L.

MECHANISMS OF COAGULATION AND

FIBRINOLYSIS
Blood extravasation

controlled or prevented by:

o blood vessels
o platelets
o plasma coagulation proteins
o physiologic and naturally occurring protease
inhibitors
o fibrinolytic system

Secondary hemostasis
o coagulation factor response

Intrinsic and Extrinsic Coagulation Pathways

o Formation of stable fibrin clot
o Clot: fibrin (2 hemostasis) and platelet plug
(1 hemostasis)
o Intrinsic activated: contact of certain
coagulation proteins with subendothelial
connective tissue
o Extrinsic activated: release of tissue factor
(HMW lipoprotein) from injured vessel

platelets
vessels
coagulation factors

Blood = prevented from leaving the vascular system (by

endothelial cell lining):
o

can be disrupted by:

o mechanical trauma
o physical agents
o chemical injury

HEMOSTATIS OVERVIEW
Hemostasis:

Primary hemostasis
o platelet and vascular response (vessel
injury)
o initiated by exposure of platelets to
subendothelial components (blood vessel):

collagen

microfilaments

basement membrane
o acute injury

small vessels constrict

platelets immediately adhere to the

exposed surfaces (release ADP and
ATP; thromboxane A2 promotes
further vasoconstriction)
o reversible primary platelet aggregation:

platelets adhere to one another

change shape

centralization of organelles

platelets may disaggregate in the

absence of further stimulation
o if with continued stimulation:

secondary, irreversible, platelet

aggregation occurs

platelet aggregation (substances released)

ADP

Promotes 2 platelet
aggregation

Recruits additional
platelets

ATP

Serotonin

Further vasoconstriction

Phospholipid (PL) becomes

available on the platelet membrane
surface

Site for fibrin formation

thrombogenesis

Stop bleeding;
Allow for vessel repair
(through formation of
a stable fibrinplatelet plug)

THE COAGULATION PROTEINS ZYMOGENS AND

SERINE PROTEASES

coagulation factors:
o enzyme precursors (zymogens)

factors II, VII, IX, X, XI, XII, and

PK

zymogens inactive; no biologic

activity

serine proteases active

exposed serine-rich active

enzyme sites

selectively hydrolyze
arginine- or lysinecontaining peptide bonds
o nonenzymatic cofactors

factors V, VIII, tissue factor, and

HMWK
o calcium
o PL (provided by platelets)
o Normally present in the plasma

Zymogen activation:
o conformational change (1)

coagulation reactions occur on the

injured surfaces
o hydrolytic cleavage (2)

on the PL surfaces of aggregated

platelets
o factors X and II

requires presence of nonenzymatic

cofactors VIII and V, respectively

must be activated (VIIIa

and Va) by small amounts
of thrombin

thrombin enhances ability

of cofactors to assist in
factors X and II activation

thrombin inhibit VIII

and V activity

Assist by either altering

zymogen conformation

(permit more efficient

cleavage by serine
proteases)

Binding the zymogen and

appropriate serine
protease on a platelet PL
surface

Enhance zymogen
activation process
Zymogen -> serine proteases
o Biochemical amplification of the coagulation
process
o Production of serine proteases - rate of
further transformation of zymogens;
activity levels of cofactors

COAGULATION AND THE KININ SYSTEM

Kinin
o Chemotaxis
o Sensation of pain
o Mediate inflammatory responses
o Increase vascular permeability
o Vasodilation and hypotension
o Induce contraction of smooth muscle
o Contact activation phase (intrinsic pathway)
o C activation
Kinin system
o contains factors that are activated by the
coagulation and fibrinolytic systems
o factors:

prekallikrein (Fletcher factor)

complex with cofactor

HMWK
o contact group

kallikrein

from PK in the presence of

factor XIIa and HMWK

conversion of HMWK to
kinin

accelerates factor XII

activation

fibrinolytic system

with XIIa = plasminogen

activator

kininogen (LMWK, HMWK:

Fitzgerald factor)

kinins: bradykinin

COAGULATION AND THE COMPLEMENT SYSTEM

C1 inactivator inhibits factors XIIa, XIIf, XIa,

plasmin, and kallikrein

THE COAGULATION FACTORS

Order of its discovery

f fragmented factor
Tissue factor
Calcium
Factor VI nonexistent

Characteristics of the Coagulation Factors

o
o

Presence or absence of factors in barium

sulfate (BaSO4)-adsorbed plasma
PK and HMWK partially adsorbed by
BaSO4

THE COAGULATION GROUPS

1.

