Trousseaus sign or syndrome

Patients with unusual or

migratory thromboses
presented with subsequent
malignancy (1865)

subsequently exhibited it
himself 2 years later
 Cancer and thrombosis
4-fold increased
Hypercoagulability
risk of thrombosis
compared to
general population

Virchows Triad 8-fold increased

Blood stasis Blood vessel risk when receiving
chemotherapy
What links cancer and thrombosis?
 Cancer cell TF-VIIa-PAR2 signaling

Florence Schaffner, and Wolfram Ruf Arterioscler Thromb

Vasc Biol. 2009;29:1999-2004

Copyright American Heart Association, Inc. All rights reserved.

How might engineered APC analogs be applied?
Thrombolytic therapy post-ischemic stroke

Ischemic strokes account for 87% of all

strokes

Early reperfusion associated with better

outcome

AlteplaseTM only approved clot busting

therapeutic

Only safe within an early window after

ischemic stroke (<4 hours)
APC makes thrombolytic therapy safer

Cheng et al. Nat Med 2003; Liu et al. Nat Ned 2004

Ongoing Phase II trial in ischemic stroke patients

 Stroke treatment and APC

Current clot busting drug, tissue

plasminogen activator (t-PA)
associated with haemorrhage
neuronal apoptosis and narrow Sham + rh-tPA
therapeutic window

Use of APC alongside t-PA resulted in

reduced haemorrhage and apoptosis
MCAO + rh-tPA
Clinical trails underway using APC to
improve thrombolytic therapy for
stroke

MCAO + rh-tPA +
APCpost-tPA
Cheng et al. Nat Med 2003
 Novel non-anticoagulant, gain-of-function APC
analog minimises t-PA induced neurotoxicity

*Current treatment *Proposed APC New state-of the-art!

therapy

Gleeson et al. Blood 2015

 Coagulation
Lecture 6: Revision and Practice Exam Qs

Dr. Roger Preston

School of Medicine, TCD
prestonr@tcd.ie
Coagulation Exam Question 2017
Test general understanding of coagulation

Know key plasma and cellular players in

mediating coagulation
PRACTISE QUESTION 1:

The coagulation pathways are activated at

the site of injury and after clotting has been
initiated, these pathways are down-
regulated and inhibited. Discuss
PRACTISE QUESTION 1:

Coagulation is activated at the site of injury

and after clotting has been initiated, these
pathways are down-regulated and inhibited.
Discuss
The coagulation pathways are activated at
the site of injury...
Key factors in initiation of coagulation:

Damage to blood vessel endothelial

barrier

1. Causes exposure of procoagulant stimuli

such as collagens, subendothelial matrix
tissue
2. Exposure of tissue factor
1. How do platelets find the site
of vascular damage?
Platelet adhesion
 Platelet adhesion and aggregation

platelet
VWF
Endothelial
cells
or platelet
GlpIb
GPVI

collagen

Platelet activation
- Release of platelet granules
- Recruitment of further platelets
Platelet aggregation
 Platelet activation

1. Change in platelet shape

2. Activation of cell surface integrins -
GpIIb/IIIa
3. Granule secretion
4. Flip/Flop of plasma membrane
 What purpose does platelet
activation serve?
Conversion from quiescent state to active
haemostatic state:

1. Adopt structural changes to help clot formation

2. Secretion of factors that facilitate clot formation

3. Activate cell surface molecules to make them

sticky for other platelets and coagulation
factors
 Tissue factor
Membrane receptor
expressed on the surface
of extravascular tissue

Not normally expressed

on cells exposed to the
blood

Aberrant expression of TF
is a major cause of
pathological thrombosis
 TF is a receptor for factor VII(a)
TF binds to FVIIa with
high affinity when
exposed by vessel
damage

Acts as the trigger for

blood coagulation
cascade
 Blood coagulation activation
Fibroblast

Endothelial
Cells
Vascular
injury
Lumen

TF Tissue factor
Initiation the thrombin spark
Fibroblast
Fibrin Clot Formation
Fibroblast

XIa VIIIa
PRACTISE QUESTION 1:

The coagulation pathways are activated at

the site of injury and after clotting has been
initiated, these pathways are rapidly down-
regulated and inhibited. Discuss
3 ANTICOAGULANT PATHWAYS:
Tissue Factor Pathway Inhibitor
Pathway

Protein C Pathway

Antithrombin-Heparin
 Tissue Factor Pathway Inhibitor (TFPI)
Works by stopping the stimulus of blood clotting

