Coagulation Medicine

Physiopathology and
monitoring

Hanady Samaha, M.D.

March 2023
 What is haemostasis?
Maintains blood in a fluid state in
circulation
and provides
defence mechanism against bleeding
when injury occurs

Vasoconstriction
of the damaged vessel

Platelet plug formation

primary haemostasis

Fibrin clot
formation Fibrinolysis
secondary tertiary haemostasis
haemostasis
Thrombus degradation following
tissue repair
Haemostasis - 3 Phases
• Primary haemostasis
- vasoconstriction (immediately)
- platelet adhesion (seconds)
- platelet aggregation (minutes)

• Secondary haemostasis: Coagulation

- activation of coagulation factors (seconds)
- formation of fibrin (minutes)

• Fibrinolysis
- activation of fibrinolysis (minutes)
- lysis of the clot (hours)
Primary Hemostasis

Vessel
Platelet plug
The vessel/intima
 Endothelial damage
Normal vessel
Platelet activation

Primary Hemostasis I: Release of serotonine

Vessel Injury causes and noradrenaline
Vasoconstriction from platelets
Vasoconstriction
Vasoconstriction after vessel injury

Local blood pressure drop Key mediator: endothelins

• Proteins
Decrease of blood loss • Potent vasoconstrictors
 Damaged or activated endothelial cells

• Secrete von willebrand factor (vwf)

Procoagulant • Synthesize adhesion molecules
• Synthesize tissue factor(TF)
functions of • Exposed smooth muscle cells and fibroblasts have
the intima TF on membrane
• Provides phospholipids for clot assembly

Exposed collagen binds platelets and vwf

The platelets
 Platelet physiology

The normal platelet count is 150–450 · 109/l.

Platelets are formed in the bone marrow from

megakaryocytes (MK), which are characteristically very
large polyploid cells, reaching up to 50 μm in diameter.

During maturation MKs undergo endomitosis to form

cells with DNA ploidy values ranging from 4 to 128 n.

Megakaryopoiesis is regulated by the cytokine

thrombopoietin (TPO) which is constantly synthesized by
the liver.
Platelet physiology

The shape and small size of

the platelet enables them to
Platelets have a mean life
flow centrifugally in blood
span in the circulation of 10
vessels, allowing them to
d.
interact optimally with
damaged endothelium.
 Platelet physiology

Upon vessel wall damage, platelets undergo a highly regulated set of functional responses
including :
• adhesion,
• spreading,
• granular release reactions,
• activation of phospholipase A2,
• aggregation,
• exposure of a procoagulant surface,
• microparticle formation
• and clot retraction.

These platelet responses enable the rapid formation of a haemostatic plug that occludes the site
of blood vessel damage and limits blood loss.
 Platelet adhesion

• Vascular damage: exposure of collagen

• Subendothelial collagen binds vWF
• vWF binds GPIb on platelets
 Platelet aggregation
• Adhesion is followed by recruitment of
additional platelets that form clumps, a
process called aggregation (cohesion).
• Mediated by GPIIb/IIIa receptor
• Most abundant surface receptor on platelets
• Platelet activation → GPIIb/IIIa changes
conformation
• Becomes capable of binding to fibrinogen
• Will not bind when platelets are inactive
• “Inside-out” signaling (cell activity → altered
receptor)
Platelet Granules

Alpha granules (most abundant)

• Fibrinogen
• von Willebrand factor
• platelet factor 4

Dense granules
• ADP
• Calcium
• Serotonin
 • Released from alpha granules
• Binds to endothelial cells
• Numerous biologic effects described
• Heparin induced thrombocytopenia
• Rare, life-threatening effect of heparin
administration
Platelet Factor 4 • Antibodies formed to PF4 complexed with heparin
• Antibodies bind PF4-heparin → platelet activation
• Diffuse thrombosis
• Low platelets from consumption
 • Stored in dense granules
• Released on platelet activation
• Basis for serotonin release assay
• Diagnostic test for HIT
• Donor platelets radiolabeled with 14C-
Serotonin serotonin
• Patient serum and heparin added
• HIT antibodies → excessive serotonin
release
 • P2Y1
Adenosine • Calcium release, change in platelet shape
Diphosphate • P2Y12
ADP • Platelet degranulation, ↑ aggregation
• Many P2Y12 receptor blocking drugs
• “ADP receptor blockers”
• Inhibit platelet activity
• Clopidogrel, prasugrel, ticlopidine,
ticagrelor
 • Powerful platelet activator
Thromboxane • TXA2 receptors found on platelets
A2 TXA2 • Basis for aspirin therapy
 Platelet aggregation and
secretion
• Metabolic pathway activation results in:
• the elevation of cytoplasmic calcium and
phosphorylation of substrate proteins,
• =>changes in the cytoskeleton, enabling platelet
shape change and spreading,
• release of a- and dense-granular contents, (ADP)
causing aggregation of further platelets
• stimulation of phospholipase A2 and liberation of
thromboxane A2 (TXA2) (Prostaglandin
metabolism)
• activation of GPIIb/IIIa receptors.
• induction of a procoagulant surface activating the
coagulation process
Secondary Hemostasis

