Initiation PHASE II: Progression PHASE III: ComplicationPHASE III: Complication

Atherosclerosis is a Chronic Inflammatory Disease With LDL-C at the CoreVulnerable plaques - inflamed, active, growing lesionsAsymptomaticRupture of vulnerable plaques sudden heart attacks

Vulnerable PlaqueVulnerable Plaque

Libby P. Libby P. J Intern Med.J Intern Med. 2000;247:3492000;247:349--358.358.

Two Paths of the Disease Cascade:

Slow Slow ProgressionProgressionSymptomsSymptoms

Rapid Rapid ProgressionProgressionAsymptomaticAsymptomatic

Glagov S, et al. N Engl J Med. 1987;316:1371-1375. Hackett D. Eur Heart J. 1988;9:1317-1323. Libby P. Lancet. 1996;348:S4-S7.

Unstable angina

Stroke

Peripheral ischemia

Unstable PlaqueRupture Can Lead to Serious Complications Including MI, Unstable Angina, Stroke, and Peripheral IschemiaMay lead to sudden cardiac death

Myocardial infarction

Stress distribution within the fibrous cap is crucial in the mechanisms of plaque ruptureCalcification commonly found in the fibrous caps

Vulnerable PlaqueVulnerable Plaque

ObjectivesAnalysis of the stresses within vulnerable plaquesEffects of fibrous cap, lipid core and calcifications to plaque vulnerability

Numerical simulations:3D time-dependent model of eccentric stenosisPhysiological flow conditionsFully coupled Fluid Structure Interaction (FSI) Arbitrary Lagrangian-Eulerian moving mesh formulation (ADINA R&D, inc.)

Hyperelastic models for the material properties Modified Mooney-Rivlin Model

vessel wall

vulnerable

plaque

vessel

lumenCalcification spot - 200μm

Fibrous cap

lipid

Transient Hemodynamics- No Calcification

Vessel Wall Stress

Pressurewithin Lumen

Blood velocity at stenosis

0

10

20

30

40

50

60

0.0 0.5 1.0 1.5 2.0 2.5 3.0

cm/s

ec

Stress distribution – effect of calcificationHigher stresses: propagate in the fibrous cap around the calcification spot

Model with no calcifications

Model with calcifications

Stress with Calc. vs. without Calc.at Peak Flow

Fibrous

Calcium

Fibro-Fatty

Necrotic Core

Vulnerable Plaque (VP) Reconstruction

MATLAB VP Reconstruction in Gambit

Vulcano IVUS System: Virtual Histology

• Vessel composition: healthy tissue, fibrous cap, lipid core

• Different material properties used for the three components (based on patients specimens) (Chau A et al., Annals of Biomedical Engineering, 2004.)

Material D1[Pa] D2

Fibrous plaque 5105.3 13Arterial Wall 2644.7 8.365

Lipid 50 5

( )2 1 3 11

D IW D e − −= ⋅

Patient Specific Reconstructed Structure

Fluid Domain

Lipid Core

Vessel Tissue

Fibrous Cap

Stress & velocity Stress & velocity –– FSI SimulationsFSI Simulations

Velocity field

Stress field

Imposed Inlet Coronary Flow

0

20

40

60

80

0.0 0.5 1.0 1.5 2.0

Time (s)

Flow

rate

(mL/

min

)

Highest stresses occurin the fibrous cap

Stresses on various locations

1

3

2

13 2

t = 0.35s

Risk of cardioembolic stroke Risk of cardioembolic stroke –– major major impediment to Prosthetic Heart Valves impediment to Prosthetic Heart Valves

(PHV) and implantable blood recirculating (PHV) and implantable blood recirculating devices (VAD, TAH, etc.)devices (VAD, TAH, etc.)

Research Approaches4 In vitro platelet studies

4 Modeling: CFD, FSI, blood damage models, multiscale modeling

4 In vivo studies

All flow phases characterized by elevated stresses (shear, deformation, and turbulent)Platelet activation by flow stresses may occur at any of the phases ⇒Free emboli formed in the wake of the valve during the forward flow phase, enhance the risk of systemic emboli

Aortic valve flow: (A) Forward flow (systole) (B) Rapid closing (C) regurgitant flow (diastole)

Mechanical Heart Valves Mechanical Heart Valves –– Flow Induced Flow Induced Emboli FormationEmboli FormationChronic Platelet activation and the initiation of thrombus formation is the salient aspect of flow induced blood trauma in MHV

