Transcript Blood Clotting

Clotting of Blood
Lecture in Basic Tissues
2004
Baynes & Dominiczak, Chapter 6
Gene C. Lavers, Ph.D.
[email protected]
©Copyright 1999-2004 by Gene C. Lavers
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Hemostasis (arrest of bleeding)
Balancing Events
Blood Clotting
Introduction
Normal hemostasis is a balance between
coagulation and fibrinolytic systems.
 VASOCONSTRICTION
 PLATELET PLUG FORMATION
 BLOOD COAGULATION
 FIBRIN CLOT FORMATION
 DISSOLUTION OF CLOT
 PROTECTING THE CLOT
 PREVENTING CLOT FORMATION
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Vasoconstriction
Blood Clotting
Platelets & Thromboxane
Overview
VASOCONSTRICTION – occurs immediately after vessel wall injury





Diminished blood flow into and out of the vessel.
Initiated by local nerve reflexes
Sustained through biochemical mediators, serotonin and histamine
released from platelets in the damaged subendothelial.
Thromboxane A2 (TXA2) – A C20 polyunsaturated fatty acid released
from the activated platelets, causes smooth muscle contraction and
prolonged vasoconstriction.
The larger the portion of vessel traumatized, the more extensive the
degree of spasm.
A sharply cut vessel (clean cut with sharp razor) undergoes less
spasm, i.e., bleeds longer, than a crushed vessel.
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Platelet Plug Formation
Collagen & von Willebrand Factor
Blood Clotting
Overview
PLATELET PLUG FORMATION – occurs within seconds after vessel wall
injury

Primary hemostasis, platelets aggregate to control blood loss from capillaries,
arterioles, and venules.

If damage is small, platelet plug can arrest bleeding completely by closing
minute ruptures in vessels that occur hundreds of times a day.

Exposure to collagen and other foreign substances (antigen-antibody
complexes, thrombin, proteolytic enzymes, endotoxins, and viruses), platelets
undergo dynamic change in appearance and begin the process of adhesion and
activation.

In adhesion, platelets swell, become sticky and adhere to collagen fibril
on the basement membrane - requires von Willebrand factor (vWF) adhere to
collagen fibrils on the basement membrane (vWF deficiency is associated bleeding
disorder).

Activated platelets release granules containing vWF, factor V, PDGF*,
heparin-neutralizing protein, and ADP (attracts other platelets)
* PDGF Platelet-derived growth factor
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Clotting Process
Blood Clotting
Zymogen Cascades
Introduction
CLOTTING – stops bleeding and hemorrhage.
 2 zymogen activation cascades – both convert inactive Stuart
factor (X) to active Stuart factor Xa.
 Xa + Va activates prothrombin (II) to thrombin IIa.
 Thrombin activates soluble fibrinogen (I) to insoluble fibrin
(Ia)
 Fibrin aggregates forming enormous numbers of fibers that
entangle one and other into a meshwork that yields a soft clot.
 Transglutaminase (XIIIa) cross-links fibrin threads to yield a
hard clot.
 Vitamin K and Ca2+ are essential for liver synthesis of effective
protein factors (II, IX, X, XI) that can bind efficiently to membrane
phospholipids exposed by wound or trauma.
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5
Clot Prevention/Inhibition
Antithrombin III
Blood Clotting
Introduction
INHIBITING CLOT FORMATION
 Prevention


unscheduled clots
clots beyond local trauma area.
 Anti thrombin III (AT III)


binds irreversibly to thrombin (IIa).
Also binds to IXa, Xa, and XIa of the intrinsic pathway.
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Clot Prevention/Inhibition
Antithrombin III
Blood Clotting
Introduction
DISSOLUTION OF CLOT – removing unscheduled clots
and fostering healing.
 Tissue Plasminogen Activator (TPA) activates plasminogen to

plasmin.
Plasmin (a protease) hydrolyzes fibrin to peptides…clot
dissolves
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C. Lavers
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Topics-subtopics
Blood Clotting
Introduction
PROTECTING THE CLOT – during healing

Antiplasmin inhibits fibrin hydrolysis.
PREVENTING CLOT FORMATION – fail safe agent

Heparin acts as a catalyst for inactivation of
thrombin by antithrombin III.


