Dr. Gemma VilahurCardiovascular Research Center

CSIC-ICCCInstituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau)

Barcelona

How big is the problem?

WORLDWHO 201217,5 million people died from CVD (mainly ISCHEMIC HEART DISEASE)LEADING CAUSE OF DEATH GLOBALLY(31% total)

G. Vilahur , L. Badimon et al Eur Heart J 2014

Thrombotic

occlusion

THROMBUS

Atheroscerotic plaque

From the culprit coronary thrombotic occlusion to platelets

Thrombus formation I: Platelet aggregationL. Badimon, R. Storey, G. Vilahur. Thromb Haemost. 2011

Foam Cell

GPIIb/IIIavWF

GPIb

Vitronectin

αvβ3

Collagen

GPVI

GPIa/IIavWF

AATXA2

ADP

P2Y

TF

Fg

PSGL

Platelet

vWF

Thrombi

n

GPIIb/IIIa PAR

Fg

CollagenP-selectin

Collagen

Initial contact

Firm adhesion

Activation

AggregationADP

vWF

Platelet recruitment: G protein–mediated signaling pathways

• ADP• Thrombin• Thromboxane A2

•Common activation of downstream effectors that lead to GPIIb/IIIa activation

L. Badimon & G. Vilahur. J Int Med. 2015

ADP

PAR-1PAR-4

TP

GPIIb/IIIa

PLC

Fibrinogen binding

Platelet aggregation

PLCPLC hydrolyzes PIP2 to

yield IP3 and DAG

G-protein mediatedactivation of PLC

ADP

ADP

P2Y1

P2Y12

GPIIb/IIIaactivation

PIP2PLC

VASP-P

Adenylyl cyclase (AC) inhibition

α AMPc

PKA

AC

VASP

AMPc decrease

Reduction PKA activation

DAG

PKC

PKC activation

GPIIb/IIIaactivation

Ca2+

Ca2+

Ca2+

Ca2+

IP3

GPIIb/IIIaactivation

Calcium cytosolincrease

TXA

2

TXA2

PLA2AA

PGH2

PGG2

PLA2 activation

TXA2 generation

PLATETL

DEGRANULATION

XIa

IX

IXa

XII PC

XI

Intrinsic pathway

Va

PLATELET ACTIVATION

X

XaVII

Extrinsic pathway

FT

VIIa

Prothrombin

TISSUE FACTOR

Fibrinogen FIBRIN

PAR-1/PAR-4

L.Badimon & G.Vilahur. Eur Heart J Vasc Biol 2015

Thrombus formation II: Activation of the coagulation cascade

VIIIa

SECONDARY PREVENTIONPrevent atherothrombosis

PRIMARY PREVENTION

What increases the risk of CVD?

YOU CAN HELP IT!

YOU CANNOT HELP IT!

Unhealthy eating

RISK RISK

Healthy eating

>45 yr >55 yr

Seven countries study

Ancel Benjamin Keys 1958Healthy diet = heatlhy heart

A food pattern shared by countires that rim the Mediterranean Sea

(no official Mediterranean diet)

Spain

Italy

GreeceTurkey

North Africa

Israel

Lebanon

What is the Mediterranean diet ?

Mediterranean

dietCheese and yogurt

Moderate in Dairy products

Beef or pork (small amounts)

Rabbit and chicken (lean meat)

more recommended

Poor in Meat

Nuts (i.e.,almonds and hazelnuts) Olive oil

Rich in ω-3/ω-6 fatty acids (PUFA)

Rich in ω -9 fatty acids (MUFA)

Walnuts

Vegetables

Fruit

Fatty fish

(anchovy, sardine, mackerel,

red tuna, etc)

Legumes

Fermented beverage

(moderate amounts)

Rich in polyphenols

Olive oil

Grains (especially wheat)

Rich in fiber

Diet characteristics of the Mediterranean dietL.Badimon & G.Vilahur. In press 2015

