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SYSMEX : HAEMOSTASIS BASIC ONLINE TRAINING
TOPIC OUTLINE
A. Haemostasis
B. Primary Haemostasis
I. Aim of Primary Hemostasis
II. Adhesion of Platelets by means of
Collagen & vWF
III. Activation of Platelets
IV. ADP as Messenger Substance
V. Thrombin as Platelet Activator
VI. Synthesis and Release of Thromboxane A2
VII. Shape Change of Platelet
VIII. Fibrinogen Receptor and Aggregation of
Platelets
IX. Platelet Plug Stabilization by Fibrinogen
X. The Von Willebrand-Jürgens syndrome
XI. The Effect of Drugs
C. Secondary Haemostasis
I. Aim of Secondary Hemostasis
II. Cascade Model
III. Cell-based Model
D. Fibrinolysis / Antifibrinolytic System
I. Activation
II. Inhibition of fibrinolysis
III. Suitable blood assays
HAEMOSTASIS
● Balance between coagulation & fibrinolysis
● Body’s physiological method of
maintaining blood circulation.
● Body’s response of stopping the bleeding
caused by vascular or external injury
● Involves formation and degradation of
Thrombin to Fibrin via Coagulation Cascade
in blood vessels to regulate the blood flow
within the body
● Phases
a. Primary Haemostasis
○ Wound closure
b. Secondary Haemostasis
○ Blood coagulation → formation of
Fibrin clot
c. Fibrinolysis
○ Degradation of fibrin clot after
wound is being healed
*** phases are occurring simultaneously
PRIMARY HEMOSTASIS
I. AIM OF PRIMARY HEMOSTASIS
● Temporarily cover wound & stop bleeding
○ Cover is made of platelets that adhere
to injured tissues
● Factor Fibrinogen
○ Interconnects platelets
○ Production of White Thrombus
STEPS (3 A’s)
1st Adhesion & Binding
2nd Activation
3rd Aggregation
Fibrinogen ○ Water-soluble precursor of Fibrin
which forms the matrix for wound
closure
○ Synthesized in the liver (hepatocytes)
and occurs predominantly in blood
○ Stored in the alpha granules of
platelets. Other storage locations are
cells of the cellular matrix and body
fluids (e.g. lymph)
○ Reference range: 1.604.0 g/L at
fibrinogen levels <0.5 g/L, bleeding
tendency increases with decreasing
fibrinogen concentrations
○ Inflammatory processes = increases
concentration
Platelet ○ Are part of the non-liquid components
of the blood. Platelets in resting state
form round discs with a diameter of
about 2-3μm.
○ Under physiological conditions, they
circulate close to the endothelial wall
without reacting with each other.
○ In its membrane are inclusions
(granules) which, among other things,
contain coagulation factors.
○ Damage to the endothelial layer
causes adhesion and aggregation of
the platelets in the area of endothelial
damage
○ Platelets play a role in primary
coagulation
○ The normal range of platelets is
140,000-345,000/μl
○ Reduced platelet counts are
associated with an increased risk of
bleeding, but can also be a sign of
increased thrombosis risk (HIT-II)
II. ADHESION OF PLATELETS (COLLAGEN & vWF)
● The inside of blood vessels are lined with a
thin layer of endothelial cells
● If injured, subendothelium (connective
tissue) is exposed
○ Subendothelium –– contains certain
proteins (mainly collagen) that only
come into contact with blood through
the injury
Subendothelium Proteins
Collagen
○ Mainly part of the
connective tissues
○ Most important fibre
component of the skin,
cartilage, bones,
tendons, teeth & blood
vessels
○ Not exposed to blood Central activator of
primary hemostasis
under physiological
conditions
○ Binds to vWF
1
 ○ Injury to endothelial Shear Rate ○ Describes the spatial change of the
layer of blood vessels, flow velocity in liquids
■ collagen is exposed ○ Since friction forces are present in
to the platelets and real fluids, shearing of a fluid, just as
triggers adhesion in the case of a solid, means a
and aggregation transmission of force