Fg, vWF

Platelet Activity State (PAS) AssayPlatelet Activity State (PAS) Assay

Agonist Activation Aggregation

––

Xa Ca2+

Va

Prothrombinase Complex

Thrombin

Fibrinogen Fibrin

–

Prothrombin

–

–

–

––

Xa

Va

Va

VaXa

Xa

AcetylationAcetylated

MHV THROMBOEMBOLISMMHV THROMBOEMBOLISMIn Vivo Studies in the Sheep Model

• Valve implantation Cardiothroracic Surgery

The effect of valve orientation and suturing techniques

Pre/Post op follow upCardiology/Neurology

Transesophageal Echocardiography (TE)

Transcranial Doppler- HITS/MES measurements of free emboli

St. Jude Medical MHV

HITS ( I > 9 dB)

0

10

20

30

40

50

60

1 2 3 4

Num

ber

of H

ITS

I > = 20 dBgaseousemboliI < 20 dbthromboemboli

1 month 2 months 14 months 15 months

Microembolic Signals Detection (HITS)

PAR Comparison between sheep with and without MHV

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

1 2

PAS

(Nor

mal

ized

)

Sheep with MHV Control sheep

Platelet Activity State (PAS) Assay in Sheep• Measurement of activation-dependent

platelet-membrane antigens with monoclonal antibodies, e.g., flow cytometry, is species-specific; antibodies are directed against human platelets

• A major advantage - PAS applicable to platelets of other species

• Confirmed PAS in four mammals: humans, mice, cattle, and sheep

Yin, W., Krukenkamp, I.B., Saltman, A.E., Gaudette, G., Suresh, K., Bernal, O., Jesty, J., Bluestein, D. (2006) The Thrombogenic Performance of a St. Jude Bileaflet MHV in a Sheep Model. ASAIO J. Vol. 52(1), 28-33.

Platelet Activity Measurements in LVADPlatelet Activity Measurements in LVAD

St. Jude tissue valve

Tri-leaflet polymeric valve

St. Jude bileafletMHV

St. Jude tissue valve

Tri-leaflet polymeric valve

St. Jude bileafletMHV

Björk-ShileyMHV

Inflow valve: St. Jude Medical MHV

LVAD – Prof. AffeldHumboldt University, Berlin - implantable part of a pneumatic heart-assist system

Harvard pulsatileblood pump: quasi-physiologic flow waveformsSV = 65 ml; 70 BPM

4 PRP bags: platelets separated from plasma by gel filtration

4 Recirculated in LVAD past MHV (incubator at 37oC)

4 Timed aliquots removed4 Factor Xa, acetylated prothrombin,

Ca++ ⇒ prothrombinase complex4 Samples assayed for thrombin

generation rates (chromogenic thrombin substrate, using a microplate reader)

Platelet ActivationBileaflet. Vs. Monoleaflet in LVAD

Bjork-ShileySt. Jude Medical

Time (min)0 10 20 30

PAS

(Nor

mal

ized

)

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

BileafletMonoleaflet

Slope: monoleaflet = 3.14E-4 bileaflet = 8.11E-4Slopes Difference: p < 0.05

Yin, W., Yared., A., Jesty, J., Affeld, K., Bluestein, D (2004) Flow Induced Platelet Activation in Bileaflet and Monoleaflet Mechanical Heart Valves in a Left Ventricular Assist Device. Annals of Biomedical eng., Vol.32, No. 8, 1058-1066.

St. Jude Medical. Vs. Bjork Shiley (tilted) Deceleration phase - time from peak systole

Yin, W., Yared., A., Jesty, J., Affeld, K., Bluestein, D. (2004) Flow Induced Platelet Activation in Bileaflet and Monoleaflet Mechanical Heart Valves in a Left Ventricular Assist Device. Annals of Biomedical eng., Vol.32, No. 8, 1058-1066.

