e d

e r
A presentation on
s t
B.Sc. Semester - II, Core Course – IV: ZOO 202 – C,
g i
Animal Physiology, Endocrinology : Unit – 4(3)
r e
n
HAEMOSTASIS3
U
. 5
(Blood clotting system, Kiningogen Kini 5
r
system, Fibrinolytic system)

r t e
v e
o n Dr O. Hemchandra
Assistant Professor
C
F
Department of Zoology

D Kumbi College, Kumbi

P
b e
d o
A
HEMOSTASIS e d
e r
- Heme = blood; -stasis = to stop
s t
g i
•
r e
It is the process of forming clots in the wall of
damaged blood vessels & preventing blood n loss while
maintaining blood in a fluid state withU in the vascular
system.
5 3
5 .
e
• Defects in hemostasis can lead r to an increased risk of
bleeding (hemorrhage) orr t
clotting (thrombosis).
v e
STAGES nOF HEMOSTASIS
o
C
F
D
Fibrinolysis
P of Platelet Plug
b e
Formation
Formation of blood clot
d o Vascular Constriction
A
 e d
Events in Hemostasis
e r
Vascular Constriction
s t
• Damaged blood vessels constrict
g i
• The vascular system has procoagulant, anticoagulant, and
r e
fibrinolytic properties and is made up of blood vessels.
n
• The innermost lining of the blood vessels is made up of U
3
endothelial cells (ECs) which form a smooth, unbroken surface
5
that promotes the fluid passage of blood and prevents
5 .
r
turbulence that may trigger activation of platelets and plasma
e
proteins.
r t
•
e
The ECs are supported by a collagen- rich basement
v
• o n
membrane and surrounding layers of connective tissues.
A breakdown in the vascular system is rapidly repaired to
C
maintain blood flow and the integrity of the vasculature.
• F
The vascular system prevents bleeding through vessel
D
P
contraction, diversion of blood flow from damaged vessels,

b e
initiation of contact activation of platelets with aggregation, and
contact activation of the coagulation system.

d o
A
 e d
Formation of platelet Plug
e r
• Platelets adhere to damaged endothelium to form platelet plug (primary
s t
hemostasis).
g i
•
r
Platelets are activated by collagen located in the basement membrane.
e
• The ECs secrete vWF, which is needed for platelet adhesion to
n
exposed subendothelial collagen in the arterioles.
U
• The ECs produce a variety of other adhesion molecules,
5 3
•
.
which include P-selectin, intercellular adhesion molecules (ICAMs), and
5
platelet endothelial cell adhesion molecules (PECAMs).
•
e r
The smooth muscle and fibroblast release tissue factor (TF), which
activates factor VII (FVII).
r t
•
v e
The vascular system provides potent anticoagulant properties, which

• o n
prevents the initiation and propagation of the coagulation process.
Coagulation is inhibited through the expression of thrombomodulin
C
(TM), which promotes activation of protein C and heparan sulfate (HS),
F
which activates antithrombin III (AT-III) to accelerate thrombin
inhibition. D
• P
Endothelial cells also release tissue factor pathway inhibitor (TFPI),

b e
which blocks activated factor VIIa (FVIIa)-TF/factor Xa (FXa) complex
o
and annexin V, which prevents binding of coagulation factors.
d
A
 e d
Blood Coagulation
e r
• Clots form upon the conversion of fibrinogen to Fibrin, and its addition

s t
•
to the platelet plug (secondary hemostasis).
The coagulation system is where coagulation factors interact to form a g i
fibrin clot. The coagulation system is involved in the conversion of
r e
soluble fibrinogen, a major component of the acute inflammatory n
exudates into fibrin. U
•
5 3
The fibrin clot reinforces the platelet plug formed during primary
hemostasis.
5 .
•
r
Various protein factors present in the inactive state in the blood
e
participate in the coagulation system.
r t
•
e
The protein factors are designated by Roman numerals according to

v
their sequence of discovery and not by their point of interaction in the
coagulation cascade.
o n
•
C
Some of the coagulation factors such as fibrinogen and prothrombin