Fibronectin vWR ○ Receptor at platelet membrane for

○ Protein found in plasma & vWF
on cell surfaces ○ Facilitates platelet adhesion to
○ For cell adhesion subendothelial cells after vessel wall
○ Absorbed by fibrin & damage
bound to Fibrin by Factor
XIIIa-mediated
cross-linking III. ACTIVATION OF PLATELETS (RELEASE OF
○ Formed in liver, Normal range (150-720 GRANULES)
endothelium & fibroblasts mg/L) ● Activation of platelets cause the release of
certain granules from the interior of the
platelet into the plasma
Laminin
○ Glycoprotein & essential
component of basal Surface of Platelets
membrane
○ For cell adhesion

vWF
○ Adhesive protein that
circulates freely in the
blood
○ Produced in the
endothelial cells and Granules Dense Bodies
platelet A. Alpha granules ○ Inter alia, Ca2+,
○ It binds to the injured (Fibrinogen, Factor V Serotonin, ADP
endothelial wall and XI)
○ Normal range B. Other
(=subendothelium),
(40-240%) coagulation-promoting
connects platelets to each
other, and binds ***varies due to blood substances (vWF)
group
circulating Factor VIII,
preventing the Factor VIII ○ Intraindividual
from prematurely fluctuations can be ADP ○ Thrombus formation
breaking down very high (CV up to (Adenosine ○ Stored in granules of platelets &
○ It therefore, occupies a 40%) Di-Phosphate) secreted into the plasma upon
kep piston in primary and activation
plasmatic haemostasis
○ vWF is synthesized in the Positive amplifier loop → activates
endothelial cells and further platelets → Additional ADP is
megakaryocytes released from the ATP platelets,
erythrocytes and other cells
↳ this & local suppression of the ADP
Bind via von Willebrand Direct bind inhibitors provides a local
Receptor (vWR) accumulation of ADP & promotes
aggregation

Factor IV ○ For activation of Factors II, VII, IX, X

(Ca2+) ○ Bridge the gap
■ (-) charged clotting factor &
(-) charged phospholipids of
platelets/cells
➢ Cannot bind to each other
w/o meditation by calcium
due to equal charge
○ Adhesion & aggregation of platelets
& muscle contraction
○ vWR Purpose –– bind ○ When shear rate not
to vWF too high
Factor V ○ Inactive form of Factor Va
○ Fusion of 4 ○ Platelets bind directly
○ Activated by Thrombin (Factor IIa)
glycoproteins to collagen of
○ Synthesized in liver & present in
○ On surface of platelet subendothelial cells
plasma (60-150%)
■ vWF binds to the ■ Can bind w/o vWF
○ Inhibitor –– Protein C/Protein S
newly exposed as mediator
complex system
collagen of ○ Adhesion phase, the
■ Leads to increased risk of
subendothelial endothelial damage is
thrombosis (APC resistance,
cells covered by platelets
Factor V Leiden)
■ Platelets adhere to
them via vWF as
Factor Va + Factor Xa = Prothrombinase
mediator 2+
Complex (w/ 𝐶𝑎 & phospholipids) w/
cofactor Factor Va
↳ Activation of thrombin is
Glycoprotein ○ Substances, consisting of a protein
accelerated a thousand fold
and carbohydrate group
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 Factor IX ○ Inactive form of Factor IXa ■ Causes polymerization of the
(Christmas ○ Synthesized in liver & present in fibrinogen & formation of fibrin
Factor / plasma (70-120%) web
Antihemophilic ○ Inhibitor –– Antithrombin
Globulin B) ○ Disorder –– Hemophilia B
VI. SYNTHESIS & RELEASE OF THROMBOXANE A2
Factor IX + cofactor VIIIa = Tenase ● Messenger substance that platelets release
2+
Complex (w/ 𝐶𝑎 & phospholipids) during and for their mutual activation is
↳ Complex of TF and Factor VIIa Thromboxane A2 (TXA2)
activates Factor IX ● Platelets have thromboxane receptor on
surface
Serotonin ○ Carried by platelets (dense bodies) &
released into plasma upon A. Synthesis of Thromboxane A2
activation ○ Formed in interior of platelet after
○ Vasoconstriction induced by
activation
serotonin reduces blood loss
○ Enhances platelet aggregation
○ ADP receptors must be activated by
triggered by Thrombin and ADP ADP to trigger the formation of
Thromboxane A2