MHV Simulations

Solve URANS (Unsteady, Turbulent Navier-Stokes) 3D non-Newtonian flow field with intermittent turbulence (Wilcox k-ω turbulence model)Two-phase calculation with particle-fluid interaction Compute platelet stress accumulation along pertinent platelet pathsFLUENT (Fluent Inc., Lebanon, NH) solver, GAMBIT & TGrid mesh generators

Valve superstructure including valve sewing ring and leafletsValve tilted at 15° – representing misalignment that frequently happens during implantationPhysiologic inlet (left ventricle) and outlet (aortic root)

Risk of cardioembolic stroke Risk of cardioembolic stroke –– major major impediment to Cardiovascular Devicesimpediment to Cardiovascular Devices

Non-physiologic flow patterns - one of the major culprits in enhancing the hemostatic response by chronically activating plateletsManufacturers likely to follow designs that are proven to perform better in long term clinical trials and animal experimentsPost the R&D design optimization stage

Can we optimize the device thrombogenicperformance during the R&D stage?

Predictive technology to facilitate a reduction of device flow-induced thrombogenicity – Device Thrombogenicity Emulator (DTE)

Optimizing the thrombogenic performance of devices by testing virtual device design modifications before prototypes are built and tested in costly preclinical trials

Comprehensive numerical methodology for modeling flow induced thrombogenicity in prosthetic devices‘Hot spot’ regions during distinct flow phases identifiedFlow trajectories and load histories within these regions computedHemodynamic Shearing Device (HSD) programmed with the stress loading waveformsBlood recirculated in the HSD and platelets activity measured

The Concept of Device The Concept of Device ThrombogenicityThrombogenicity Emulator (DTE)Emulator (DTE)

Interfacing numerical modeling with Interfacing numerical modeling with in vitro in vitro measurementsmeasurements

Input of dynamic shear stress waveforms - τ(t)from CFD

Design modifications aimed at reducing thrombogenicity computed in the virtual domainResultant load waveforms programmed and tested in the HSDProcedure iterated to achieve optimization

0

5

10

15

20

25

0 5 10 15 20 25 30

distance along platelet path [cm]

stre

ss a

ccum

ulat

ion

[dyn

e-s/

cm2 ]

Damage Model MethodologyStochastic particle tracking model for particle trajectoriesActivation potential of a platelet -cumulative effect of total stresses (shear, turbulent, deformation) and exposure time max

0

tba

ii t

( )tτ=

× Δ∑

j ' 'iij i j

j i

12

scalar ii jj ii jj ij ij1

6

uu u ux x

( ) ( ) ( )

τ μ ρ

τ τ τ τ τ τ τ

⎛ ⎞∂∂= + −⎜ ⎟⎜ ⎟∂ ∂⎝ ⎠

⎡ ⎤= − − +∑ ∑⎣ ⎦

Computation of damage accumulation along a turbulent platelet trajectory in MHV

(*) Bludszuweit C. Three-dimensional numerical prediction of stress loading of blood particles in a centrifugal pump. Artif Organs. Jul 1995;19(7):590-596.

Heart pulse

-0.10

0.10.20.30.4

0.50.60.70.80.9

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Time (sec)

Velo

city

(m/s

ec)

(*)

0

0.25

0.5

0.75

1

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6time [sec]

Dam

age

Single passage:Incorporates effect of damage history (D0 - initial damage)

Damage accumulation behavior (experimental):Initial lag phaseConstant accumulation phaseRefractory effect approaching full activation

Platelet Activation Platelet Activation –– Cumulative Damage ModelCumulative Damage Model• Cumulative platelet damage during repeated passages• Initial damage (D0) to account for platelet senesencePhenomenological Damage Index (Yeleswarapu et al., 1995):

senescence SS SSG (loading rate)

D = Do + f(τ, D) + g(τ)

[ ]∫ −⎟⎟⎠

⎞⎜⎜⎝

⎛+=

t

tk

r

tDdtttDtD

0)(1

)()()(0

0 σσ

[ ]max,,2,1,

)(1)(

)()(10

1 Κ=−

Δ⎟⎟⎠

⎞⎜⎜⎝

⎛+=

−− i

tDtt

tDtD ki

r

iii σ

σ

[ ]

r

k0

1( t )1 D( t )

D σσ

⎛ ⎞= ⎜ ⎟

−⎝ ⎠&

Yared, A. and Bluestein, D. (2007) Flow induced platelet activation and damage accumulation in a mechanical heart valve – numerical studies. Artificial Organs, 31(9):677–688.

Repeated passages - randomized trajectoriesRepeated passages – comparing two repeated trajectories (assumes ‘perfect memory’)

Comparison of ATS Open Pivot Valve and St Jude Regent Valve using a CFD-FSI model

Dumont, K., Vierendeels, J., van Nooten, G., Verdonck, P., Bluestein, D. (2007) Comparison of ATS Open Pivot Valve and St Jude Regent Valve using a CFD model based on fluid-structure interaction. J. Biomech. Eng., 129; 558-565.