Citrate acts to chelate* Ca 2+
O
Oxalate also chelates Ca
C
2+
R
HO
COO–
COO–
Ca2+
COO–
Citrate
Chelate: claw
Ca2+
C
g-carboxylate group C
COO–
Ca2+
COO–
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Oxalate
C. Lavers
O–
O–
O
8
Four Principle Components
Blood Clotting
Introduction
 Intact endothelial cells – inhibit blood clotting by displaying
proteins that inhibit the clotting system, and they form
prostacyclin (PGI2) which they release (t/2 ~ 3 min) to prevent
platelet aggregation, adhesion, and thrombus formation.
 Subendothelial tissues – contain membrane proteins normally
not in contact with the blood. When exposed after injury,
platelets and clotting factors adhere to components of the
subendothelial tissue, where they become activated.
 Platelets – bind to components of the subendothelial tissue.
Subsequently, clotting factors bind and are activated on their
surface.
 Clotting factors – are soluble plasma proteins, most are serine
proteases, that circulate as inactive zymogens awaiting
proteolytic cascade activations that trigger insoluble fibrin
formation from soluble fibrinogen.
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Blood Clotting
Coordinated Four Functions
Thrombosis vs. fibrinolysis
4 stages  Clotting
3
2
4
1
1. Arteriolar vasoconstriction can
transiently reduce blood flow, and
can give some platelet aggregation.
2. Activation of blood platelets for
adhesion to traumatized vessel
wall.
3. Activation of coagulation cascade
systems for activation of thrombin.
4. Activation of fibrin polymerization
and crosslinking of fibrin soft clot
to stable hard clot after
vasoconstriction lessens or
traumatized tissue is disturbed;
clot is anchored.
Dissolution  peptides
1.
2.
TPA activates plasminogen.
Plasmin proteolytically digests clot
 peptides  amino acids.
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C. Lavers
Fig. 6.1 Overview of hemostatic mechanisms
Bleeding Disorders
Congenital and acquired
Blood Clotting
Hemophilia
Clotting is slow, slower, absent
 Hemophilia A
deficient VIII
 Hemophilia B
deficient IX
(Christmas)
Fig. 6.2 Congenital and acquired causes of excessive bleeding.
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Blood Clotting
Classic Hemophilia
Factor VIII deficiency
Fig. 6.7 Severe bruising from minor fall in a 3-yr old
child with classic hemophilia.
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Genetic Disorders
Blood Clotting
Clinical
 Hemophilia A (recessive, X-linked) - Females carry the
defective gene, males express the phenotype of the
disorder.
 85% of cases, Factor VIII (intrinsic pathway) is missing.
 Because antihemophilic factor is missing, clotting is blocked.
Interaction with Ca-dependent IXa that yields Xa does not
occur. Thus, inactve IX fails to act on prothrombin (II) to yield
thrombin (IIa).
 Occurs in about 1 in 10,000 males.
 Treat with factor VIII or IX concentrates.
 Hemophilia B - Factor IX (Christmas) is missing, thereby
limiting normal blood clotting.
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Blood Clotting
Vessel Wall
Four layered structure
Functional Roles
 Thrombo-resistant endothelial cell lining:
produces two vasodilators and inhibitors of
platelet function: prostacyclin (prostaglandin I2,
PGI2,
 nitric oxide (NO, also called Endothelium-Derived
Relaxing Factor, EDRF) produced from arginine by
eNOS (synthase) controlled by intracellular [Ca2+].

Vasoconstriction after vascular injury: mediated
in part by serotonin and thromboxane A2 (TXA2)
released from activated platelets.
 Disruption of the endothelial layer exposes
subendothelial
 Tissue factor – activates the extrinsic pathway of
blood coagulation.
Fig. 6.3 Structure of blood
vessel wall.
 Collagen – activates the intrinsic pathway of blood
coagulation.
TXA2 is a potent mediator
of platelet activation and
vasoconstriction
 Stimulates platelet activation
 Stimulates platelet binding to exposed collagen via
von Willebrand factor (vWF) released from
endothelial cells also bound to platelet GPIb-IX
receptor.
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Blood Clotting
Platelet Activation
Antiplatelet drugs
drugs
Eicosanoids
 Platelets (150-400 x 109/L)
 Activated by
 ADP from endothelial, RBC, and
Exposed
collagen
drugs
Platelet
TXA2
platelets
 Epinephrine
 Collagen
 Thrombin
 Platelet Activating Factor, PAF
 Immune complexes (infection)
 Shear stress from blood flow
 Platelet shape: disc to sphere
 Release: TXA2 (t/2, 30sec) and
vWF
 Aggregation via vWF and
Exposed
collagen
fibrinogen binding to GPIbIX and
GPIIa-IIIP receptors
 Adhesion to vessel wall linked by
Endothelial lining
vWF
O
COOH
CH3
OH
© Copyright 1999-2004 by Gene TXA thromboxane 15
Fig. 6.4 Platelet activation
2
Fig 36.16
pathways & antiplatelet drugs C. Lavers
Blood Clotting
Coagulation Factors
zymogens
Plasma soluble proteins
prothrombin = II  IIa = thrombin
fibrin I  Ia = fibrinogen