Prevents efficient activation of coagulation

Blocks efficient interaction between tissue factor, factor VIIa

and factor Xa

HOWEVER, only works where stimulus (tissue factor

exposure) is low

Dampening response
Thrombin functions as an anticoagulant on Endothelial Cells

BLOOD

Lumen
TM EPCR FIIa binds to TM on endothelial cells

Tunica
Intima

Tunica
Media
 BLOOD

Protein C

Lumen Protein C binds to EPCR

on endothelial cells

Tunica
Intima

Tunica
Media
 BLOOD

Protein C
FIIa:TM activates
Lumen
Protein C

Tunica
Intima

Tunica
Media
 BLOOD

Activated Protein C:Protein S

inhibit thrombin generation at APC-mediated
Lumen the thrombus edge inhibition
occurs on EC
XX X XX (& platelet
Tunica membranes)
Intima

Tunica
Media
Antithrombin functions as a scavenger of
thrombin

T
Thrombin (active proteinase)
AT

Antithrombin
 Antithrombin functions as a scavenger of
thrombin
Thrombin (active proteinase)
T
Antithrombin

AT

AT T
Vessel wall heparan sulphate

Endothelium
Thrombin-antithrombin
complex (inactive)
 Antithrombin is a SERPIN
Forms a 1:1 irreversible complex with all serine
proteases (XIIa / XIa / Xa / IXa / thrombin)

However affinity is highest for thrombin

Most important direct inhibitor of thrombin

Antithrombin accounts for 70% of
antithrombin activity in plasma

Inactive inhibitor / protease complexes removed

by liver / spleen
 SERPIN mode of action
Involves two steps
1. Initial complex
Formation of a stable bond between reactive site
of antithrombin and the serine residue in reactive
site of serine protease (e.g. thrombin)

AT acts as a pseudo substrate forming 1:1

complex

2. Stable complex
Initially the reactive bond of the inhibitor acts as
a substrate for the enzyme
With cleavage of the bond, a conformational
change occurs and traps the enzyme
Huntington lab, University of Cambridge
PRACTISE EXAM QUESTION 2:

Describe the structure and function of plasma

proteases that control procoagulant and
anticoagulant reactions after blood vessel
injury
 Coagulation glycoproteins

Despite the fact that opposite functions, procoagulant

and anticoagulant factors share many similarities:

1. Synthesised by liver and secreted into plasma

2. May be dependent upon vitamin K for normal function

3. Composed of similar domains

4. Activation may involve cleavage of an activation peptide

Coagulation factors use four common domain modules

Epidermal growth factor

Gla (EGF)
domain

Serine protease
(chemotrypsin)

Kringle
domain
 Coagulation factor domain comparison
Gla EGF Serine protease
domain

Factor IX

Factor X

Factor VII

Protein C

Protein S

Prothrombin
Kringle
domain
Similar domain structure, diverse roles

Factor VII

Factor IX Procoagulant

Factor X

Protein C Anticoagulant
FVII, FIX, FX and PC

Homologous modular structure

consisting of four domains
Significant sequence identity with
each other

Circulate in plasma as a zymogens,

- activated by proteolytic cleavage
Serine proteases

Gla domain is involved in binding to

phospholipid surface

EGF domain is involved in protein-

protein interactions
eg EGF1 FVII binds TF
Initially, coagulation factors are synthesized with
glutamic acid residues in their Gla domains

Nascent clotting factors,

II, VII, IX, X CH-CH2-CH2-COOH

(glutamic acid or Glu)

Conversion of Glu residues to Gla residues prior to
secretion into plasma

Nascent clotting factors,

II, VII, IX, X CH-CH2-CH2-COOH
(glutamic acid, Glu)

Vitamin K g Glutamyl-
Carboxylase

COOH
Secreted clotting factors,
II, VII, IX, X CH-CH2-CH
COOH
(g carboxyglutamic acid or Gla)
 Modified Gla residues can interact with Ca2+

Nascent clotting factors,

II, VII, IX, X CH-CH2-CH2-COOH

(glutamic acid, Glu)

Vitamin K g Glutamyl-
Carboxylase

COO--
Secreted clotting factors,
II, VII, IX, X CH-CH2-CH Ca 2+
COO--

(g carboxyglutamic acid, Gla)

Modified Gla residues can interact with Ca2+ and
mediate phospholipid binding

Nascent clotting factors,

II, VII, IX, X CH-CH2-CH2-COOH

(glutamic acid, Glu)

Vitamin K g Glutamyl-
Carboxylase

COO--
Secreted clotting factors, Negative
Ca 2+
II, VII, IX, X CH-CH2-CH platelet
membrane
COO-- phospholipid

(g carboxyglutamic acid, Gla)

Describe the structure and function of plasma
proteases that control procoagulant and
anticoagulant reactions after blood vessel
injury

vitamin K dependent proteases:

where they are synthesised, common gene and

protein structure, vitamin K dependence

key roles in coagulation cascade and in protein C

pathway
EXAMPLE EXAM QUESTION 3:

Thrombin generation is central to both clot

formation and regulation of clot growth
Discuss
PROCOAGULANT activity of thrombin