Coagulation
Coagulation
Factors
• Proteins synthesized in liver
• Soluble in plasma
• Activate when triggered by
endothelial damage
• Form an insoluble protein:
Fibrin
• Fibrin mesh prevents blood loss
Coagulation Factors

Named by Roman numerals: I, V, X

Circulate as inactive enzymes (zymogens)

Activated forms: Ia, Va, Xa

Many activate to become serine proteases

• Serine: amino acid

• Protease: cleaves proteins
• Serine protease: protein cleavage enzyme, contains
serine
 No
No
bleeding
bleeding

Variable bleeding
Variable bleeding
Severe bleeding
Severe bleeding
 ⚫Upon vessel wall injury,
tissue factor (TF) is exposed
to circulating endogenous
factor VII/VIIa – leading to
the TF/VIIa complex, which
initiates coagulation 
⚫A limited amount of
thrombin activates factors
V, VIII and platelets 
⚫Activation of factor X leads
to the formation of the prothrombinase
complex Xa/Va which subsequently
generates large amounts of thrombin 
⚫This “thrombin burst”
Adapted from Hoffman M et al., 2001.1 induces the generation of a haemostatic
plug that
prevents further blood loss 
Thrombin-The key enzyme in haemostasis

Optimal thrombin generation is crucial because of it’s multiple actions:

Converts fibrinogen into fibrin

Activates platelets

Activates FVIII and FV

Activates FXIII necessary for the formation of fully stabilized fibrin clots/plugs

Activates FXI (feed-back loop leading to more thrombin formation via FIX)

Activates TAFI (thrombin activatable fibrinolytic inhibitor

Impaired Thrombin generation Results in the formation of a:

• More porous clot
• More permeable clot
• Less dense fibrin hemostatic plug that is easily susceptible to fibrinolytic dissolution
 Formation of the Clot

• Thrombin clips two peptides

(fibrinopeptide A and B) from fibrinogen.
• This produces the fibrin monomer with
exposed polymerization sites that can
bind to other fibrin monomers.
• The monomers polymerize to form a
loose clot.
 Formation of the Clot
• Factor XIIIa then solidifies the bond by
forming glutamyl-lysine bridges between
the side chains of the fibrin monomers.

• Factor XIII is the only coagulation enzyme

that is NOT a serine protease.
Cell-based model of coagulation: termination
Once a fibrin clot forms over the injured area, the coagulation process
must be limited

Naturally occurring anticoagulants in the blood regulate this step

• Antithrombin (ATIII) - inhibits the activity of thrombin and other enzymes, such as FIXa,
FXa, FXIa and FXIIa
• Proteins C and S - inactivate the cofactors FVa and FVIIIa
• TF pathway inhibitor (TFPI) – inactivates FXa and TF/FVIIa

Fibrinolytic system disposes of excess fibrin and the fibrin clot

• Includes plasminogen, plasminogen activator and plasmin, all of which bind specifically to
the fibrin in clots
1. Hoffman M, Monroe DM. Hem/Onc Clin N America. 2007 21:1–11
2. Konkle B. In: Harrison’s Hematology and Oncology; 2010
3. Riddel JP et al. J Ped Oncol Nursing. 2007;24:123–131
Tertiary Hemostasis

Inhibitors of coagulation cascade or teriary

hemostasis
Is it all over when the
bleeding stops?
NATURAL INHIBITORS OF THE COAGULATION CASCADE