IV. ADP AS MESSENGER SUBSTANCE AA + PL → TXA2

● Released from granules when activated COX1
● Activation of platelets ↳ COX1 –– catalyst
○ ADP receptors on surface (P2Y1 &
P2Y12) ○ When formed, TXA2 can activate
■ ADP binds to leading to other platelets via Thromboxane
mobilization of 𝐶𝑎
2+ receptors
➢ Change of the platelet
shape and aggregation B. Synthesis of Thromboxane A2
○ TXA2 binds to Thromboxane receptors
Release of ADP → positive feedback → triggers chain of other platelets when activated
reaction of platelets ○ Platelets release granules
○ Release of Ca2+ inside cell causes
● Activated platelets further seal the area of change in shape
injury
● Serotonin is released AA ○ Unsaturated fatty acid
○ Acts as a coactivator for platelet (Arachidonic Acid) ○ Synthesized from phospholipids
aggregation of the cells by phospholipase A2
○ Third step of primary hemostasis ○ Synthesized from platelets
■ Catalyzed by (COX1) to
thromboxane A2 (TXA2)
V. THROMBIN AS PLATELET ACTIVATOR
➢ Promotes platelet
● Plays a central role in primary & secondary activation
hemostasis ○ Synthesised from endothelial
● Has signaling effect in cells of vascular cells
system ■ Becomes Prostaglandin I2
○ Mediated by protease-activated (PGI2)
receptors (PARs) on surface of ➢ Inhibits platelet
platelets & smooth muscle cells activation
■ Via PARs platelets can also be
COX1 ○ Enzyme that converts AA into
activated by Thrombin
(Cyclooxygenase-1) TXA2 & PG12
○ Occurs in all tissue types
PAR ○ G-coupled receptor at surface of ○ AA inhibits the activity of
platelets, endothelium, and muscle cyclooxygenase irreversibly
cells
○ Activated by cleavage parts of their PL ○ A class of lipids & major
extracellular domain (Phospholipids) component of all cell
○ Types : membranes
■ PAR-1, PAR-2, PAR-3, PAR-4 ○ Forms lipid bilayers (amphiphilic)
○ PARs activated by thrombin (PAR-1, ■ 2 hydrophobic fatty acids & a
PAR-3, PAR-4) on surface of hydrophilic phosphate group
platelets contribute to the activation
of platelets. TXA2 ○ Mainly in the platelets
○ Contribute to the regulation of (Thromboxane A2) ○ Product of thromboxane
vascular tone and permeability, as synthesis
well as inflammatory responses. ○ It belongs to the group of
prostaglandins & activates
Thrombin ○ Central component of plasmatic platelet aggregation via its
(Factor IIa) coagulation receptors
○ Cannot be found in the
bloodstream
VII. SHAPE CHANGE OF PLATELET
○ Inactive form of thrombin
○ Synthesized in the liver ● Release of Ca2+ inside cell causes change in
○ Activates coagulation factors, shape
potent platelet activator, activates ● Formation of Pseudopods
antithrombotic systems ○ Thin extensions
○ Acts on fibrinogen & cleaves ○ Allow a more stable connection with
fibrinopeptides A and B wound as well as the platelets
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 ○ Variable plasma bulges of mobile ■ Must be stabilized by
body cells secondary haemostasis
○ Present in protozoa

X. VON WILLEBRAND-JÜRGENS SYNDROME

● Most common genetic disease affecting
VIII. FIBRINOGEN RECEPTOR & AGGREGATION OF primary hemostasis
PLATELETS ● Classifications :
A. Quantitative effects
○ Too little vWF is formed
B. Qualitative defects
○ Present but not sufficiently
functional
● Common symptom –– pronounced bleeding
tendency
● Laboratory tests
vWF : AG vWF : FVIII