2.

3.

Contact group

PK, HMWK, Factor XII, Factor XI

Adsorbed by contact (negatively charged

surface: collagen or subendothelium)

Causes slow conversion of factor XII to XIIa

o Initiates intrinsic pathway and
fibrinolysis
Prothrombin or vitamin-K dependent group

Synthesized in the liver in the presence of

vitamin K (acts as cofactor)

No substantial storage of vitamin K in the

body

Vitamin K
o Necessary to -carboxylate the
preformed enzyme precursors of
factors II, VII, IX, and X
o -carboxylation

allows factors to binds to

Ca and form calcium
bridges (with acidic PL
surface of activated
platelets)

Vitamin K-dependent -carboxylation

reactions may be inhibited:
o dietary vitK deficiency
o administration of antibiotics that
sterilize the GI tract (normal flora)
o oral anticoagulant therapy (ie.
warfarin)
Fibrinogen group

factor I, V, VIII, and XIII

highest MW

most labile

consumed in coagulation

only group that act as substrates for the

fibrinolytic enzyme plasmin

found in platelets (-granules)

o except: factor XIII = general
platelet cytoplasm
o factor VIII:C coagulant portion;
not in platelets

factor VIII (VIII/vWF)

o VIII:C coagulant portion

cofactor in intrinsic
pathway

produced in liver

antihemophilic factor
o VIII:vWF important in normal
platelet function
o HMW portion:

synthesized by the
endothelial cells and
megakaryocytes

antigenic portion (factor

VIIIR:Ag)
von Willebrand portion
(VIII:vWF)

Factor XII activation

PHOSPHOLIPIDS CONTRIBUTING TO
COAGULATION
Tissue Factor

thromboplastin (PL and protein)

extrinsic pathway (binding its PL portion to factor
VII, converting VII to VIIa
extrinsic necessity of adding a tissue extract (PL) to
plasma
o prothrombin time (PT) test

rabbit brain or lung tissue

thromboplastin

Ca activate factor VII

Partial Thromboplastin and Platelet Phospholipid

Partial thromboplastin rgt; platelet substitute

(intrinsic pathway)
o partial thromboplastin time

partial rgt only has PL for factor

X activation in vitro
In vitro tests of intrinsic pathway platelet-poor
plasma (avoid test variation)

PHYSIOLOGIC VARIATIONS OF THE COAGULATION

FACTORS
Newborns deficient in vitK

THE PROCESS OF FIBRIN CLOT FORMATION

Earlier Theories
Morawitzs classic coagulation theory
coagulation = 2 stages
o first stage: prothrombin -- thrombin
(by thrombokinase in Ca presence)
o second stage: fibrinogen to fibrin (by
thrombin)
Modern Theory
cascade and waterfall
difference in intrinsic and extrinsic:
o mechanism of initial activation
o mechanism of activation of factor X
Intrinsic Coagulation Pathway
dominant
intrinsic - all of the components in the system are
found in circulating blood
exposure of the factors to negatively charged surfaces
(ie. glass, damaged vascular endothelium)
begins with contact phase of coagulation

contact phase begins with factor XII absorption to the

negatively charged surface of vascular collagen
o PK in complex with cofactor HMWK (also
absorbed)
o factor XI complexes with HMWK
o once assembled, factor XII undergoes
conformational change (presence of PK)
factor XIIa (enhancement by HMWK)
o factor XIIa autoactivate (slow rxn)
o enzymes in basophils and endothelial cells
activate factor XII
o factor XIIa cleaved to XIIf (by plasmin and
kallikrein)
o HMWK enhances the proteolytic effect of
kallikrein on factor XIIa
Factor XIIa (contact activation):
o initiates the intrinsic pathway