Shear stress histories calculated for 15,000 plateletsReleased during

Forward flow phaseRegurgitation flow phase

Trajectories computed using Lagrangian approach of particulate two-phase flow Stress accumulation was higher than 35 dyne·s/cm2 (Hellumscriterion) platelets considered activated

ATS SJM

Stress Acumulation during Forward Flow

0

10

20

30

40

50

60

70

80

90

100

0 - 5 5 -15 15 - 25 25 - 35

accumulation intervals in dynexs/cm2

perc

enta

ge o

f par

ticle

s

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

perc

enta

ge o

f par

ticle

s

ATS

SJM

ATS

SJM

Stress Accumulation during Regurgitation Flow

0

10

20

30

40

50

60

70

80

90

100

0 - 5 5 - 15 15 - 25 25 - 35 35 - 45 45 - 55 55 - 65

accumulation intervals in dynexs/cm2

perc

enta

ge o

f par

ticle

s

0

0.5

1

1.5

2

2.5

3

perc

enta

ge o

f par

ticle

s

ATS

SJM

ATS

SJM

Rgurgitation through the closed valves (diastole)

Different scales in percentageaxes

Highly resolved mesh for closed and open valves DNS

–Laminar, Newtonian, unstructured grid - 6,250,950 finite volumes

–Clustering in areas of gradients and vortical activity: smallest grid size is seven times smaller than the smallest Kolmogorov scale (20-70 μm)

Hinges

Valve housing

Leaflets

Platelet trajectories and stress history in ‘hot spots’

Stress load waveforms (dynes/cm2)

A

CD B

A

C D

B

platelet trajectories experiencinghigh stress accumulation

Stress levels even during forward flow reaching160 dynes/cm2 (laminar viscous shear) in platelet trajectories in the hinges regions

Vorticity

τ(t)

Computer controlled

~R e

• CPV can be used to study both turbulent and laminar flows in the same device by controlling modified = (r2ωα2)/(12ν) *

• Re < 0.5, primary or laminar flow• 0.5 < Re < 4, transition or mixed flow• Re > 4, turbulent flow (core region characterized by homogeneous turbulence)• Annular Couette region: dynamic instabilities (characterized by Taylor Number) -

optimization is achieved by radius and aspect ratio

• Fluid inertial effects may be minimized by reducing the cone angle in the CPV, and by optimizing the radius ratio of the Couette region

• Both primary and turbulent flow for the same time-averaged shear stress can therefore be investigated *

* Sdougos et al., 1984; Einav et al., 1994

Cone & Plate + CouetteViscometry

Uniform shear stress in cone-plate:

Shear stress in Couette part:αωμτ =

⎟⎠⎞

⎜⎝⎛

−= 222

22 12rRiRo

RiRoωμτ

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛−≡

2

121

RoRiα

Fluid dynamic considerations for HSD

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60 70Time (min)

PAS

(Pla

tele

t Act

ivat

ion

Stat

e)

T2 = 10 s

T2 = 30 s

T2 = 60 s

1. 5

20

Time (s)

1s T2

She

ar s

tress

(dyn

e/cm

2 )

Time (s)

0.00 0.05 0.10 0.15 0.20

Stre

ss (d

yne/

cm2 )

0

20

40

60

80

100

120

( ) ( ) ( ) φφ⎥⎦

⎤⎢⎣

⎡χ

+ξξχα= αβ

−ααφαβ

∫ ∫ dτtPASdτPAS /t

t

/

t

/1

0

1

0

0

Damage based on model proposed by Grigioni et al.

Model parameters identified with Genetic Algorithms (GAs)Nobili, M., Sheriff, J.F., Morbiducci, U., Redaelli, A., Bluestein, D. (2008) Platelet Activation Due to Hemodynamic Shear Stresses: Damage Accumulation Model and Comparison to in vitro Measurements. ASAIO J., 54(1), pp. 64-72

Platelet Damage Accumulation Under Dynamic Shear Stress Waveforms in HSD

Combines advanced numerical modeling with in vitro testingCompact programmable HSD interfaced with numerical simulations – recreates the stress loading environment of blood flowing within a device A test-bed for optimizing design modifications aimed at reducing the device thrombogenicity to a level that will minimize the need for anticoagulants

