PIASTRINE nel SANGUE

Piastrine
 Cellule discoidi anucleate
» Diametro 2-3 m
» Vita media 9-12 gg
 Livello normale: 150 000 – 400 000 /nl
 Attivatori:
» ADP, epinefrina, collageno, trombina, PAF (platelet activating factor), complessi Ab-
Ag, shear stress
 Quando attivate:
» Adesione al collageno tramite fattore di von Willebrand (vWF, glicoproteina
plasmatica)
» Cambiamento conformazione discoide  sferica
» Polimerizzazione
» Aggregazione
» Rilascio di fattori (ADP, trombossano, fattori della coagulazione, serotonina
(vasocostrittore), fattori di crescita (riparazione dei vasi)
Diagrammatic Representation of the Platelet
Peripheral Blood Film
Magnification x100
adhesion, shape change, spreading and secretion
ORIGINE delle PIASTRINE
FILAMINA UNISCE (CROSS-LINK) FIBRE DI ACTINA A
FORMARE GEL TRIDIMENSIONALI
STRUTTURA CHE SOTTENDE LA MEMBRANA PLASMATICA IN DIVERSI TIPI
CELLULARI
CITOSCHELETRO PIASTRINE

OCS: Open Canalicular System

DTS: Dense Tubular System
SMF: Filaments of Membrane Skeleton
MT: Microtubules
DB: Dense Bodies
Gly: Glycogen Granules
G: α granules
GZ: Golgi Zones
M: Mitochondria
ATTIVAZIONE DELLE PIASTRINE
CITOSCHELETRO PIASTRINE

OCS: Open Canalicular System

DTS: Dense Tubular System
SMF: Filaments of Membrane Skeleton
MT: Microtubules

DB: Dense Bodies

G: α granules
Gly: Glycogen Granules
GZ: Golgi Zones
M: Mitochondria
DEPOLIMERIZZAZIONE E RI-POLIMERIZZAZIONE DI ACTINA
NEL MOVIMENTO DELLE CELLULE (LAMELLIPODIO)
INTERAZIONI FRA PIASTRINE ED ENDOTELIO
INTERAZIONI FRA PIASTRINE ED ENDOTELIO
INTERAZIONI FRA PIASTRINE ED ENDOTELIO
PIASTRINE E COAGULAZIONE
PIASTRINE E LEUCOCITI
TRASDUZIONE DEI SEGNALI DI ATTIVAZIONE NELLE PIASTRINE
TRASDUZIONE DEI SEGNALI DI ATTIVAZIONE NELLE PIASTRINE
ATTIVAZIONE DELLE PIASTRINE (SCHEMA ATTIVAZIONE DA PARTE DI ADP
GENERICO).
PAF
RECETTORE DELLA TROMBINA
SINTESI PGI2 e TXA2
RECETTORI DEL TROMBOSSANO
VASOPRESSINA V1 RECETTORE
RECETTORI DEL
COLLAGENO

GPIa/IIa
GPIb/IX/V Leucine Rich

N-Terminale

Zona Anionica

Oligosaccaridi
VON WILLEBRAND FACTOR
GPIIb/IIIa
 Platelet Aggregation
Fibrinogen binding to Glycoprotein IIb-IIIa on
activated platelets
 Normal Function of Platelets

Haemostasis
• Preventing bleeding from wounds

• Integrity and repair of the vessel wall

 Haemostatic role of platelets in health:
how do they work?

 Platelets circulate in a resting, inactive state

 Must become activated

 Must stick together = Aggregation

Factors that activate platelets

ADP
Thrombin
Collagen
5-hydroxytryptamine (serotonin)
Thromboxane A2
Mechanical stimuli

Many stimuli
Several different receptors
Multiple signalling pathways
 Platelet Function
•Effective primary hemostasis requires three critical
events--platelet adhesion, granule release, and platelet
aggregation.