[HMWK, XIIa] factor XI to XIa

o initiates fibrinolysis *[ presence

[XIIa, kallikrein] complex reqd

for conversion of plasminogen to
plasmin
o initiates kinin and complement system

kallikrein formation (by XIIf and

HMWK) -> conversion of HMWK to
kinins (ie. bradykinin)
Kallikrein:
o perpetuates factor XII activation and own
production
o initiates kinin system
o initiates fibrinolytic and C systems together
with factor XIIa
o directly activates factor IX (on its own)
Plasmin:
o result of contact activation
o promotes clot dissolution (limits coagulation
process)
o activates C
o cleaves factor XIIa to XIIf

Factor XI activation

factor XIIa (with HMWK) -> factor XI to XIa

Factor XIa = factor IX to IXa ( + Ca as cofactor)
directly activates by contact activation
XIa also activates plasminogen
XIIa and XIa involved in initiation of fibrinolytic
and C systems

Factor IX activation

factor IX to IXa (by factor XIa and Ca)

completes contact activation phase
kallikrein (activating factor IX)
IXa + VIII (VIIIa cofactor) + Ca (on PL platelet
surface) = activation of factor X

Extrinsic Coagulation Pathway

tissue factor

o tissue thromboplastin
o from cell membranes into plasma
factor VII
o -carboxyglutamic acid residue of factor VII
binds the PL portion of tissue factor in the
presence of Ca

bridge between factor VII and tissue

factor
o VII to VIIa
o VIIa-Ca-tissue factor complex (platelet PL
surface)

converts factor X to Xa (common

pathway)
Ca

Alternate Pathways Linking the Extrinsic and Intrinsic

Pathways

factor XIIa activate factor VII

factor VIIa two-chain form
o greater effect on factor X activation
factor IXa and kallikrein activate factor VII in
plasma (has been exposed to glass surfaces)
VIIa-Ca-tissue factor complex
o can slowly activate facot IX to IXa
o with subsequent activation of X to Xa

alternate pathway

bypass contact activation

phase
large concentrations of factor Xa cleave facor VII
into a three-chain molecule; INACTIVE

Common Coagulation Pathway

Extrinsic Pathway Activation

VIIa-Ca-tissue factor complex

o X to Xa

Intrinsic Pathway Activation

Factor VIII (cofactor)

o VIIIa by thrombin to take factor X activation
IXa-Ca-factor VIIIa
o binds with platelet PL
o converts X to Xa
o conversion rate of X accelerated thousand
times
o Xa prevented from diffusing away from the
complex
o prothrombinase complex

Xa-Va-Ca-PL

V to Va by thrombin

prothrombin to thrombin
o common pathway

thrombin activation of fibrinogen to

fibrin

The Thrombin Feedback Mechanism

Thrombin factor IIa

o feedback mechanism
o control of coagulation process

activator:

autocatalytic

small amounts, enhances reactivity

of factors V and VIII

stimulates platelet aggregation

acts on fibrinogen

activation of XIII to XIIIa

soluble fibrin -> stabilized firbin

clot (by factor XIII and activated by
thrombin)
inhibitor

prevent excessive clotting

thrombin amount: opposite effect

on factors V and VIII -> destroys
them

activates protein C (potent

anticoagulant)

enhanced by Ca

thrombomodulin

cofactor protein S

protein C-S complex

inhibits factors Va and

VIIIa

stimulates fibrinolysis (
plasminogen activator
activity)

Activated protein C

inhibits tissue
plasminogen activator
inhibitor (TPAI)

prevention of interference
with plasmin production

thrombin + thrombomodulin = no
longer enhance factor V or VIII
activity, stimulate platelet
aggregation, or fibrinogen
conversion to fibrin