SummarySummary

• Various design parameters (PHV, VAD) optimized numerically• Loading waveforms of optimized designs programmed into the

HSD for testing the resulting thrombogenicity• Optimized designs fabricated and thrombogenicity measured to

establish whether a significant reduction was achieved• Ideally- reduced to a level that does not require anticoagulation (as

measured and established in reference bioprothetic valves)This study is supported by the National Institutes of Health (1R01 EB008004-01), and an

Established Investigator Award from the American Heart Association (DB)

Device Device ThrombogenicityThrombogenicity Emulator (DTE)Emulator (DTE)A methodology for reducing the A methodology for reducing the thrombogenicthrombogenic potential of CVS devices:potential of CVS devices:

MHV design parameters (Shu et al., 2003)

Optimization conducted in the virtual domain Optimization conducted in the virtual domain ––tested in an iterative process:tested in an iterative process:

AcknowledgementsStony Brook University

Jolyon Jesty, Ph.D.- HematologyShmuel Einav, Ph.D. – Biomedical Engineering

Gaurav Girdhar, Ph.D. – Biomedical EngineeringMichalis Xenos, Ph.D. – Biomedical Engineering

Wei Yin, Ph.D. – Biomedical EngineeringSulan Xu, M.Sc. – Biomedical Engineering

Yared Alemu, M.Sc. – Biomedical EngineeringJawaad, Sheriff, B.E. – Biomedical Engineering

John Ricotta, MD – Chair, SurgeryLuis Gruberg, MD – CardiologyMarlene Zawin, MD - Radiology

CalTechMory Gharib, Ph.D.Idit Avrahami, Ph.D.City College of New York (CUNY)Shelly Weinbaum, Ph.D.Yuliya VenGrenyuk, Ph.D.Florida International universityRichard Schoephoerster, Ph.D.Siobhan Gallocher, M.Sc.Tel Aviv University, IsraelShmuel Einav, Ph.D.Sagi Raz, M.Sc.

Humboldt University, Berlin Klaus Affeld, Ph.D.Ghent University - BelgiumKris Dumont, Ph.D., Jan Vierendeels, M.Sc., Patrick Segers, Ph.D., Guido van Nooten, MD, Pascal Verdonck, Ph.D.Politecnico di Milano, ItalyAlberto Redaelli, Ph.D., Matteo Nobili, M.Sc., Umberto Morbiducci, M.Sc.

THANK YOU

Time (minutes)

0 5 10 15 20 25 30 35

PA

S (n

orm

aliz

ed %

)

-10

0

10

20

30

40

50

60

70

Unlit Sidestream Loops"Strong" Sidestream Loops"Mild" Sidestream Loops

Sidestreamsmoke (SHS)

Cigarette smoking is a leading cause of heart diseaseSecond hand smoke (SHS) increases heart diseaseSHS has nonlinear potency as a risk factor

Time (minutes)

0 5 10 15 20 25 30 35

PA

S (n

orm

aliz

ed %

)

-10

0

10

20

30

40

50

60

70

Unlit Mainstream Loops"Strong" Mainstream Loops"Mild" Mainstream Loops

Mainstream smoke

EFFECTS OF SMOKING ON CARDIOVASCULAR RISKEFFECTS OF SMOKING ON CARDIOVASCULAR RISK

The "light" designation appears to refer only to the mainstream smoke

High-tar: Marlboro 100's, 16mg tar, Philip Morris, Richmond, VALow-tar: Kent III ultralight 100's, 5mg tar, Lorillard, Greensboro, NC

Nicotine reduction in cigarettes – cardiovascular risk in smokers, and in non-smokers exposed to second hand smoke (SHS)

(PI and FAMRI grantee: Dr. Danny Bluestein)Biomedical Engineering

State University of New York, Stony Brook

Background

• Platelet activation was found to be significantly elevated in smokers relative to non-smokers¶

• Cigarette smoke, irrespective of nicotine content, has been identified as primary risk factor for cardiovascular disease (CVD)†

• Potentially reduced-exposure products (PREPs) that would be less harmful and/or less addictive when compared to conventional tobacco products (including nicotine reduction), have been recently suggested‡

• Nicotine, in-vitro, has been shown to significantly decrease platelet activation in the presence of multiple agonists (e.g., shear and either mainstream smoke or SHS)*