Secondary Prevention(AMI-patients)

Lyon Diet Heart StudyDe Lorgeril et al Lancet 1994

FREE CANOLA OIL BASED MARGARINE: high omega-3 PUFA

Mediterrenan dietFrench diet (control)

Primary Prevention(Asymptomatic high-risk patients)

PREDIMEDRamon Estruch et al New Eng J Med 2013

PR

IMA

RY

EN

D-P

OIN

T:

AM

I, s

tro

ke o

r d

eath

fro

m C

VD

(3

0%

↓)

PRIMARY PREVENTION: Beneficial effects of a healthy food

IMPROVED CARDIOVASCULAR HEALTHREDUCED CVD MORBI/MORTALITY

Improves vascular functionReduced inflammationEnhance NO availabilityImproved reactive hyperemea indices

Reduces blood pressure

Improved vascular functionReduces inflammationEnhanceNO availabilityReduces ROS

Reduces platelet reactivity

Improved platelet functionEnhance NO availability

Improves lipid profiles

Reduces LDL-cholesterolReduces TGReduces total Cholesterol

Reduces oxidative stress

Improves anti-oxidant capacityReduces LDL-oxidation

Reduces weight

Reduces ROSRuduce lipid profilesReduce blood pressureImproves exercise capacity

Adapted from R. Jay Widmer et al American Journal Med 2015

THROMBOTIC RISK

REDUCTION

G.Vilahur & L.Badimon Vasc Pharmacol 2014

Food stuff that known to reduce platelet reactivity

Energy 885 kcal

Carbohydrates 0 g

Protein 0 g

Fat

saturated 14 g

polyunsaturated 11 gMonounsaturated

(oleic acid) 73 g

Heatlhy properties of olive oil: MUFA and PHENOLYIC COMPOUNDS

Control cholesterolImproves blood pressureImproves lipid profileAnti-inflammatory effects

Badimon & Vilahur . Cardiovasc Therapeut 2010

Delgado Lista et al. Curr Pharnaceut Des 2011

Covas MI. Pharmacol Res 2007

Improves endothelial functionReduces LDL oxidationeImproves lipid profile

Lowers platelet reactivity

Polyphenols

A3A1

A3A1 RGD

EPINEPHRIN

vWF

Fibrinogen

PAR-1/PAR-4

P2Y12

GPIIb/IIIa

GPIIb/IIIa

TXA2

PLA2

AA

PGH2

PGG2

GPVI

GPIb-IX-V

GPIIb/IIIa

SEROTONIN

5-HT2

α cAMP

PKAVASP-P

VASP

β

ACCa2+

Ca2+

Ca2+

Ca2+

PLCβ

PIP2

IP3

DAG

PKC

PI3K

A3A1

A3A1 RGD

PLATELET

GRANULES

ADP

vWF

Fibrinogen

ADP

PLATETL

DEGRANULATION

PLCβ

Ca2+

Ca2+

Ca2+

VITRONECTIN

GPIa/IIa

ATP

COLLAGEN

MAPK

MAPK

TP

OLIVE OIL

GPIIb/IIIa

inhibition

activation

vWF

OLIVE OIL?

Rubio-Senent Eur J Nutr 2014

Mezzano et al Pathophysiol Thromb Haemost 2003

Turpeinen et al. Net Met Cardiovasc Dis 1999

COLLAGEN

OLIVE OIL

OLIVE OIL

OLIVE OIL

Differential proteomics: ADP-stimulated platelets

Alperujo extractNon-alperujo

Phenolyc compounds in olive oil protect against platelet activation

Alperujo extract

GPIIb

Rho GDP-dissociationinhibitor

Annexin A5

1. Regulation of platelet structure2. Anticoagulant effects3. GPIIb/IIIa

Roos et al Eur J Nutr 2011

- Hydroxytyrosol (HT)