Final Clot Formation and Stabilization

fibrinogen A, B, and (disulfide bonds)

o and fibrinopeptides A and B

Conversion of Fibrinogen to Fibrin

STEP 1: cleavage of the four fibrinopeptides ( and

ends) by thrombin
o chains remain intact; do not hydrolyze
during fibrin formation
o A and B fibrinopeptides removed = soluble
fibrin monomer or unstable gel
STEP 2: fibrin monomers = aggregate spontaneously
(electrostatic bonds only)
o fibrin polymers = soluble; dissolved in vitro
in 5M urea or weak acids (1%
monochloroacetic acid)

vulnerable to plasmin
STEP 3: clot stabilization
o requires factor XIII, Ca, and thrombin
o thrombin activates XIII

XIII fxns as transamidase

(covalent bonds)

and chains formation of the stabilized

fibrin clot

insoluble in 5M urea and weak acid

THE FIBRINOLYTIC SYSTEM

fibrinolysis bodys defense against occlusion of

blood vessels; dependent on plasmin
activation of plasminogen to plasmin
o plasmin

digest and destroy fibrinogen,

fibrin, and factors V and VIII

coagulation

activates kinin and C

o plasminogen

to plasmin by plasminogen
activators

liver

stored and transported in

eosinophils

increased concentrations
inflammation

intrinsic activation substances

normally present in plasma

extrinsic activation substances

entering plasma from outside
source
o Intrinsic Plasminogen Activation

Factor XIIa, kallikrein, HMWK, and

proactivator(activated by kallikrein)

activate plasminogen
intrinsically
o Extrinsic Plasminogen Activation

plasminogen activators present on

organ tissues

endothelial cells (proteases.. veins)

o Exogenous Plasminogen Activation

urokinase

therapeutic destruction of
thrombi

purified from urine

activates plasminogen

induce fibrinolysis

streptokinase
plasminogen part of any clot; tendency of fibrin to
absorb plasminogen
excessive free plasmin antiplasmin is depleted;
plasmin begins destroying components other that
fibrin

o
o

fibrin(ogen) degradation products (FDP)

fibrin(ogen) split products (FSP)
plasmin cannot distinguish fibrinogen from fibrin;
degrades both
A and B chains remain intact in fibrinogen
fragments; removed by RES and etc

Four principal products:

o Fragment X

Plasmin acts on specific sites of each fragment

Pathologic Effects of Fibrin Degradation Products

FDP
Inhibit coagulation
Form incoagulable or slowly coagulable
complexes with fibrin monomers or
fibrinogen

Detectable by protamine sulfate and

ethanol gel tests

Fibrin monomer test

More accurate test for

detection of these
complexes
o Antithrombin activity
o Interference with polymerization of the
fibrin monomer
o Interference with platelet activity
o Fragments X, Y, and intermediate FDPs

Most important in exerting

anticoagulant effects
o Fragments Y and D

Inhibit fibrin polymerization

o Fragment E

Powerful inhibitor of thrombin

All four fragments (particularly low-molecular weight
FDP)
o Affinity for coating platelets membranes
o Cause a significant platelet dysfunction by
inhibiting aggregation
o
o

Fibrin(ogen) Degradation by Plasmin

early degradation product

first and largest fragment formed
Result of plasmin cleavage of the
terminal portions of alpha chains
(isolated fibrin strands)

Then cleaved by plasmin to form

two fragments (YY) and an
intermediate complex (DXD)

Complex is further cleaved DED

and DY/YD (intermediate
complexes) until fragments E and D
(D-D dimer) are formed
Fragment Y early degradation product
Fragment D (D-D dimer)

late degradation product

presence is a specific indicator of in

vivo fibrinolysis (intravascular
thrombin formation -> fibrin
formation -> degradation)

indicative only of fibrin (NOT

fibrinogen) degradation products
Fragment E late degradation product

NATURALLY OCCURING COAGULATION AND

FIBRINOLYTIC INHIBITORS

COAGULATION
o Antithrombin III (AT-III)