F
are referred to by their common names, whereas others such as factors

D
VIII and XI are referred to by their Roman numeral nomenclatures.
•
P
Activation of a factor is indicated by the addition of low case “a” next to

e
the Roman numeral in the coagulation cascade such as VIIa, Xa, XIIa.
b
d o
A
 e d
• Some of the common names were derived from the original
e r
patients in whom symptoms leading to the determination of the
s t
factor deficiency were found.
g i
• Examples are the Christmas factor and Hageman factor.
r e
• The coagulation factors may be categorized into substrates,
n
cofactors, and enzymes. Fibrinogen is the main substrate.
U
•
3
The cofactors accelerate the activities of the enzymes, which are
5
involved in the coagulation cascade.
5 .
•
r
Examples of cofactors include tissue factor, factor V, factor VIII,
e
t
and Fitzgerald factor. With the exception of factor XIII, all the
r
• v e
enzymes are serine proteases when activated.
The coagulation factors may also be categorized into 3 groups on

o n
the basis of their physical properties.
• C
These groups are the contact proteins comprising of factors XII,
F
XI, prekallikrein (PK), and high molecular weight kininogen
D
(HMWK); the prothrombin proteins comprising of factors II, VII,
P
IX, and X; and the fibrinogen or thrombin sensitive proteins

b e
comprising of factors I, V, VIII, and XIII.

d o
A
 e d
Fibrinolytic System
e r
s t
i
• Fibrinolysis is the physiological process that removes insoluble fibrin
clots through enzymatic digestion of the cross-linked fibrin polymers.
e g
•
r
Plasmin is responsible for the lysis of fibrin into fibrin degradation
products, which may have local effects on vascular permeability. n
• U
Plasmin digests fibrin and fibrinogen through hydrolysis to produce
smaller fragments.
5 3
• .
The gradual process occurs at the same time that healing is occurring,
5
r
and eventually cells of the mononuclear phagocytic system

e
• r t
phagocytize the particulate products of the hydrolytic digestion.
Recent evidence suggest that the renin-angiotensin-aldosterone
system
v e
• n
(RAAS) may participate in the regulation of fibrinolytic function.
o
C
• Angiotensin II (Ang II) is the primary candidate to mediate this
interrelationship, since this peptide is capable of stimulating
F
D
plasminogen activator inhibitor-1 (PAI-1) in vitro and in vivo.
•
P
It has been suggested that aldosterone may also modulate fibrinolysis,

•
b e
possibly by interacting with Ang II.
Fibrinolysis is controlled by the plasminogen activator system.

d o
A
 e d
• The proteolytic activity of this system is mediated by plasmin,
e r
which is generated from plasminogen by 1 of 2 plasminogen
s t
activators.
g i
• Inactive plasminogen circulates in plasma until such a time that
r e
an injury
n
• occurs. Then, plasminogen is activated by means of a number of
U
proteolytic enzymes known as plasminogen activators.
5 3
•
.
These activators are present at various sites such as the
5
r
vascular endothelium. Some of the activators include tissue-type
e
plasminogen activator, urokinase,
r t
streptokinase, and acyl-

• v e
plasminogen streptokinase activator complex.
Inhibitors of fibrinolysis include α2- plasmin inhibitor, tissue

o n
plasminogen activator inhibitor, and plasminogen activator
inhibitor-1 (PAI-1). C
• F
Individuals with reduced fibrinolytic activity are at increased risk
D
for ischemic cardiovascular events, and reduced fibrinolysis may
P
underlie some of the pathological consequences of reduced nitric

b e
oxide (NO) availability.

d o
A
Coagulation Factors
e d
Factor Common name Function Pathway
e r
s t
Participation
I Fibrinogen Thrombin substrate,
g i
Common
polymerizes

r e
n
II Prothrobin Serine protease Common
III Tissue factor Cofactor
U
IV Ionic calcium Mineral