Normal blood flow

hepatic degradation of
dilutes the activated
clotting factors
clotting factors below
washed away from the
the level required to
site of clot formation.
propagate the cascade.
 Antithrombin: (AT; previously called antithrombin
III [AT III])
NATURAL
• is the most important physiologic inhibitor
INHIBITORS OF of activated coagulation factors.
THE • is synthesized in the liver and endothelial
cells.
COAGULATION • It irreversibly binds to and inhibits thrombin,
CASCADE factor Xa, and other activated clotting
factors.
• Heparin (or heparan sulfate on endothelial
cells) binds to and activates AT.
• By itself, AT has a low affinity for thrombin;
however, complexing with heparin increases
the activity of AT ~ 1,000-fold.
 • Protein C and protein S: vitamin K–dependent
NATURAL inhibitors of the coagulation cascade that control
coagulation by inactivating factors Va and VIIIa.
INHIBITORS OF • Protein C is activated by the binding of thrombin to
THE thrombomodulin on endothelial cell surfaces;
COAGULATION • When thrombin binds to thrombomodulin, thrombin
is no longer able to convert fibrinogen to fibrin;
CASCADE instead, it enzymatically cleaves and activates protein
C.
• Activated protein C, in combination with protein S,
inactivates factors Va and VIIIa.
• Protein S circulates in two forms: free (active) protein
S and protein S complexed with a protein involved in
the complement system, the C4b binding protein.
35
 hepatic degradation of clotting factors washed
away from the site of clot formation.
• Complexes with Factors VIIa/TF/Xa; inactivates Xa

Antithrombin /Heparin Cofactor II/Heparin

COAGULATION • Binds and Inactivates Enzymes

INHIBITORS Protein C/Protein S/Thrombomodulin

• Cleaves & Inactivates Cofactors (Va & VIIIa)

Plasminogen - 3º hemostasis

• Cleaves Fibrin
 plasminogen/plasmin

THE
FIBRINOLYTIC t-PA.

SYSTEM
Plasmin inhibitors: α2-antiplasmin,
and inhibitors of plasminogen
activation.
Plasminogen/Plasmin

Plasmin is the enzyme that digests fibrin and thus dissolves

clots.

Plasmin circulates as an inactive precursor, plasminogen.

Plasminogen is activated to plasmin primarily by t-PA, which is

secreted by endothelial cells.

Plasminogen can also be activated by the contact activation

pathway (factor XII, HMWK, and PK). (minor activator in vivo)
 • localization of plasmin activity to the surface of fibrin clots.
• Plasminogen is bound into fibrin clots as they are formed.
Control of the • Tissue plasminogen activator has a much higher affinity for
plasminogen that is localized on the surface of a fibrin clot
fibrinolytic than it does for free plasminogen, and this helps to specifically
system localize fibrinolyis to the clot.
• circulating inhibitor of plasmin, α2-antiplasmin: inactivates
plasmin that is free in circulation. Plasmin bound to fibrin is
protected from inhibition by α2-antiplasmin
Inhibition of Plasminogen Activation

plasminogen activator inhibitor-1 (PAI-1). Most

important

plasminogen activator inhibitor-2 (PAI-2). The

concentration of PAI-2 is high during pregnancy and is
present in high concentration in placental circulation.
Otherwise, it plays a relatively minor role.
Fundamental properties of the hemostatic system

Regulation and Control Mechanisms

• Each system is balanced by an opposing or inhibiting
system
• There are multiple negative feedback loops
• A system and its opposing system are often initiated
simultaneously.
• A system often initiates its own inhibiting system.
Fundamental properties of the hemostatic system
Surface Dependence
• Hemostasis is designed to occur on a phospholipid surface ( Platelet)
• Calcium dependence.
• link between the platelet phospholipid and the coagulation
proteins
• platelet activation and aggregation.
Hemostasis

Testing and monitoring

Contents

How we detect what goes wrong

•Tests for primary haemostasis

•Tests for secondary haemostasis
•Test for overall haemostasis
Bleeding time (BT)
(Ivy's method modified by Mielke)

a blood pressure cuff is An incision The wound is blotted

first placed on the approximately 1cm with filter paper every
upper arm of the deep on the forearm is 30 seconds and the
patient, and inflated to then created using a total bleeding time is
40mm Hg. special knife. recorded.
Bleeding time (BT)
(Ivy's method modified by Mielke)
 Normal range (depending on technique)
 Normal 2-7 minutes
 “Grey zone” 7-10 minutes
 Prolonged (abnormal) >10 minutes

 Possible causes for abnormal BT

 Low platelet count
 Platelet function disorders
 Von Willebrand disease
 Aspirin-like platelet disorder
 Bleeding time cannot detect single coagulation factor deficiency
 Secondary haemostasis consists of

• The activation of various coagulation factors

on the surface of activated platelets leading
to thrombin burst
• Thrombin activates fibrinogen to fibrin, and
Tests for FXIII to FXIIIa, forming a stable clot which
secondary plugs the ruptured vessel
haemostasis
Tests for secondary haemostasis

• Global tests
• Measurement of single coagulation factor
• Plasma mixing test
Global tests
Measure the haemostatic components in plasma