- Dihydroxyphenylglycol

(DHPG)

Platelet aggregationPlatelet activation (AE: 40mg/mL)

Platelet inhibition

MUFAs reduce the activation of the coagulation cascadeL. Badimon & G.Vilahur. Eur Heart Journal ACC 2012 (minimally modified)

Francisco Perez-Jime nez et al Public Health Nutr 2006 MUFA vs SAFA

In a prospective study of 43,757 male health professionals followed for 6 years, a relatively small

increase in ALA intake (1% of total energy) was associated with a 59% decrease in the risk of acute MI

PUFA: Essential fatty acids

Alpha-linolenic acid (ALA)

Omega-3 Fatty Acids: ALA, EPA, DHA

Eicosapentaenoic acid (EPA)

Docosahexaenoic acid (DHA)

Must be obtained from the diet

Omega-6 Fatty Acids

Linoleic acid (LA)

Arachidonic acid (AA)

Weak anti-inflammatory & anti-thromboticmediators

PG3 LTB5 TXA3

Potent Pro-inflammatorypro-thromboticmediators

PG2 LTB4 TXA2

Elongase

6-desaturase

5-desaturase

COX

Omega-6

Linoleic acid

Gamma linoleic acid

Arachidonic acid

(AA)

Dihomo GLA

Omega-3

Alpha-linoleic acid

Stearic acid

Eicosatetraenoic acid

Eicosapentaenoic acid

(EPA)

Preferred

pathway

As the Omega-6/Omega-3 ratio decreases, so does the platelet aggregationFreese et al Thromb & Haemost 1994

The OMEGA-PCI (OMEGA-3 fatty acids after PCI to modify

responsiveness to dual antiplatelet therapy) study

Addition of omega-3 ethyl esters to aspirin and

clopidogrel therapy modifies antiplatelet

response in patients with stable CAD

undergoing PCI

63 patients- Stable CAD- Elective PCI- Aspirin and clopidogrel on board

Platelet function test

ASA+clopidogrel (600mg) +EPA+DHA (1gr)ASA+clopidogrel (600mg) +placebo

Platelet function test(12h)

ASA+clopidogrel +EPA+DHAASA+clopidogrel +placebo

Platelet function test3-5 days1month

Platelet function test3-5 days1month

G. Gajos, et al JACC 2010

1 month

Lycopene? Adenosine?

Tomatoes : red, green, sauce, boiled…are they all the same?

MaximalAggregationADP Collagen

MaximalAggregation TRAP6

MaximalAggregation AA

MaximalAggregation

E. Fuentes et al 2013

Green vs red tomatoes

Fresh vs pomace (heated)

Do tomatoes exert antiplatelet effects via lycopene-related effects?

Red-colored pulp more than 40 times higher lycopene content than that in the green pulp (lycopene is directly related to color)

MaximalAggregationADP Collagen

MaximalAggregation

1. Lycopene partially contributes to

inhibit platelet activation

2. Heat does not affect antiplatlet

function of tomatoes

GPIIb/IIIa

P2Y12

Platelets

A2A

Platelet inhibition

A1A2aA2bA3

Adenosine receptors

- Vasodilation- Cardioprotection

(post-conditioning)- Modulation of inflammation

ADENOSINE

Potential adenosine-related antiplatelet effects

1 kg of fresh tomato contains about 30-times more adenosine than lycopene.

Garlic and onions

Antiplatelet properties are lost with cooking (aliinase is deactivated by heat)

Bordia A, et al. Effect of garlic (Allium sativum) on blood lipids, blood sugar, fibrinogen and fibrinolytic activity in patients with

coronary artery disease.

Prostaglandins Leukot Essent Fatty Acids. 1998.

>>

Kiesewetter H, et al Effect of garlic on thrombocyte aggregation, microcirculation, and other risk factors.