5 3
V
VI
Labile factor
Stable factor 5 .
Cofactor
Serine protease
Common
Extrinsic
VII Antihemophiliac factor
e r
von Willebrand Cofactor
r t Intrinsic
factor (vWF)
v e
IX
X o n
Christmas factor
Stuart Prower factor
Serine protease
Serine protease
Intrinsic
Common
XI C
Plasma thromboplastin Serine protease Intrinsic
F
antecedent (PTA)
XII D Hageman factor Serine protease Intrinsic
XIII P Fibrin stabilizing factor Trasglutaminase Common

b
High moleculare Fitzgerald factor, HMWK Cofactor Intrinsic

o
weight kininogen

d
Platelet factor 3 Phospholipids, PF3 Assembly molecule
A
Platelets
e d
• Platelets are anuclear fragments derived from the bone marrow
e r
s t
i
megakaryocytes. They have a complex internal structure, which
reflects their hemostatic functions.
e g
•
r
The 2 major intracellular granules present in the platelets are the α-
granules and the dense bodies. n
• U
The α-granules contain platelet thrombospondin, fibrinogen, fibronectin,
platelet factor 4, vWF, platelet derived 3
growth
5
factor, β-
thromboglobulin, and coagulation factors V and VIII.
5 .
•
r
The dense granules contain ADP, adenosine triphosphate (ATP), and

e
•
serotonin.
r t
When stimulated, platelets release both the α-granules and the dense

v e
bodies through the open canalicular system.
• n
When platelets aggregate, they expend their stored energy sources,
o
C
lose their membrane integrity, and form an unstructured mass called a
syncytium.
F
D
• In addition to the plug formation, platelet aggregates release micro-

P
platelet membrane particles rich in phospholipids and various

b e
coagulation proteins which provide localized environment that support
plasma coagulation.

d o
A
• Platelets and ECs have biochemical pathways involving the
e d
metabolism of arachidonic acid (AA), which is released from
e r
membrane phospholipids by phospholipase A2.
s t
• Subsequently, cyclooxygenase converts AA to cyclic endoperoxides.
g i
• The endoperoxides are then converted by thromboxane synthetase to
r e
thromboxane A2.
n
• U
Thromboxane A2 is a potent agonist that induces platelet aggregation.
3
Endothelial cells also contain AA and preferentially convert cyclic
5
.
endoperoxides to prostacyclin, which is a potent inhibitor of platelet
5
r
aggregation.
•
t e
During primary hemostasis, platelets interact with elements of the
r
damaged vessel wall leading to the initial formation of the platelet plug.
•
v e
The platelet/injured vessel wall interaction involves a series of events

o n
that include platelet adhesion to components of the subendothelium,
activation, shape change, release of platelet granules, formation of
C
fibrin stabilized fibrin platelet aggregates, and clot retraction.
• F
In this process, the activation of platelets with exposure of negatively
D
P
charged phospholipids facilitates the assembly of coagulation factors

b e
on the activated platelet membrane, leading to the generation of
thrombin and subsequent fibrin deposition.

d o
A
 e d
Platelet Function
e r
s t
• In some disorders, platelets may be normal in number, yet hemostatic
plugs do not form normally, and therefore, bleeding time will be long. g i
•
r e
Platelet dysfunction may stem from an intrinsic platelet defect or from an
n
extrinsic factor that alters the function of otherwise normal platelets.
• U
Defects may be hereditary or acquired. Tests of coagulation phase of

5 3
hemostasis such as activated partial thromboplastin time (APTT) and

5 .
prothrombin time (PT) are normal in most circumstances but not all.
•
r
When a patient’s childhood history reveals easy bruising and bleeding
e
t
after tooth extraction, tonsillectomy, or other surgical procedures, the
r
v e
finding of normal platelet count but a prolonged bleeding time suggests
a hereditary disorder affecting platelet function.
•
o n
The cause is either vWD (which is the most common cause of

C
hereditary hemorrhagic disease) or a hereditary intrinsic platelet
disorder.
F
•
D
Whatever the cause of platelet dysfunction, drugs that may further

P
impair platelet function should be avoided such as aspirin and other non-

e
steroidal antiinflammatory drugs (NSAIDs).
b
d o
A
Thrombocytopenia e d
e r
s t
• Thrombocytopenia may be the consequence of failed platelet
production, splenic sequestration of platelets, increased platelet
g i
destruction, or dilution of platelets.
r e
• n
Regardless of the cause, severe thrombocytopenia usually results in a
U
typical pattern of bleeding such as multiple petechiae in the skin,