•Within a few seconds of injury, platelets adhere to collagen

fibrils in vascular subendothelium via a specific platelet
collagen receptor.
Activation/secretion

Adhesion Aggregation
Platelet Aggregation/adhesion
 The Hemostatic System
Accidental injury vs. pathological injury
hypercholesterolemia, diabets,
hypertension
Coagulation cascade – platelet activation and coagulation

vasospasm platelets (5HT, TXA2)

platelet plug adhesion, activation, aggregation

fibrin plug extrinsic, intrinsic (humoral)

Recanalization fibrinolysis
Hemostasis reactions
1. Initiation phase

Injury of vessels wall

leads to contact
between blood and
subendothelial cells

Tissue factor (TF) is

exposed and binds to
FVIIa or FVII which
is subsequently
converted to FVIIa

The complex between

TF and FVIIa activates
FIX and FX

FXa binds to FVa on the

cell surface
2. Amplification 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.
FXIa converts FIX
to FIXa

Activated platelets
bind FVa, FVIIIa
and FIXa
3. Propagation phase

The FVIIIa/FIXa complex

activates FX on the
surfaces of activated
platelets

FXa in association with

FVa converts large
amounts of prothrombin
into thrombin creating a
“thrombin burst”.

The “thrombin burst”

leads to the formation
of a stable fibrin clot.
a

• Haemostasis starts with the interaction between TF and

FVIIa on the surface of subendothelial cells .

• The small amount of thrombin generated during the

amplification phase activates platelets locally on whose
surface the subsequent reactions take place.

• The resulting thrombin burst results in the formation of

a stable clot.
b
Stimulus e.g. damaged vessel wall

• Adhesion to exposed vWF via glycoprotein (GP Ib) receptor,

or collagen (GPIa, GPVI receptors)
• Signalling events, Shape Change
• Exposure of Fibrinogen binding site on GPIIb/IIIa
• Secretion from granules
• Exposure of anionic phospholipids

Platelet Aggregation

• Promotion of clotting on activated platelet surface

 NORMAL HAEMOSTASIS

 formation of the platelet plug

 coagulation = fibrin formation
 clot retraction
 fibrinolysis
 RESOLUTION
 3 Major systems involved

 Vessel wall
» Endothelium
» ECM = BM (type 4 collagen + other proteins)
 Platelets
 Coagulation cascade
» Coagulation factors (proteins)
– Names and numbers
– Active and inactive forms (zymogens)
 Vessel injury

Antithrombogenic Thrombogenic
(Favors fluid blood) (Favors clotting)
 Phases of Hemostasis
Primary Secondary
 
Platelet-mediated Fibrin-formation
Endothelial cell-mediated mediated

•Results in clinicians obtaining a clinical history using a two-

compartment classification system:

--primary defects result in immediate bleeding;

--secondary defects cause delayed bleeding.

 Overview of Hemostasis:
Clot Formation & Vessel Repair

Figure 16-11: Overview of hemostasis and tissue repair

 Anticoagulant Properties of the
Endothelium
• Anti-platelet properties
– Covers highly thrombogenic basement membrane
– Uninjured endothelium does not bind platelets
– PGI2 (prostacyclin) and NO from uninjured
endothelium inhibit platelet binding
– ADPase counters the platelet aggregating effects of
ADP
 Vascular Endothelium Function

Prostacyclin Vasodilation, inhibition of platelet

aggregation

Thromboxane A2 From platelets, muscular arteries

constrict

ELAMs, ICAMs Cytokines induce synthesis to promote

leukocyte adhesion

von Willebrand factor Promote platelet-collagen adhesion to

exposed sub-endothelium
 Vascular Endothelium Function

Tissue factor pathway Anticoagulant- Inhibits coagulation

inhibitor extrinsic pathway

Thrombomodulin Anticoagulant- Inhibits coagulation by

activating protein C system

Tissue plasminogen activator Anticoagulant- Inhibits coagulation by

activating fibrinolysis

Heparan sulfate proteoglycansAnticoagulant- Inhibits coagulation by

activating antithrombin
Tissue factor Procoagulant- Inflammatory cytokines
(IL-1, TNF) induce expression
 Anticoagulant properties of the endothelium

*HEPARIN-LIKE MOLECULES: activate anti-

thrombin III (inactivates active proteases)

*THROMBOMODULIN: changes specificity of

thrombin (activates protein C , which inactivates
factors Va and VIIIa

*Endothelial cells produce t- PA which activates

fibrinolysis via plasminogen to plasmin
Prothrombotic Properties of the
Endothelium
•Synthesis of von Willebrand factor
•Release of tissue factor
•Production of plasminogen activator inhibitors (PAI)
•Membrane phospholipids bind and facilitate activation of
clotting factors via Ca bridges
 Coagulation Cascade