Int J Clin Pharmacol Ther Toxicol. 1991

Kiesewetter H, et al Effects of garlic coated tablets in peripheral arterial occlusive disease.

Clin Investig. 1993

Steiner M, Lin RS. Changes in platelet function and susceptibility of lipoproteins to oxidation associated with administration of aged

garlic extract.

J Cardiovasc Pharmacol. 1998

Barrie SA, et al. Effects of garlic oil on platelet aggregation, serum lipids and blood pressure in humans.

J Orthomolecular Med. 1987

Two cloves/day

Garlic and onions majorly interfere with AA metabolism

Sulfur compounds from gralic and onions

Reduces platelet

aggregation

induced by ALL

agonists

Decreases

functional

GPIIb/IIIa

receptors

Interferes with

membrane viscosity

(inner layer)

Rendu et al Biochem Pharmacol 1989

FRAMINGHAM

Heart study

Friedman, 1986

Honolulu Heart

Study

Blackwelder,1980

Caerphilly Scotish

Study

Renaud, 1993

The Copenhagen City

Heart Study

Tjionneland,1999

Thun et al N Eng J Med 1997

Rimm et al. Br Med J 1999

Hennekens et al, JAMA 1979

Fuchs et al N Eng J Med 1995

Camargo et al Arch Intern Med 1997

Stampfer et al N Eng J Med 1988

Yano et al N Eng J Med 1977

Cardiovascular effects of moderate intake of fermented beverages

Mortality reduction in around 33%

Coronary and cerebrovascular risk reduction in around 20-40%

U or J - SHAPE

Benefitial effects of wine intake

Moderate alcohol consumption:30g/day men: 2-3 beers, 2-3 glasses of wine20g/day women: 1-2 beers, 1-2 glass of wine

•↓ Coagulation(PAI) •↓ Fibrinogen, VWF•↑ Fibrinolysis (tPA)•↑ HDL•↓ LDL oxidation•↓ ICAM, VCAM, E-selectin•↓ PCR y IL-6•↑ VEGF y adiponectin•↓ Homocysteinemia↑ PKC-ε•↑ Paraoxanase-1•↑ Insulin sensitivity

Platelet

aggregation

NO

PGI2

TxA2

MCP-1 VEGF

TF

TF

MMPs

CRP

LDLox

LDLox

LDLox

HDLHDL

+

Monocyte

adhesion

LDL

Lipoprotein

oxidation

Wine

WineBadia,2004

++ Wine

Leikert,2002

Wine

Kondo,1994

Gaziano,1993

Guivernau,1989

Wine

LDLox

Beneficial effects of moderate intake of red wineModified B.C.Choi, G.Vilahur, JJ. Badimon. Current Molecular Medicine, 2006

Hyperlipemiccontrol

Hyperlipemic + HD wine(40g alcohol/día)

Hyperlipemic + LD wine(20g alcohol/día)

Platelets

Fibrin

Wine reduces thrombus formation and fibrin depositionL.Casani, G. Vilahur, L. Badimon et al Circulation 2004

GDP

GDPInactive Rho A

GEF

GTPActive Rho A

GTP

resveratrol

PLATELET AGGREGATION

eNOS

PLATELET AGGREGATION

[AMPc][GMPc]

PDEAMPGMP

Resveratrol-related antiplatelet mechanisms of actionL.Casani, G. Vilahur, L. Badimon et al Circulation 2004

Dark chocolate: the “sweetest” antiplatelet food

The fruit of the Theobroma cacao tree

Source Flavanol Content(mg/kg or mg/L)

Chocolate 460–610

Green tea 100–800

Red wine 80–300

Black tea 60–500

Corti R et al. Circulation 2009Catehins and Epicatechins

Dark chocolate: Polyphenol content 30% to 80% cocoaMilk chocolate: Polyphenol content 7% to 35% cocoaWhite chocolate: does not contain polyphenols.