5 3
scattered small ecchymoses at the sites of minor trauma, mucosal
bleeding, and excessive bleeding after surgery.
5 .
•
r
Heavy gastrointestinal (GI) bleeding and bleeding into the central
e
t
nervous system (CNS) may be life threatening.
r
•
e
However, thrombocytopenia does not cause massive bleeding into

v
tissues, which is characteristic of bleeding secondary to coagulation
disorders.
o n
•
C
Adult idiopathic thrombocytopenic puerperal (ITP) usually results from

F
development of an antibody directed against a structural platelet
antigen.
D
•
P
In childhood ITP, viral antigen is thought to trigger synthesis of an

b
surface.
e
antibody that may react with viral antigen associated with the platelet

d o
A
• Platelet count is usually maintained in a range of 150,000 to 400,000/
e d
μL and counts of 100,000 to 150,000/μL are regarded as borderline for
e r
thrombocytopenia while counts that are less than 100,000/μL are
s t
considered abnormal.
g i
•
r
Symptoms do not usually develop until the platelet count is less than
e
n
50,000, at which time easy bruising may be evident and petechiae may
appear on the skin.
U
•
3
Surgeons usually do not perform routine surgery on patients whose
5
5 .
platelet counts are <50,000/μL because the risk of prolonged bleeding
after dental procedures or childbirth will be increased.
•
e r
When the platelet count reaches 10,000 to 20,000/μL, the risk of
spontaneous and serious bleeding rises.
r t
•
v e
This includes strokes, GI bleeding, and prolonged nose bleeds. When

the bleeding. o n
these conditions develop, platelet transfusions are often used to stop

• C
Unfortunately, transfused platelets are short-lived and cannot be used
F
indefinitely as antibodies may develop against the platelets.
D
P
• Platelet transfusions are most appropriate when the cause of
thrombocytopenia is a temporary lack of production such as after

b e
intensive
• o
chemotherapy.
d
A
 e d
Keninogen Kinin System
e r
• The kinins are peptides of 9 to 11 amino acids of which the most
s t
important
g i
e
• vascular permeability factor is bradykinin (BK).
• The kinin system is activated by coagulation factor XII.
n r
U
• Bradykinin is also a chemical mediator of pain, which is a cardinal
feature of acute inflammation.
•
5 3
Therefore, bradykinin is capable of reproducing many of the

5 .
characteristics of an inflammatory state, such as changes in local blood

r
pressure, edema, and pain, resulting in vasodilation and increased
e
microvessel permeability.
r t
•
e
Human HMWK, a single-chain protein with a molecular weight of
v
n
120,000 daltons, is cleaved by human urinary kallikrein (HUK) to

o
release kinin from within a disulfide loop and form a 2-chain protein that

C
retains all the procoagulant activity of the native molecule.
•
F
It is a multifunctional protein, a parent protein of bradykinin, and serves

D
as a cofactor for FXI and PK assembly on biologic membranes.
• P
The docking of HMWK to platelet and EC membranes requires its
e
binding by regions on both its heavy and light chains.
b
d o
A
 e d
Serine Protease Inhibitors
e r
• It is becoming increasingly clear that coagulation augments

s t
inflammation and that anticoagulants, particularly natural
anticoagulants, can limit the coagulation induced increases in the g i
inflammatory response.
r e
• n
The latter control mechanisms appear to involve not only the inhibition
U
of the coagulation proteases but interactions with the cells that either

5 3
generate anti-inflammatory substances or limit cell activation.
•
5 .
Recent studies have demonstrated a variety of mechanisms by which

r
coagulation, particularly the generation of thrombin, FXa, and the
e
t
TF/FVIIa complex, can augment acute inflammatory responses.
r
•
e
Many of these responses are due to the activation of 1 or more of the
protease activated receptors.
v
•
o n
Activation of these receptors on endothelium can lead to the expression

C
of adhesion molecules and platelet activating factor, thereby facilitating

F
leukocyte activation.
•
D
Therefore, anticoagulants that inhibit any of these factors would be

P
expected to dampen the inflammatory response.
•
e
The 3 major natural anticoagulant mechanisms seem to exert a further
b
inhibition of these processes by impacting cellular responses.