Intrinsic pathway (•): activated by

Clinical tests
 APTT (30-50 sec) Activated Partial
plasma components
Thromboplastin Time, also known as the
KCCT (kaolin-cephalin clotting time),
intrinsic pathway.
 PT (10-15 sec) Prothrombin time, extrinsic
pathway
 Thrombin time

Extrinsic pathway (o): activated by
nonendothelial ‘surface’ tissue factor
 Vitamin K deficiency reduces hepatic
synthesis of II, VII, IX, and X. Treat
with vit K injections
 Oral anticoagulents – warfarin reduces
hepatic factor synthesis. Excessive
bleeding with warfarin treated by
stopping the drug, giving vit K or
replacing II, VII, IX, and X with fresh
frozen plasma or concentrates.
Fig. 6.5 Coagulation factors and some
of their properties
 Liver Disease – Factor syn reduced;
acetaminophen overdose  hepatic
failure  longer prothrombin time
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Topics
Blood Clotting
Introduction

CLOT FORMATION

INHIBITING CLOT FORMATION

DISSOLUTION OF CLOT

PROTECTING THE CLOT

PREVENTING CLOT FORMATION
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Activation Cascades
Intrinsic, Extrinsic, Final pathways
Blood Clotting
Die XII CO2 Intrinsic
Extrinsic
Final
Prekalikrein
HMWK
XI
XIa
Tissue Factor(III)
VII + Ca2+
IXa
IX
Xa
X
VIII
VIIIa
XII
Prothrombin
V
thrombin
Va
XIIa
Fig. 6.6 Blood Coagulation
fibrinogen
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1999-2004 by Gene
Stable
fibrin
fibrin C. Lavers
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Intrinsic and Extrinsic Converge at Xa
Blood Clotting
Regulation Cascade*
Damaged Surface
Trauma Damage
Intrinsic Pathway
Prekallikrein
Kininogen
Kallikrein
Hageman
XII
Extrinsic Pathway
1
XIIa
XI 2
Plasma
thromboplastin
IX
Convertin
XIa
3
Stuart
VII
VIIa
Christmas
IXa
Antihemophilic VIIIa
X
Preconvertin
4
II
Tissue
factor
Trauma Damage
Fibrin
X - + -
X
Xaa
Ia
IIa
Prothrombin Thrombin
* Other Cascades?
Glycogenolysis
cAMP
- - I
- + - + - + - + - + -
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Fibrinogen
C. Lavers
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Activation of Thrombin
I  IIa
NH2
NH2
10
10

Blood Clotting
Molecules
274 Arg
275Thr
C 274 Arg
275 Thr
A
NH2

323 Arg
324 Ile
S
S
NH2
A
SS
C323 Arg
B
II
C 582
324Ile
IIa
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B
C 582
20
Fibrinogen, Factor I
–4
–8
Blood Clotting
Molecules
–4
Properties
 Synthesized in the liver, t/2 = 4 days
 0.3 mg% in human plasma
 Dimeric heterotrimer: 2 [3 different polypeptides] = [ g]2




joined by S-S bonds, the middle disulfide knot.
Each half contains an g subunit mostly in a rod-like triplehelix where the halves join, but with random coils at the outside
ends (globular).
Fibrinogen is about 9 nm wide and 46 nm long with two halves
of 23 nm (230Å).
Fibrinogen is trinodular (three-globule dumbbell-shape), central
knob has three clusters of charges: -4 at each end and -8 at the
central knob:
-4
-8
-4
Charge repulsion prevents fibrinogen from self-associating, thus
it remains a soluble monomer the circulating blood.
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Fibrinogen (I)  Fibrin (Ia)
Via thrombin (IIa)
Blood Clotting
Molecules
-4