Free Thrombin

Procoagulant serine protease

Activate platelets via PAR1
Feedback amplification (activates
FV, FVIII, FXI)
Cleaves fibrinogen to form fibrin
Activate FXIII to strengthen fibrin clot
Thrombin - Protease-activated receptor (PAR-1)
EXAMPLE EXAM QUESTION 3:

Thrombin generation is central to both clot

formation and regulation of clot growth
Discuss
 Key points about coagulation
cascade.
Each step is the formation of a complex consisting of
enzyme-cofactor-substrate

Think in terms of the complexes and what each

complex does

Not linear

thrombin generation is central to

functionthrombin generates fibrin
 EXTRINSIC TENASE COMPLEX
THE TRIGGER
FXa
FX FVIIa (vessel damage)

TF TF (cofactor): FVIIa (enzyme)

complex

phospholipids FXa FX (substrate)

Extrinsic Xase complex:

TF-FVIIa / phopholipids / Ca and FX
 PROTHROMBINASE COMPLEX
THE ENGINE
thrombin FXa generated
FVIIa
by tenase complex
FVa
prothrombin FVa (cofactor): FXa (enzyme)
complex

phospholipids thrombin prothrombin

(substrate)

Prothrombinase complex:
Factor Xa/factor Va/phopholipids/prothrombin
PROBLEM EXTRINSIC TENASE COMPLEX
RAPIDLY INHIBITED BY ANTICOAGULANTS
FXa
FX FVIIa (vessel damage)

TF TF (cofactor): FVIIa (enzyme)

complex

phospholipids FXa FX (substrate)

Tissue factor pathway inhibitor
 PROTHROMBINASE COMPLEX
FORMATION BLOCKED

thrombin FXa generated

FVIIa
by tenase complex
FVa
prothrombin FVa (cofactor): FXa (enzyme)
complex

Tiny amount of prothrombin

phospholipids
thrombin generated
(substrate)

Not enough thrombin gets generated to convert fibrinogen into

fibrin no clot
 THE THROMBIN SPARK

thrombin FXa generated

FXa
by tenase complex
FVa
prothrombin FVa (cofactor): FXa (enzyme)
complex

Tiny amount of prothrombin

phospholipids
thrombin generated
(substrate)

THROMBIN SPARK
XII XIIa FXII not needed
The thrombin spark

XI XIa Tissue factor / FVIIa

(vessel damage)
IX IXa
VIIIa
Pl
Ca 2+

X Xa X
Va
Pl
Prothrombin THROMBIN
 INTRINSIC TENASE COMPLEX
THE AMPLIFIER
FXI activated by thrombin spark from
FXa extrinsic pathway
FIXa

FVIIIa
FX FXIa FIXa

phospholipids
FIXa (enzyme)
+ FVIIIa (cofactor)
FXa (product)
+ FX (substrate)

Intrinsic Xase complex:

FIXa / FVIIIa / phospholipid / Ca and FX
 THE THROMBIN BURST

thrombin LOTS OF FXa generated

FVIIa by intrinsic tenase complex
FVa
prothrombin FVa (cofactor): FXa (enzyme)
complex

phospholipids thrombin prothrombin

(substrate)

Fibrinogen Fibrin
How does protein C get activated?

T T
T
T
T
T
T T
T T
platelet platelet

T
VIIa
Excess thrombin binds thrombomodulin
on intact endothelial cells

Thrombomodulin

T T
platelet platelet
T

VIIa
Excess thrombin binds thrombomodulin
on intact endothelial cells
Thrombin

Thrombin binds to TM with

high efficiency

Thrombomodulin (TM) Endothelium

 2. Thrombin : TM complex activates PC

APC
Thrombin cleaves
protein C to
release the
activation peptide
& generate APC

Protein C

Thrombin: TM complex
Thrombin generation is central to both clot formation
and regulation of clot growth Discuss

Procoagulant role of thrombin:

Activate platelets
Feedback amplification (activates FV, FVIII, FXI)
Cleaves fibrinogen to form fibrin
Activate FXIII
Cell signalling (via PAR molecules)

Anticoagulant role of thrombin:

Initiates the protein C pathway
 In vitro laboratory testing of coagulation
in patients plasma
Intrinsic pathway

Activated partial
thromboplastin time
(APTT) Extrinsic pathway

Prothrombin time
(PT)
 What test would pick up haemophilia A?

Intrinsic pathway

Activated partial
thromboplastin time
(APTT) Extrinsic pathway
What test would pick up factor VII
deficiency?
Intrinsic pathway

Extrinsic pathway

Prothrombin time
(PT)
 What test would pick up
Hypofibrogenemia?
Intrinsic pathway

Activated partial
thromboplastin time
(APTT) Extrinsic pathway

Prothrombin time
(PT)
 What test would be most impacted in
warfarin overdose patient?
Intrinsic pathway

Activated partial
thromboplastin time
(APTT) Extrinsic pathway

Prothrombin time
(PT)
 Any further questions
prestonr@tcd.ie

Good luck!