 Enzymatic cascade (amplification)

 Several serine proteases
» Produced by liver (most)
» Require Vit K (several)
 3 protein cofactors (not enzymes)
 Requires Ca 2+
 Localized to site of injury
 Reversible (via production of plasmin)
Formazione del reticolo di fibrina
Sistema intrinseco Sistema estrinseco
Contatto con superfici con cariche negative lesione tissutale
In assenza di fattori intracellulari Fattori intracellulari

Fattore Xa (Stuart)

Coagulo di fibrina

 Distinzione solo storica

 Tutti i fattori presenti nel plasma in forma
inattiva (proenzimi o zimogeni), attivati a
cascata
» Indicati con numeri romani in ordine di scoperta
Fattori della coagulazione
Le vie della coagulazione del
sangue
 La via estrinseca
 III (TF, proteina transmembrana) + Ca++
 VIIa
 X  Xa
 Test clinico: tempo di protrombina
» Tempo necessario per la
coagulazione dopo esposizione a un
fattore tissutale standard: cefalina
(fosfolipide)
» Normale: 10-15 s
 Tissue Factor Pathway
NB: activation of IX by VIIa
IX

TF
Prothrombin
IXa VIIa
VIII VIIIa Xa
Va V

Soft clot
Fibrinogen Thrombin Fibrin
XIIIa Hard clot
Fibrin
 Tissue Factor Pathway
 VIIa forms as usual via binding of VII to TF
 VIIa activates some XXa
 Xa converts a small amt of prothrombin to thrombin; this
thrombin is used to produce small amts of VIIIa and Va
 As the conc of TF-VIIa-Xa-thrombin increases, Tissue Factor
Pathway Inhibitor inactivates this complex stopping further
production of thrombin.
 VIIa also activates IXIXa
 IXa, with VIIIa (produced as above), produces Xa; this Xa with Va
(produced as above) produces new thrombin; this thrombin
produces more VIIIa and Va and now we get lots of thrombin and
fibrin!
 La via intrinseca
 Lesione cellulare con esposizione di una
superficie con carica negativa (anche vetro)
 XII + superficie  XIIa
 Prekallikreina  kallicreina
 XI  XIa
 IX  IXa
 X  Xa
 Test clinico: Tempo di tromboplastina
» Tempo necessario per la coagulazione dopo
contatto con una superficie standard
(caolino)
» Normale: 30-50 s
Interazione con cariche negative
 Via comune

 Fattore X converte protrombina

in trombina
 Trombina converte fibrinogeno
(solubile) in fibrina (insolubile)
 Test clinico: tempo di trombina
» Tempo necessario per la
coagulazione dopo contatto
con trombina
» Normale: 10-15 s
 VIII V

Thrombin (IIa)

VIIIa Va
 Protrombina  Trombina
 150 mg/l in plasma, sintetizzata in fegato
 T1/2=24 h
 Fattore Xa converte trombina
» Fattore limitante del processo di coagulazione
» Tempo di protrombina (11-15 s): test di funzionalità epatica
 Dipende da Vitamina K
trombin
 Fibrinogeno  fibrina
 3 g/l in plasma, sintetizzato in fegato, ()2
 T1/2=4 g
 Trombina converte fibrinogeno solubile in fibrina insolubile
 Stabilizzazione con XIII
Stabilizzazione del coagulo di fibrina con Fattore
XIII (transglutamidasi), attivato da trombina
 Hemostasis: Coagulation & Clot Stabilization

 Prothrombin
 Ca++
 Fibrinogen
 Fibrin
 Polymerization

Figure 16-13: The coagulation cascade

 Siti di controllo della
coagulazione
 Trombina (protrombina >> trombina)
» Antitrombina III (inibisce anche IXa, Xa, XIa e XIIa)
» 2-macroglobulina
» 1-antitripsina
 Prostaciclina (endotelio)
» Inibisce aggregazione piastrinica
 Proteine C e S (Disattivano V e VIII)
 TFPI (INIBISCE VIA ESTRINSECA)
 PROTEIN C, S, TROMBOMODULIN

 Eparina
» Potenzia azione di antitrombina III
 Aspirina:
» Potenzia azione di antitrombina III
» Inibisce cicloossigenasi e formazione di trombossano
» Inibisce prostaciclina
ANTITROMBIN
PROTEIN C,S,TROMBOMODULIN
 Physiologic Inhibitors of
coagulation