Cocoa and chocolate have higher levels of polyphenolsthan in many fruits:

20 times higher than in tomatoes2 times higher than in garlic3 times higher than in grapes

Dark chocolate: is it all about flavanols?Rull et l Vasc Pharmacol 2015

6 weeks intake 50g/day

Improves vasodilation

PDE inhibitor

AMPc

GMPc

PDE

AMPGMP

Platelet inhibition

Kamruzzaman SM et al Phytochem Res 2011

Kamruzzaman SM et al Vasc Phamacol 2013

Mechanisms involved in platelet inhibition

G.Vilahur. & L.Badimon Vasc Pharmacol 2014

The impact of foods and dietary supplements onnormal hemostasis and antithrombotic therapyshould be taken into consideration in patients

prescribed antithrombotic drugs

Many foods may exert beneficial effects in primaryprevention by modulating platelet

function…moreover…foods may offer new leads forthe development of antiplatelet agents..yet…

1.- En el proceso de adhesión y activación plaquetar:

1. La exposición de contenido aterogénico al torrente circulatorio es el único desencadenante de la

adhesión / activación plaquetar

2. El factor de Von Willebrand juega un papel clave en la adhesión plaquetar bajo cualquier condición de

flujo sanguíneo

3. El receptor plaquetar GPIIb/IIIa es el efector final en el que convergen todas las vías de señalización

plaquetar

4. El ADP liberado por las células blancas induce activación plaquetaria

2.- En cuanto a la influencia de la dieta y/o nutrientes sobre la función plaquetar:

1. Todos los alimentos poseen propiedades antiplaquetares

2. Las propiedades antiplaquetares de determinados alimentos (cacao, vino, aceite) viene determinada

exclusivamente por su contenido en antioxidantes

3. Los ácidos grasos monoinsaturados (MUFA) w-6 presentes en el pescado desplazan los ácidos grasos

poliinsaturados (PUFA) w-3 de la membrana de las plaquetas lo que deriva en última instancia en una

menor reactividad plaquetaria.

4. Sólo las formas crudas del ajo y la cebolla ejercen efectos antipalquetares.

Las preguntas……..

3.- En el contexto del vino como producto natural capaz de inhibir la activación plaquetar:

1. El contenido en alcohol modula de manera dosi-dependiente la función plaquetaria

2. El vino interfiere en múltiples vías de activación plaquetar

3. Su contenido en resveratrol (polifenol presente en la pulpa de la uva) es quien determina el grado de

inhibición plaquetar.

4. Sólo el vino blanco ejerce efectos antiplaquetares.

4.- Los siguientes componentes bioactivos ejercen efectos antiplaquetares:

1. Hidroxitirosol presente en el aceite de pescado

2. Los ácido eicosapentanoico y ácido docosahexaenoico presentes en el aceite de pescado

3. Compuestos ricos en sulfuro presentes en las setas

4. El licopeno presente en el chocolate negro

EPINEFRINA

FvW

Fibrinógeno

PAR-1/PAR-4

P2Y1

P2Y12

GPIIb/IIIa

GPIIb/IIIa

TP

ATP

TXA2

PLA2

AA

PGH2

PGG2

GPVI

GPIb-IX-V

SEROTONINA

5-HT2

P2X1

α cAMP

PKAVASP-P

VASP

β

ACCa2+

Ca2+

Ca2+

Ca2+

PLCβPIP2

IP3

DAG

PKC

PI3K

A3A1

A3A1 RGD

Gránulos

plaquetares

ADP

FvW

Fibrinógeno

FvW

ADP

Degranulación

plaquetar

PLCβ

Ca2+

Ca2+

Ca2+

COLLAGEN

VITRONECTIN

GPIa/IIa

ATP

COLLAGEN

MAPK (JNK,

p38MAPK)

MAPK (Erk)TOMATOES

TOMATOES

GPIIb/IIIaTOMATOES

inhibición

activación

MAPK

(Erk)

PLCγ