d o
A
 e d
r
• Antithrombin has been shown in vitro to increase prostacyclin responses and
activated protein C has been shown to inhibit a variety of cellular responses
t e
including endotoxin induced calcium fluxes in monocytes, a key step in the
i s
•
generation of the inflammatory response.

e g
Serine proteases (such as thrombin, FXa, elastase, trypsin) are implicated in

n r
many clinical disorders such as emphysema, arthritis, and cardiovascular
diseases.
U
3
• Naturally occurring serine protease inhibitors (such as antithrombin) which are

5
involved in thrombin inhibition regulate these enzymes in normal physiological
.
•
conditions.

r 5
Serine protease inhibitors attach to various enzymes and inactivate them.
•
t e
Antithrombin was the first of the plasma coagulation regulatory protein to be
r
identified and the first to be assayed routinely in the clinical laboratory.
•
v e
Other members of the serine protease inhibitor family are heparin cofactor II, α1-

• o n
antitrypsin, and α2-macroglobulin.
More than 90% of the antithrombin activity of normal plasma is derived from AT-
III. C
•
F
Antithrombin-III has been shown to exert marked anti-inflammatory properties

D
and proven to be efficacious in experimental models of sepsis, septic shock, and

P
disseminated intravascular coagulation (DIC).
•
e
Antithrombin- III also inhibits factors XIIa, XIa, IXa, protein S, protein C, plasmin,
b
and kallikrein.

d o
A
 e d
Complement System
e r
• Complement has an important role in inflammation and in the
s t
normal function of the immune system.
g i
• Activated complement fragments have the capacity to bind and
r e
damage self-tissues. n
• On their surfaces, cells express regulators of complement U
5 3
activation that protect the cell from the deleterious effects of
cell-bound complement fragments.
5 .
•
r
Abnormalities in these regulators may participate in the
e
r t
pathogenesis of autoimmune diseases and inflammatory
disorders.
v e
•
n
The complement system consists of approximately 22 serum
o
proteins, which together with antibodies and clotting factors
C
perform an essential role as mediators of both immune and
allergic reactions. F
D
•
P
Complement protein are involved in reactions which lead to the

e
lysis of cells.
b
d o
A
 e d
• This is due to the production of the membrane attack complex
e r
(MAC). The activation of complement may follow the classical
s t
pathway or the alternative pathway.
g i
• Complement is activated by plasmin through the cleavage of C3
r e
into C3a
n
• and C3b. U
• C3a is an anaphylotoxin that causes increased vascular
5 3
.
permeability via degranulation of mast cells leading to the
5
r
release of histamine. C3b is an opsonin that causes immune
e
adherence.
r t
•
e
During reperfusion, complement may be activated by exposure
v
intermediate filaments. o n
to intracellular components such as mitochondrial membranes or

• C
In order to protect themselves from the complement attack, cells
F
express several regulatory molecules including the terminal
D
complex regulator CD59 that inhibits assembly of the large
P
b e
MACs by inhibiting the insertion of additional C9 molecules into
the C5b-9 complex.

d o
A
Activated Partial
e d
Thromboplastin Time
e r
s t
• Activated partial thromboplastin time was developed from the
observation that hemophiliacs have prolonged clotting time. g i
r e
• However, when tissue thromboplastin is added, the plasma
n
clots as normal plasma does.
U
•
3
Thromboplastins are lipoproteins. They may be classified as
5
.
either complete or partial, which means that they consist of
5
only phospholipids.
e r
•
t
Addition of negatively charged activators to the system results
r
e
in significantly shorter clotting times.
v
•
n
It is the most widely used test for screening for factor
o
deficiencies in the intrinsic and common pathways.
• C
The APTT reflects the activity of PK, HMWK, and factors XII,
F
D
XI, VIII, X, V, II, and I. The APTT may be prolonged due to