 Antithrombin III (serpin)

 Activated Protein C + protein S
» Inactivates Va and VIIIa (via proteolysis)
» NB: Factor V Leiden
 Thrombomodulin (EC glycoprotein)
» Binds to thrombin
» Decreases ability to produce fibrin
» Increases ability to activate Protein C
 Dissoluzione del coagulo
(fibrinolisi)

 Nel coagulo di fibrina è presente plasminogeno

 Plasminogeno  plasmina
» Catalizzatori: Tissue Plasminogen Activator (tPA) e
urokinasi
» Inibito da 2-antiplasmina
 Plasmina  fibrina (proteolisi)
 Farmaci fibrinolitici (infarto del miocardio)
» tPA ricombinante
» Streptokinasi
» Urokinasi
Dissolving the Clot and
Anticoagulants

Figure 16-14: Coagulation and fibrinolysis

 The Plasminogen Network

Plasminogen Activators (t-PA, u-PA)

Plasminogen Activator Inhibitors (PAI-1)

Plasminogen Plasmin

 2-Antiplasmin
TAFI, pCPB

Non-fibrinolytic Functions Fibrinolytic Function

♦ extracellular matrix degradation ♦ blood clot dissolution
♦ matrix metalloproteinases activation
♦ growth factor activation
♦ cell migration
GLA CONTAINING PROTEINS
 Vitamina K
 Liposolubile
 Non passa attraverso
placenta
 K1: vegetali verdi
 K2: deriva da K1 ad opera
di batteri intestinali
 K3 o menadione:
preparato industriale
 THIOREDOXIN

DICUMAROL WARFARIN DICUMAROL WARFARIN

THIOREDOXIN

WARFARIN
DICUMAROL
Antimetaboliti di Vitamina K

Dicumarolo

Warfarina
 Role of vitamin K
Some clotting factors require a post-translational
modification before they are active in clotting

These factors are II, VII, IX, X, proteins C and S

This PTM involves the addition of a COO- to

certain Glu residues in the clotting factors

This PTM results in the formation of several -carboxy

glutamates = Gla

This PTM requires vitamin K

PLATELETS IN DISEASE
 THROMBOSIS

Predisposing factors: “Virchow’s Triad”

abnormalities of
vessel wall

blood flow blood constituents

 Activating Factors for Platelets in Cardiovascular
Disease
 Damage to endothelium :

Atherosclerosis  Loss of inhibitory

function of
endothelium

 Exposure of von
Willebrand Factor and
collagen
 How do we know platelets are important in CVD?

 Platelets are present in atherosclerosis, thrombosis,

embolism i.e. at early and late stages of cardiovascular
disease

 Activated platelets are present in circulation of patients

with cardiovascular disease

 Modification of platelet activity affects the development

and progression of cardiovascular disease
Activating Factors for Platelets in Cardiovascular Disease

 Disturbed blood
flow -
 shear stresses,
narrowing of artery,
turbulence

Others
Low density lipoprotein or oxidised LDL
 Bacteria - myocardial infarction?
 Immune Complexes - present in MI
 Le piastrine nei difetti
coagulativi
 Anomalie congenite
» Sindrome di von Willebrand
» Sindrome di Bernard-Soulier: deficit del recettore
 Anomalie acquisite
» Trombocitopenia (basso livello di piastrine)
» Distruzione delle piatrine circolanti: leucemia, sindromi mieloproliferative,
eccesso di aspirina etc)
 Farmaci antipiastrinici:
» Aspirina (inibisce cicloossigenasi e riduce TXA2
» Dipiramidolo (inibisce effetto di ADP
 Patologie della coagulazione
Emofilia A o emofilia classica

 Deficit di Fattore VIII (150 differenti

mutazioni descritte)
 X-linked, frequenza 1:5 000-10 000 nati
maschi
 Emorragie
 Trattamento con infusione di VIII