†Glantz and Parmley, Circ., 1991

* Rubenstein et al., Circ., 2004¶ Hawkins, Nature, 1972

‡ NIH PA-04-103, May 2004

In-vitro Evidence: Protective Effects of Nicotine on Platelets

Conditions: Gel-purified platelets exposed to intermittent shear stress and smoke in a circulation flow loop

50 nM nicotine decreasedplatelet activation by ~50%

Time, minutes

0 5 10 15 20 25 30

PA

S, n

orm

aliz

ed

0.0

0.1

0.2

0.3

0.4

0.5

050 nM5 μM

( )

( )

[nicotine] B: FLOWEXPT

Ramachandran et al., Nico. Tob. Res., 2004

Quest Medium Nicotine (QUEST-1): 0.6 mg nicotine. 10 mg tar (per cig)Quest Zero Nicotine (QUEST-3): Nicotine free. 10 mg tar (per cig)

KEY Objective:

Smoker Study: 32 healthy chronic smokers (at least one pack a day)To demonstrate what happens on acute reduction in nicotine

SHS Study: 32 healthy non-smokers (no history of smoking)To demonstrate effects of nicotine in SHS

To test the “inhibition of platelet activation” by nicotine in cigarettes, inboth cigarette smoking and non-smoking human subjects

Studies

Cigarettes

(For comparison: Marlboro Red has 1.9 mg nicotine per cig.)

The SHS study32 Non-smoker Adult Human Volunteers

BLEED

161.5 HR; ZER0-NICOTINE SMOKE

BLEED

161.5 HR; MEDIUM-NICOTINE SMOKE

BLEED

AFTER 4 HRBLEED

AFTER 4 HRBLEED

MEASURE PLATELET ACTIVATION STATE (PAS)

Note: Procedure repeats exactly over 2 consecutive days for the same subjects

Quantifying Platelet Activation: Flow Cytometry

Vacutainer pre-loaded with citrate and 1% PFA(instantaneous fixation)*

PRP (Platelet-rich-plasma)

Centrifugation

P-Selectin-PE antibody

* Hagberg and Lyberg, Platelets, 2000

Determination of PAS (Platelet Activity State)

M2 = PAS (% platelets to the right of arbitrary baseline)

M1

M2

M1

M2Baseline

PAS

TRAP treated

Un-activated

Time (h)0 2 4 6 8 22 24 26 28 30

Δ P

AS

(per

cent

act

ivat

ed)

0.0

0.2

0.4

0.6

0.8p<0.05

p<0.05

Results: SHS Study

Zero Nicotine

Medium Nicotine

No difference in ∆ PASbetween the two (p > 0.5)

Smoke exposure period

32 Smoker Adult Human Volunteers

The Smoker Study

1 HR MEDIUM-NICOTINE CIG. SMOKING(ESTABLISH COMMON BASELINE)

BLEED

151.5 HR SMOKING

ZER0-NICOTINE CIG.BLEED

161.5 HR SMOKING

MEDIUM-NICOTINE CIG.BLEED

MEASURE PLATELET ACTIVATION STATE (PAS)

1

3

5

7

0

2

4

6

8- nicotine

+ nicotine

1 2.5 1 2.5time (hours) Mid-Nicotine Zero-Nicotine

0 1

Δ P

AS

0.0

0.5

1.0

1.5

2.0

2.5Zero NicotineMean+/- SE(n=15)

Mid NicotineMean+/-SE(n=16)

p < 0.02

Smoking zero-nicotine cigarettes activates platelets MORE than medium-nicotine cigarettes

Results: Smoker Study

PAS (% activated)

Nicotine content in Plasma of SmokersCorrelation with Platelet Activation

Nicotine reduction INCREASES platelet activation

+nicotine

- nicotine

1

3

5

7

0

2

4

6

8- nicotine

+ nicotine

1 2.5 1 2.5time (hr)

PAS (% activated)

0

50

100

150

200

250

300

1 12.5 2.5time (hr)

Plasma nicotine (nM)

+ nicotine

- nicotine

Conclusions

SMOKER STUDY

• Nicotine reduction in cigarettes SIGNIFICANTLY enhances platelet activation in smokers

• PREPs should be scientifically scrutinized for safety before use

SHS STUDY

• Smoke exposure causes increasein platelet activation (P-selectinexpression)

• Any effect of nicotine in SHS isnot detectable in this study

OVERALL

• Cigarette smoke increases platelet activation

• Removal of nicotine from cigarettes is dangerous for smokers and may increase risk of CVD