Hydrolysis by Thrombin (IIa) at Arg-gly site yields
Fibrin (Ia) monomer + fibrinopeptides A and B
Fibrin is insoluble because its - 4
+5
- 4 charges allow it to
aggregate to form fibers.
The aggregated intertwined fibers trap cells yielding a soft clot.
The soft clot is not stable. It is held together by electrostatic charges.
Soft clot is cross-linked by factor XIIIa with Ca2+ to yield hard clot
fibrin fiber.
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-4
+5
22
Hard Clot Formation
Transglutaminase (XIIIa)
Blood Clotting
Cross-linking
Gamma chains cross-linked


The soft clot continues enlarging as platelets, other blood cells, and
debris from the wound, become enmeshed.
Factor XIIIa (transglutaminase) cross-links the fibrin monomers
comprising the thread-like fibers by forming sets of peptide (amide)
bonds between adjoining C-terminal peptides of the g-chains thereby
stabilizing and strengthening each fibrous thread along its length.
– COOH region:
g
etc ------------------------Gly
Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val COOH
|
|
COOH Val Asp Gly Ala Gln Lys Ala Gly Gly Leu His His Gln Gln Gly ------------------------------- etc of

As cross-linking proceeds, the soft clot becomes more stable and a
hard clot develops: clot contracts, water is squeezed out.
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23
g
Topics
Blood Clotting
Outline

CLOT FORMATION

INHIBITING CLOT FORMATION

DISSOLUTION OF CLOT

PROTECTING THE CLOT

PREVENTING CLOT FORMATION
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Antithrombin III
Blood Clotting

Antithrombin III – always present in the serum.

Binding between Antithrombin III and Thrombin (IIa)
prevents thrombin from proteolytically activating fibrinogen
(I) to fibrin (Ia).

Antithrombin III counteracts any unscheduled clot
formation.

Impact injury triggers clotting at the injury-site by activating
thrombin.

Any thrombin that might escape the local clot is inactivated,
thus, clotting does not spread throughout the body.
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Inhibiting Intrinsic Clot Cascade
Blood Clotting
Process
 Anti thrombin III binds irreversibly to
– IIa (thrombin) active site.
– also binds to IXa (Christmas)
– also binds to Xa (Stuart)
– also binds to XIa (Plasma Thromboplastin)
Thus, the active factors exist briefly!
* All four have proteins contain N-terminal g-carboxyglutamate clusters with Ca2+
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Inhibitors of Coagulation
Blood Clotting
Preventing harmful thrombi that cause strokes & infarcts
Die  CO2
.
Fig. 6.8 Sites of inhibitors
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Topics
Blood Clotting
Outline

CLOT FORMATION

INHIBITING CLOT FORMATION

DISSOLUTION OF CLOT

PROTECTING THE CLOT

PREVENTING CLOT FORMATION
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Blood Clotting
Coagulation Inhibitors
Fibrinolytic System Components
Fig. 6.9
Hemostasis
Fig. 6.10
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Blood Clotting
Fibrinolytic System
Plasminogen  plasmin
Clot Dissolution
Fibrin  Fibrinogen Degradation Products (FDP peptides)
 Dissolving clots
– Unscheduled
 Use tPA and streptokinase
– Wound healing
 Plasminogen  plasmin(active)
 Fibrin proteolyized 
Fibrinogen degradation
peptides
 Saving clots:
 2 antiplasmin inhibits
plasmin, thus the hydrolysis
of fibrin clots is prevented.
Fig. 6.11 Fibrinolytic system. Plasminogen
to plasmin by uPA, tPA or
streptokinase
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Topics
Blood Clotting
Outline

CLOT FORMATION

INHIBITING CLOT FORMATION

DISSOLUTION OF CLOT

PROTECTING THE CLOT

PREVENTING CLOT FORMATION
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Anticoagulants

Blood Clotting
Molecules
Vitamin K Antagonists - Inhibit g-carboxylase thereby
lessening g-carboxyglutamate formation.
– Dicumarol - present in hay, mimics Vitamin K.
– Warfarin (Coumadin)- a clinical anticoagulant (strokes/infarcts).
– Chelated Ca2+ normally promotes binding of factors IIa, VIIa,
IXa, and Xa to trauma-exposed membrane phospholipids.
– The fewer or absence of the g-carboxyglutamate cluster at the Ntermini of these clotting factors slows or prevents these events
and interferes with effective clotting.

Heparin - A carbohydrate polymer that promotes thrombin
inactivation by antithrombin IIIa.

Citrate or oxalate - Chelates Ca2+ thereby inhibiting clotting.
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END
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C. Lavers
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