Test principle

• Only a certain part of the coagulation system is activated to form a clot

• The time from activation to clot formation is recorded
• In cases of prolonged clot formation time, the causative coagulation factor can be
identified by the single factor assay

Common global tests

• Prothrombin time (PT)

• Activated partial thromboplastin time (aPTT)
THE PT REFLECTS THE FACTORS IN THE EXTRINSIC /INITIATION
PATHWAY

Detects lack of FII, FV, FVII, (FIX), FX, Fibrinogen

Principle:

•Clot formation is initiated by adding tissue factor

(thromboplastin) to plasma

Normal range (depends on test system):

•PT: 10-14 seconds Used to monitor Warfarin by converting to INR

•Quick: 70-120% INR= {Patient PT/Control PT}ISI
•International normalised ratio (INR):0.8-1.2 ISI = International Sensitivity index (from reagent manufacturer)
INR permits comparison of PT results between Laboratories
THE aPTT REFLECTS THE FACTORS IN THE INTRINSIC/
AMPLIFICATION-PROPAGATION PATHWAY

Used to monitor heparin therapy

• Problem: different people have vastly

different PTT responses to heparin.
• The factor Xa inactivation assay is an
alternative test to monitor therapy with
unfractionated heparin
 Activated partial thromboplastin time, aPTT

• Detects lack of FVIII, FIX, FXII, FXI, FX, FV, FII and fibrinogen (Prolonged in
deficiencies of all clotting factors except VII).

• It is prolonged in liver disease, vitamin K deficiency, therapeutic warfarin and

heparin anticoagulation, Lupus anticoagulants, DIC, and with high levels of FDPs.
• Principle:
• Clot formation is initiated by adding “contact activator” to plasma
• Contact activator activates FXII to FXIIa

• Normal range (depending on assay): 26-36 seconds

Thrombin Time

Thrombin Time is sensitive for the detection of defects in fibrin generation

Thrombin Time starts the reaction by adding thrombin

• This thrombin is able to generate fibrin in patient plasma by activating fibrinogen

Highly sensitive for heparin, hypofibrinogenaemia or functional defects of fibrinogen

Normal range 18-21 sec

It is not affected by deficiencies in the intrinsic or extrinsic pathways

Plasma mixing assay
Principle

• Patient plasma is mixed with normal plasma in a ratio of 1:1

• Clotting time measured immediately after mixing is normal because FVIII or
FIX in normal plasma compensates for the inactive FVIII or FIX (inhibited by
inhibitor) in patient's plasma
• The mixture is incubated in 37°C for 2 hours
• During this time the inhibitor inactivates FVIII or FIX in normal plasma as
well
• The clotting time which was initially normalised is now prolonged

Results are expressed in Bethesda Units (BU)

• 1 BU = quantity of inhibitor that results in loss of 50% factor activity in a

normal plasma sample after 2 hours incubation at 37°C
55

Plasma mixing assay

Inhibitor Clotting factor

Incubate at 37°C
for 2 h
Plasma mixing assay

Prolonged TP or PTT

Repeat the test

Mixing test

Corrected Not corrected

Factor deficiency Circulating Ab

Measurement • Principle
of single • Assays are based on the same principles as PT or
coagulation aPTT tests

factor activity • BUT plasma which is deficient of a specific

coagulation factor is added to patient’s plasma

• The formation of clot depends on the activity of

this factor from patient’s plasma
 Measurement of single coagulation factor activity

Example Results are expressed in various ways

• FVIII-deficient plasma is added to • Normal value of all coagulation factor is

patient’s plasma around 100%
• aPTT test is carried out
• Normal aPTT – patient’s plasma contains
normal FVIII
• Prolonged aPTT – patient’s plasma
contains low level of FVIII
• All other factors are at ~100% by added
plasma
 How we detect what goes wrong
• Tests for secondary haemostasis
• Tests for primary haemostasis • PT sensitive to FV and FVII
• Platelet count • aPTT sensitive to FVIII and FIX
• Bleeding time • Thrombin time for Fibrinogen function
• PFA • Fibrinogen for Fib-level
• Advanced Tests: • Plasma mixing test detects inhibitor
• Induced platelet to FVIII or FIX
aggregation • Test for overall haemostasis
• Multiplate Analyzer
• Flow cytometry • Thromboelastography
• Thrombin generation assays
 Thank You

T: +961 1 577055
Youssef Sursock Street – Rmeil, Beirut
P.O. BOX: 16-5146 – Ashrafieh, Lebanon
Email: info@sgub.edu.lb

www.sgub.edu.lb