● Fibrinogen receptors vWF : RiCo RiPA

○ Only become functional through
activation of platelet vWF : CB APTT
○ Following a conformational change
■ Change in spatial vWF : multimers FVIII : C
arrangement of its structural
elements XI. EFFECT OF DRUGS
■ Fibrinogen receptors are
altered
○ Bind to fibrinogen molecules from Aspirin ○ Active substance ASA
○ General analgesic & platelet
plasma
anti-aggregation agent
○ Can bind to vWF ○ Irreversibly blocks COX1
■ Allows platelet to bind to
enzyme
Collagen fibers of
■ TXA2 is not formed
subendothelium
● GPIIb / IIIa
○ Complex of the proteins GPIIb and Clopidogrel & ○ Bind irreversibly to ADP
GPIIa Prasugel receptor P2Y12 on platelet
○ Most common receptor on surface of membrane
platelets ■ Prevent platelet
○ Receptor for Fibrinogen and vWF activation
○ Fibrinogen binds to this receptor at
the surface of the platelets
■ Allowing a robust gluing of
ASA ○ Widely used analgesic,
platelets
(Acetylsalicylic Acid) anti-inflammatory,
antipyretic, antiplatelet drug
IX. PLATELET PLUG STABILIZATION BY
○ Inhibits COX1 irreversibly
FIBRINOGEN

SECONDARY HAEMOSTASIS

I. AIM OF SECONDARY HAEMOSTASIS

● Tight sealing of vascular injury by fibrin clot
● Transformation & reinforcement of white
thrombus
● Produced at the end of primary
hemostasis into a red thrombus
● Driven by coagulation factors which
● Each Fibrinogen molecule has 2 binding
domains predominantly belong to proteases
○ Can bind to 2 activated Fibrinogen
receptors Proteases ○ Enzymes that perform
● Additional stabilizers –– Laminin & proteolysis (cleaves)
Fibronectin ○ cleavage serves to activate the
● End result –– white thrombus (clot) related coagulation factor
○ Preliminary coverage of wound can ○ Thrombin –– serine protease
still be removed relatively easily
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 ■ Catalytic center consists of Factor VI ● Historically, independent
residue of amino acid serine coagulation factor
○ Coagulation factors activated by ● Identical to Factor V
cleavage activate further ■ Not used anymore
coagulation factors,
■ Resulting in a complex, Factor XII ● Pro-enzyme of serine protease
multi-step activation process (Hageman Factor) Factor XII
○ End of process –– fibrin net ■ Activates Factor XI to Factor
■ Stabilize red thrombus XIa & Factor VII ro Factor VIIa
● Synthesized in liver
Fibrin ● Final product of plasmatic ● Concentrations: 70 - 120%
coagulation from the cleavage of
Fibrinopeptide A and B
C. Common Pathway
■ From the E-fragment of
○ Final phase
fibrinogen molecule induced
○ Leading to a fibrin clot
by thrombin
● Biological glue sealing the area
of the endothelial damage
covered by platelets

Serine Protease ● Enzymes that cleave peptides in

proteins
● Amino acids serves as the amino
acid at the active site

II. CASCADE MODEL a. ACTIVATIONS BY OTHER COAGULATION

● Like cascades of the waterfall FACTORS
● Drawback –– certain limit of presentation and ● For example: Factor IX
explanation ■ Must be activated first to become
effective
● Progressive activations of coagulation factors ■ Cleaving off a part of its molecule by
follow one another specialized enzyme
● Pathways –– Extrinsic & Intrinsic ➢ Enzyme –– activated
■ Lead to activation of Factor X coagulation factor acting
(Thrombokinase) as activator (ex. Factor XIa
– “a” = “activated”)
A. Extrinsic Pathway ➢ Cleavage portion
○ “Exogenous Activation” converts inactive clotting
○ Usually triggered by bleeding factor into its active form
■ Endothelial injury & internal
tissue injury (vascular Cleave enzyme (inactive form) → active form
Aneurysm) Activated enzyme
○ Extrinsic Tenase Complex ● 12 coagulation factors are numbered I to XIII
■ Endothelial injury → TF contact
w/ blood → activates Factor VII
to Factor VIIa → TF & Factor
VIIa complex
■ Can activate Factor X to Factor
Xa