Protein (2 globulin) synthesized in

the liver and endothelial cells

Half-life: 2.7 days

Inhibits thrombin (1:1 complex),

XIIa, XIa, Xa, and IXa (by forming
enzyme inhibitor complexes with
these activated factors ->
neutralizing and preventing their
action

Inhibitory effect on plasmin and

kallikrein

Vital role in monitoring the

coagulation, fibrinolytic, kallikreinkinin, and C systems

Enhanced by cofactor heparin

(attaches to AT-III)

Conformational change

Heparin

rate of factor
inactivation, NOT
magnitude

Accelerates rate of binding

of AT-III with serine
proteases

Treatment may deplete

available plasma AT-III

Heparin-like substance

Acidic
mucopolysaccharide

Naturally-occuring heparin

Mast cells and basophils

Enhance AT-III activity

Heparan sulfate

Surface of platelets and

vascular endothelium

+ AT-III -> protects

uninjured vessels against
abnormal thrombus
formation (neutralizing
serine proteases)
Alpha-2-Macroglobulin

Binds with various proteolytic

enzymes including thrombin (does
NOT COMPLETELY inhibit them)

Rate of inhibition is slower than

AT-III

Protect its bound enzymes against

other circulating inhibitors

Inhibits fibrinolysis

Not totally eliminating

plasmins function in
fibrinolysis

Inhibits kinin system by inhibiting

kallikrein
Alpha-1-Antitrypsin

Potent inhibitor of factor XIa

Inactivate thrombin at a slow rate

Inhibits fibrinolytic system ->

inactivation of plasmin

Least significant of the three

naturally occurring fibrinolytic
system inhibitors
C1 Inactivator (C1 Esterase Inhibitor)

Affects coagulation, fibrinolytic,

kinin, and C systems

Important inhibitor of factor XIIa

and XIIf

Inhibits factor XIa

Inhibits plasmin and kallikrein

o Proteins C and S

Vitamin K-dependent

Cofactor S

Destroy factors Va and VIIIa

Complex: enhances fibrinolysis

(inactivating inhibitors of
plasminogen activators ->
enhanced plasmin formation)

Protein C activated slowly by

thrombin circulating in the plasma

Greatly enhanced:
thrombin +
thrombomodulin ; (1:1)
with protein S

Thrombin specificity is
altered (when +
thrombomodulin)

Deficiencies are similar to AT-III

(before 30 yo)
FIBRINOLYSIS
o Alpha-2-Antiplasmin

Most important naturally occurring

inhibitor of fibrinolysis

Principal inhibitor of fibrinolysis

(1:1) -> neutralizing plasmin

Prevents plasmin from binding to

fibrin

Prevents plasmins premature and

uncontrolled digestion of fibrin,
fibrinogen, and factors V and VIII

Permits slow and orderly

dissolution of clot

Plasmin + 2-antitrypsin
similar to thrombin-AT-III
complex
inactivating serine
protease

inhibits serine proteases XIIf, XIa,

IIa, and Xa

inhibits clot-promoting activities of

plasma kallikrein

inhibition of tissue plasminogen

activator
o Alpha-2-Macroglobulin

Inhibits components (fibrinolytic

and coagulation systems)

Effectively inhibits plasmin after

2-antiplasmin depletion
o Alpha-1-Antitrypsin

Third most important

Inactivates plasmin slowly

Does not bind plasmin until both

2-plasmin and 2-macroglobulin
are saturated

Inhibition of coagulation by its

potent inhibitory effects on factor
XIa
o Other

AT-III inhibits fibrinolysis

(inhibiting plasmin and kallikrein)
C1 inactivator inhibits plasmin

Rapid blood flow

o Prevent excessive propagation of a thrombus
and dilute any excess procoagulants or
profibrinolytic components at sites of injury

Fibrin
o

Restricts active coagulants to the interior of

the fibrin clot
Platelets and endothelium
o Restrict coagulation
Hepatic clearance