I Romanov e il principe Alexis

Pedigree di Emofilia A nelle
famiglie reali europee
 Patologie della coagulazione
 Emofilia B, deficit di Fattore IX (300 mutazioni conosciute)
 Deficit di Fattore XIII (autosomici recessivi)
 von Willebrand Disease, deficit di Fattore VIII
» La patologia ereditaria più diffusa (125 nati per milione, doppio di
emofilia A)
» Adesione piastrinica difettosa
 Deficit di Fattore XI, o emofilia C, 3 mutazioni conosciute
» Mancata attivazione da contatto con superfici cariche
negativamente
» Identificata in 1953, comune negli ebrei Ashkenazi
 Deficit di antitrombina
» Subsintomatico, autosomico dominante (1 per 2000-5000)
» Sintomatico (trombosi venose profonde, embolismo polmonare) in
associazione con interventi chirurgici, traumi e gravidanza
 Patologie del fibrinogeno
 Eccesso di fibrinogeno in
» Pazienti con ipertensione, diabete, iperliproteinemia,
ipertrigliceridemia e coronaropatie
» Gravidanza, ipercoleresterolemia, contraccettivi orali, menopausa,
fumo
 Malattie genetiche
» Afibrinogenemia (aborto spontaneo da emorragie dal cordone
ombelicale e interne)
» Ipofibrinogenemia, acquisita o ereditaria
» Disfibrinogenemia (fibrinogeno non funzionale, con emorragie,
aborti e tromboembolismo)
 Major Anticoagulants

 Heparin
 Warfarin
 Hirudin
 Ancrod
 Protein C
 Dicoumerol
How does it work? Aspirin efficacy
Aspirin irreversibly inhibits
platelet COX enzyme
Platelets cannot synthesize new
COX (no nucleus)
No thromboxane
(procoagulant, vasoconstrictor)
synthesis
Low dose aspirin (80-160 mg)
does not inhibit endothelial
COX
Prostacyclin (anticoagulant,
vasodilator) formation not
affected Aspirin reduces clots by 15%, on average. 2% have a bleed,
that is serious each year. Use in high risk clotters.
 Heparin

 Proteoglycan - make up of large carbohydrate

component, so more like a poly-sugar than a
protein
 injected drug
 found in mast cells in endothelium of blood
vessels
 activates antithrombin (plasma protein)-
inactivates clotting factor
 One of few carbohydrate product with therapeutic
application
 Anticoagulants - Heparin
 Heparin is a glycoasminoglycan – alternating
glucuronic acid and N-acetyl-D-glucosamine residues
– sulfate and acetyl groups.
Avg mol. wt - 12,000 daltons

Heparin HEPALEAN
 Heparin is negatively charged
 Heparin & LMW Heparins circulates in the plasma - rapid
difference in action inhibits thrombin only in the
presence of heparin
Heparin
~ 45 saccaharide units
MW ~ 13,500
This reaction goes
1000 to 3000 times
faster with heparin.
Antithrombin
inhibits thrombin,
Xa, IXa and to a
lesser extent VIIa
Low Mol. Wt.
Heparin
~ 15 saccaharide
units
MW ~ 4,500
 Vitamin K Antimetabolites

 Dicoumarol and warfarin- given orally

 prevent vitamin K-dependent carboxylation
of factors II, VII, IX and X
 hinders effective functioning of the coag
cascade
 Side effect: prolonged bleeding
 need to choose dosage carefully
 Hirudin

 Directly inhibits thrombin by binding to it

 isolated from leech saliva in Hirudo medicinalis
 short 65 aa polypeptide
 does not require a co-factor
 weak immunogen
 not as much bleeding side effects
 Trade name: Refludan (purifying scheme in
Figure 9.15, page 378)
 Thrombolytic Agents

 Thrombosis function to plug damaged blood

vessel
 after repair, blood clot is removed via
enzyme degradation (fibrinolysis)
 when inappropriate clot forms, damage
minimized if you can speed up removal of
clot
 Agents developed to do so
 Thrombolytic Agents

 tPAs Activase (Genetech)

» Ecokinase (Galenus Mannheim)
» Retavase (Boehringer-Mannheim/Centocor)
» Rapilsyn (BM)
 Streptokinase
 Urokinase
 Tissue plasminogen activator

 Plasmin catalyzes degradation of fibrin in

clots (dissolving clot)
 plasmin derived from plasminogen
 plasminogen synthesized and released from
kidneys; 90 kDa protein
 tPA potently activates plasminogen
 Recombinant tPA

 Several modified forms to improve efficacy

(faster- acting, prolonged half-life)

 produced in E. coli (Ekokinase, Retavase

and Rapilysin)

 no glycosylation leads to prolonged serum

half-life