Factor III ● Protein of cell membrane

(Tissue Factor) ● Activates Factor VII to Factor VIIa
● Expressed on inner blood ■ Tissue factor –– activator of secondary
vessel coagulation (don’t need activation)
● Coagulation factor that ■ Calcium –– not a protein & don’t need
doesn’t need to be activated activation
● Formed & stored in CT cells
● Ex. fibroblasts b. ACTIVATIONS BY ENZYME COMPLEXES
● For example: Factor X
■ Not cleaved
B. Intrinsic Pathway
■ Activated by multiple coagulation
○ “Endogenous Activation”
factors
○ Activation process
➢ Combined to a specific
■ Factor XIIa → Factor XI → Factor
enzyme complex (ex.
XIa → Factor IX → Factor IXa
Factors VIIIa & IXa for
○ Intrinsic Tenase Complex
Factor X)
■ Factor IXa + cofactor VIIIa
➢ 2nd factor of a complex
■ Can activate Factor X to Factor
can act as a catalyst for
Xa
cleavage & activation

Kaolin ● Negatively charged substance in Main factor + cofactor → active form

platelet granular 2nd factor (catalyst)
Page 5
 Fibrinopeptide ● Short amino acid sequences on
Factor VIII ● Antihemophilic Factor A, soluble Fibrinogen molecule
● Pro-enzyme of Cofactor VIIIa +
serine protease Factor IXa, Ca2+ &
e. FORMATION OF FIBRIN CLOT
phospholipids
● Thrombin activates coagulation Factor XIII
● Synthesized in liver & carried in
blood bound to vWF
● Concentration: 50-150% Factor XIII ● Fibrin-stabilizing factor
● Pro-enzyme Factor XIIIa
Factor X ● Vitamin K dependent ● Activated by Thrombin (Factor
(Stuard-Prower pro-enzyme of serine protease IIa)
Factor) Factor Xa ● Provides cross-linking of D
■ Activating Factor II domains of Fibrin polymers
(Prothrombin) to Factor IIa ● Ensures fibrin net contracts
(Thrombin) in subsequently
prothrombinase complex w/ ● Makes fibrinogen in insoluble
Cofactor Va, Ca2+ & state
phospholipids ● Synthesized in Megakaryocytes,
Macrophages, placenta & liver
● Concentrations: 70-140%
c. FORMATION OF PROTHROMBINASE

Factor Xa + Cofactor Va → Prothrombinase

● Can cleave Prothrombin to Thrombin

Prothrombinase ● Complex of coagulation factors

activating Factor II (Prothrombin)
to Factor IIa (Thrombin)
● Formed by Factor Xa, Factor Va,
Ca2+ & phospholipids

Factor II ● Vitamin K dependent

(Prothrombin) pro-enzyme of serine protease
Thrombin ● Stable Fibrin clot
● Central component of plasmatic ■ Mainly consists of platelets,
coagulation cross-linked Fibrin strands &
● Not found in free form in Erythrocytes
bloodstream ➢ Erythrocytes are
● Synthesized in liver randomly entangled in
fibrin net & don’t
contribute to solidify/seal
d. FORMATION OF FIBRIN POLYMERS
clot
● Thrombin generated by Prothrombinase
complex can convert Fibrinogen to Fibrin
f. RETROACTIVE EFFECTS
● Fibrinogen molecule
● Not only linear processes but also reactive
■ 1 E domain –– has Fibrinopeptides A &
effects
B
● Advantage –– Self-reinforcing process
➢ Fibrinopeptides are
■ Generated Thrombin causes
cleaved off by Thrombin
Thrombin production to be boosted
to convert Fibrinogen to
● Disadvantage –– Model is inconsistent
Fibrin
■ Thrombin must activate other factors
■ 2 D Domains
before it has been produced

● Fibrin monomer are capable if binding

together to form Fibrin polymer

■ Resulting fibrin polymers are more

stable than soluble Fibrinogen

Page 6
***Ex. to activate Factors VIII & V, Thrombin is ■ Relatively small amount of Thrombin
required; thrombin is self-generated formed in initiation phase binds to
PAR on platelet membrane
g. ANTI-THROMBOTIC SYSTEM ➢ Supports activation of
● Prothrombotic System platelet
■ Form oversized clots & unwanted ➢ Activation produces
thrombosis w/ fatal consequences negatively charged
● Anti-thrombotic System phospholipids expressed
■ Counter-regulating system on the outside of platelet
■ Balance between coagulation & membrane
anticoagulation ➢ Greatly accelerates the
■ Regulated by endothelial cells & coagulation process
certain plasma proteins
■ Opposite effects
➢ Anti-thrombotic
subsystem
➢ Partially included in
cascade model