P
either a factor decrease or the presence of circulating
e
anticoagulants.
b
•
o
The normal APTT is less than 35 seconds.
d
A
 e d
Prothrombin Time
e r
• Prothrombin time is the routine test used to screen for
s t
deficiencies of factors I, II, V,VII, and X. It is the test of choice for
g i
monitoring anticoagulant therapy by vitamin K antagonists.
r e
•
n
Three of the 5 factors measured by PT (II, VII, X) are sensitive to
and depressed by these anticoagulants.
U
•
3
4 Prothrombin time is widely utilized for evaluation of diseases
5
severe liver dysfunction and DIC. 5 .
with single or multiple coagulation factors disorders, such as

e r
•
t
However, its standardization of reagents and method is not
r
v e
established yet for universal purpose except International
Normalized Ratio (INR) for control of oral anticoagulant therapy
(OAT).
o n
•
C
Oral anticoagulants have been widely employed to decrease

F
thrombotic risk by reducing the levels of vitamin K-dependent
D
clotting factors.
• P
The use of oral anticoagulants also decreases the levels of

b e
natural anticoagulants such as protein C and protein S.

d o
A
 e d
• The PT test investigates the production of thrombin and the
e r
s t
formation of fibrin via the extrinsic and common pathway.
• In the presence of calcium ions, tissue thromboplastin g i
complexes with and activates FVII. This provides surfaces r e
n
for the attachment and activation of factors X, V, and II.
U
Normal values for PT range from 10 to 13 seconds.
5 3
• Values for the INR are preferable to the PT because
5 .
different thromboplastin reagents have
e r different

r t
sensitivities to warfarin-induced changes in levels of
clotting factors.
v e
n
• The INR corrects for most but not all of the reagent
o
C
differences expressed as international sensitivity index

F
(ISI), which is a correction factor assigned by the
D
manufacturer of the thromboplastin reagent.
P
• The problems associated with the INR are that the concept

b e
and reasons for use are poorly understood and the value is

d o
generally misused.
A
 e d
e r
Fibrinogen and Fibrinogen
s t
Degradation Products
g i
r e
• Fibrinogen levels are useful in detecting n deficiencies of
fibrinogen and alterations in the conversion U of fibrinogen to
fibrin. The normal value for fibrinogen3ranges from 200 to
. 5
400 mg/dL. 5
• This may be decreased in liver rdisease or the consumption
r t
of fibrinogen due to accelerated
e intravascular coagulation.
v
• An increased concentration e of fibrinogen degradation

o n
products (FDPs) is commonly used in conjunction with other
C
hemostatic test abnormalities to identify patients with DIC.
F
D
P
b e
d o
A
 e d
e r
Investigation of Disseminated
s t
Intravascular Coagulation
g i
r e
• A variety of tests are used to detect DIC. The most n sensitive tests are
markers of endogenous thrombin generation. U
• In the practical management of patients, cruder
5 3 measures of DIC are
often used. .
• Some of these tests include screening5tests such as PT and APTT,

e r
r t
which may be prolonged reflecting consumption of many coagulation

e
proteins.

n v
• Plasma concentrations of coagulation
as fibrinogen, FV, and FVIII,
proteins consumed in DIC, such
all show decreases in concentration.
o
C and fibrin monomer may be present.
• Fibrinogen/FDPs or D-dimers
increased in concentration
(a fragment from fibrin alone) are both

thrombinF clotting
• The
D and the presence of FDPs.20
time may be prolonged reflecting

P
hypofibrinogenemia

b e
d o
A
Conclusion
e d
• Hemostasis involves the stoppage of bleeding following an
e r
s t
•
injury to the vasculature.
The various systems work together to maintain the integrity of g i
this process and prevent what would otherwise be a traumatic r e
n
reaction.
U
•
3
A delicate balance is maintained between all of the systems that
5
•
are involved in the hemostatic process.
5 .
A derangement of this delicate balance may result in adverse
outcomes for the patient.
e r
• r t
For centuries, there have been conceited efforts to understand

v e
the coagulation process and design accurate methods for
n
evaluation and monitoring of this complex process.
o
•
C
New understanding has been acquired, and today clinical

F
laboratories provide physicians with very useful information with

D
which to diagnose cases of coagulation derangement.
• P
It is hoped that the clinical laboratory will continue to be at the
e
forefront in the elucidation of the intricate processes that
b
o
constitute hemostasis.
d
A