III. CELL-BASED MODEL

● Most recent explanatory model of secondary
hemostasis
● More powerful than the cascade model ● Activation of Factors XI, V, VIII
● Provides better explanations of processes “in ○ Cofactor VIII bound to vWF is released
vivo” (ex. Injured vessels) by Thrombin activation
● Aim –– describe haemostasis among real ■ Factor VIIIa sticks to platelet
physiological conditions surface
■ vWF binds to vWR and
promotes further platelet
PHASES adhesion
1st Initiation

2nd Amplification

3rd Propagation

a. INITIATION
● On smooth muscle cell & fibroblast exposed
to blood by injury
● Formation of Intrinsic Tenase
● Reaction of factors occur on surfaces of
○ Activation process
damaged cell membranes
■ Factor XIa → Factor IX → Factor
■ Most coagulation factors circulating in
IXa + Factor VIIIa (Intrinsic
the blood would have been
Tenase) → Factor X → Factor Xa
deactivated by certain antagonists
➢ Factor Xa is bound to
immediately after activation
negatively charged
surfaces of platelet via
1. TF released by vessel wall/cell damage
Ca2+
activates Factor VII –– forms Extrinsic Tenase
Complex
2. Extrinsic Tenase activates Factor X
3. w/o Cofactor Va, activated Factor Xa can
activate a small amount of Prothrombin to
Thrombin
4. Thrombin attaches to surface of adjacent
platelets
○ Small amount of Thrombin triggers
much more powerful intrinsic system

● 1st Thrombin Generation ○ Intrinsic Tenase is 50x more effective

○ In extrinsic system serves as initial than Extrinsic Tenase in activating
spark Factor X
○ Thrombin is not immediately ■ Expansion of Secondary
deactivated in open blood plasma Haemostasis to platelet
membrane results in dramatic
Factor VIIa ○ Vitamin K dependent serine
amplification effect
protease activating Factors IX, X, VII,
c. PROPAGATION
II, X
● Thrombin Burst
○ Synthesized in liver
○ Factor Xa binds to Cofactor Va
○ Concentration: 60-170%
○ Resulting Prothrombinase complex
activates Prothrombin to further
b. AMPLIFICATION Thrombin on platelet surface
● Activation of Platelet Membrane
Page 7
 ■ Prothrombin is up to 1000x
more effective than Factor Xa
in Extrinsic System (w/o
cofactor)
○ Process ^^^
demonstrates the
activation of Cofactors VIII & V
contributing the essential complexes
to boost coagulation process
○ Result –– more Thrombin is generated
on platelet membrane = “Thrombin
burst”
○ Thrombin cleaves corresponding
amounts of Fibrinogen to Fibrin ––
Fibrin strands
■ New Thrombin activates
additional quantities of factors
indirectly required for its
generation
➢ Results in continuous
propagation
(self-reinforcing process)
● Formation of Fibrin Thrombus
○ Fibrin polymerization leads to a Fibrin
net that connects platelets &
Erythrocytes to form a red thrombus
○ Thrombin activates Factor XIII
■ Lead to cross-linking of Fibrin
polymers
*** Cell-based model does not have a common pathway (ex.
Waterfall model) since the low volumes of Thrombin produced
from the Extrinsic System is not able to convert sufficient volume
of Fibrinogen into Fibrin. Extrinsic pathway serves mainly to
activate much more powerful intrinsic pathway.
d. JOSSO LOOP
● Extrinsic Tenase w/ TF & Factor VIIa is directly
activates Factor X & Factor IX (part of Intrinsic
Tenase)
● Activation path
○ “Short-cut” for acceleration
○ Directly activated Factor IX can form
Intrinsic Tenase complex more rapidly
w/ Cofactor VIII
■ Bypassing Factor XI
● Intrinsic Tenase activates Factor X at platelet
membrane
○ Accelerating self-reinforcing loop of
Thrombin generation
e. NEGATIVE CHARGE OF PLATELET MEMBRANE
● When activating the platelet, membrane is
rebuilt so it receives a negative charge
● Platelet membrane
○ Phospholipid bilayer
○ Non-activated platelets:
■ Outer layer: uncharged lipids
■ Inner layer: negatively charged
lipids
○ Due to activation –– negative charged
lipids swap w/ uncharged lipids
(Flip-flop mechanism)
■ Leads to negative charge on
entire outside of membrane
during platelet activation
f. ADVANTAGES OF BINDING
● Negative charge –– binding certain
coagulation factors
*** Activation of coagulation factor by separation of a molecule
requires a certain specific angle of the Factor & its activator
● Bound to platelet membrane, factors are
aligned accordingly
○ Activation & coagulation process on
2D plane is faster
■ Meeting on the 2D plane is
more efficient than right angle
by accident in 3D space
g. CALCIUM AS MEDIATOR
● Binding of 2 negatively charged substances
requires a positively charged mediator
● Calcium –– circulating in blood
○ 1 pole: binds to surface of platelet
○ 1 pole: binds to coagulation factor
● Included as Factor IV in series of coagulation
factors
● Factors IXa & VIIIa utilize Ca2+ to bind to
negatively charged phospholipids of cell
membrane
● Prothrombinase complex of Factor Xa & Va
contains Ca2+ & phospholipids
h. FORMATION OF NEGATIVE CHARGES
● Factors X, IX, VII & II are synthesized in liver &
initially not negatively charged
○ The become negatively charged
retrospectively by Post-Translational
Modification
■ Factors is supplemented by a
Carboxy group (COOH) →
separation of hydrogen cation
→ negatively charged
■ Carboxylation
➢ Adds negatively charged
carboxy group to existing
Glutamate chain end of
factor
Page 8
 inhibit Cofactors Va &
VIIIA
■ Proteolysis: Inactivates
coagulation Factor Va by
cleaving the molecule
into 2 inactive parts
■ Inactivates Factor VIIIa
○ Converts the central Factor
Thrombin into anticoagulant
■ Triggered by
Thrombomodulin on
surface if adjacent,
uninjured endothelium

i. IF CARBOXYLATION FAILS
● Carboxylation of coagulation factor results
OTHER PHYSIOLOGICAL ANTICOAGULANTS
from enzyme Gamma-Glutamyl Carboxylase
● Process requires Vitamin K cofactor
Protein ○ Most important inhibitor
○ No Vitamin K → No carboxylation →
Antithrombin ○ Inhibits Factor Xa & Thrombin
coagulation is inhibited
■ Absence causes failure in
α1 - Antitrypsin ○ Inhibits Thrombin & Plasmin
carboxylation → failure to
produce negatively charged α2 - Macroglobulin ○ Inhibits Thrombin & Plasmin
group → coagulation factor
○ Synthesized in large volumes
can’t bind to negatively
in liver & maintaining
charged phospholipids via Ca2+
secondary haemostasis

Plasmin ○ Serine protease cleaving

Fibrin net into the
water-soluble Fibrin
degradation products
○ Interacts w/ Factor V,
Fibrinogen & Factor VIII
○ Activated from Plasminogen
by t-PA & u-PA

k. HAEMOPHILIA
● Most important disease
● Genetic condition mainly in men
j. ANTI-THROMBOTIC SYSTEM ● Patient has pronounced bleeding tendency
● Counterbalancing System –– to prevent body due to a coagulation disorder
from uncontrolled Fibrin formation ● Laboratory Tests:
○ To localize Fibrin formation at area of Haemophilia A Haemophilia B
vessel wall damage (Factor VIII (Factor IX Deficiency)
Deficiency)
Thrombomodulin ○ Anticoagulant
■ Thrombomodulin
FVIII Assay FIX Assay
modifies Thrombin from
pro-coagulant to an l. APC RESISTANCE
anticoagulant enzyme ● Congenital disorder
○ Cofactor of Thrombin ● Factor V is modified, once activate, it can be
○ Protein acting as receptor for significantly degraded by physiological
Thrombin on surface of anticoagulant APC
uninjured endothelial cells ● Excessive thrombosis formation
○ Binds & inactivates Thrombin ● Laboratory Tests:
preventing the conversion of Protein C APC Assay FV Assay
Fibrinogen to Fibrin Assay
■ Reduces the amount of
free Thrombin in blood
m.EFFECTS OF DRUGS
Protein C/S System ○ Complex of Protein C &
Protein S to inhibit Cofactors Marcumar, Falithrom ○ Depending on the dose,
Va & VIIIA & Warfarin they block Vitamin K &
○ Thrombomodulin modifies prevent carboxylation of
bound Thrombin by a Factors VII, IX, X &
Thrombin
conformational change that
Thrombin activates free ○ PT Test
circulating Protein C ■ Therapeutic
○ Complex of Protein C monitoring by
(APC) & Protein S to measuring

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 a. ACTIVATORS OF FIBRINOLYTIC SYSTEM
Prothrombin Time
(PT Test) A. tissue Plasminogen Activator (t-PA)
● Formed in the endothelial cells
Direct-acting Oral ○ Rivaroxaban & Apixaban ● Converts Plasminogen to Plasmin,
Coagulation Inhibitors ■ Direct inhibitors of which degrades Fibrin
(DOAC) Factor Xa
B. Urokinase (u-PA)
■ Anti-Xa Assays
● First detected in urine
● In the blood, it acts as a general
○ Dabigatran activator of Plasminogen to Plasmin
■ Direct inhibitors of ● Manufactured by pharmaceutical
Thrombin companies as a drug to be used for
■ Direct Thrombin
thrombolytic therapy
Assays

b. ANTI-FIBRINOLYTIC SYSTEM
● Antagonists of the fibrinolytic system

Plasminogen ○ Prevents the Fibrin

Activator Inhibitor degradation
type 1 (PAI-1) ○ Formed in the endothelium
and in the platelets

Alpha ○ Serine protease produced in

2-Antiplasmin the liver
(α2-Antiplasmin) ○ Forms a complex with
FIBRINOLYSIS
Plasmin
● Breaking down Fibrin clot after the wound ○ Prevent Plasmin from
healed breaking down the Fibrin net
● Fibrinolytic system ○ Irreversible
- Enzymes triggering and proceeding
the degradation of the fibrinolysis Thrombin-activata ○ Produced in the liver
ble Fibrinolysis ○ Activated by Thrombin
- continuously degrades no longer
Inhibitor ○ Inhibits the binding of t-PA
required / excessive Fibrin
(TAFI) and Plasminogen to Fibrin
● Secondary haemostasis
● Promotes formation of Fibrin Alpha ○ Inhibitor of Plasmin and
● Occurs simultaneously with 2-Macroglobulin Thrombin
fibrinolysis in the vascular system (α2-Macroglobulin) ○ Appears as acute phase
● Regulated by activating or inhibiting the protein
Plasmin
c. SUITABLE BLOOD ASSAYS
Plasmin
● D-dimer assay
○ Key enzyme
○ Quantity of cross-linked Fibrin
○ Activated from its precursor
currently degraded in the body
Plasminogen by t-PA and u-PA
○ Elevated levels = increased level of
○ Serine protease cleaving the
coagulation activity
Fibrin polymer or Fibrin net into
■ Exclusion of thrombosis
soluble peptide fragments
➢ The most common
○ These peptide fragments give rise
indication to determine the
to D-dimers, the E-Fragments,
D-dimer level
and the larger complexes
➢ Not often used as the single
○ Also interacts with Factor V,
criterion
Fibrinogen, and Factor VIII
■ Blood tests
D Dimer ○ Product of the Fibrin cleavage by ➢ Related to the
Plasmin anti-fibrinolytic system
○ Elevated levels = pro-coagulation ➢ PAI assay, alpha
activities in the human body 2-Antiplasmin assay, TAFI
assay

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 TEST YOURSELF

PRIMARY HAEMOSTASIS SECONDARY HAEMOSTASIS - CASCADE

SECONDARY HAEMOSTASIS - CELL-BASED FIBRINOLYSIS

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