Hema Chapter 24_Hemostasisnnnnnnnnnnnnnnnn (2).ppt

Chapter 24
Hemostasis and
disorders of coagulation

Objectives
At the end of this chapter, the student will be able to:
 Describe normal and abnormal hemostasis
 Discuss how the components of normal hemostasis interact
with each other to bring about normal blood flow with in the
vascular system
 Explain the intrinsic and extrinsic pathways of blood
coagulation
 Discuss the normal control of the clotting process and the
fibrinolytic system

Objective cont’d
 State the principles of the different tests of the bleeding
disorders
 Perform the different tests of the bleeding disorders
 Indicate the normal values of the different tests of the bleeding
disorders

outline
 Introduction to hemostasis
 Components of coagulation
 Vascular system
 Platelets
 Coagulation factors
 Fibrinolysis
 Bleeding and coagulation disorders
 Laboratory diagnosis of Bleeding and Coagulation Disorders
 Bleeding time test
 Whole blood coagulation time test
 Clot retraction time
 Prothrombin time test with INR
 Partial thromboplastin time
 Thrombin time
 Fibrinogen Assay
 D-Dimer

1 Introduction
Hemostasis (Haima= blood and stasis=arrest)
 is a complex process which continually ensures prevention of
spontaneous blood loss
 is the arrest of bleeding
 stops hemorrhage caused by damage to the vascular system.
 is initiated by vascular injury and culminates in the formation
of a firm platelet-fibrin barrier that prevents the escape of
blood from the damaged vessel

Definition…
 Hemostasis is defined as the process that maintains
the flowing blood in a fluid state and confined to the
circulatory system.
•The hemostatic mechanism is not a single biological
pathway, but the product of the complex interactions
of a number of distinct systems:
 vascular system;
 blood platelets;
 blood coagulation system;
 fibrinolytic system;
 inhibitors of the above systems.

COMPONENTS OF COAGULATION
 The vital physiological function involves three
components:
vessels and vascular coagulation factors
thrombotic: platelets and platelet coagulation
factors
 humoral: plasma factors, coagulation and
fibrinolysis activators and inhibitors.

phases
Generally, this is described as a three –phase
process
 division is arbitrary since the various phenomena are
strongly interrelated:
I . primary hemostasis
II. secondary hemostasis-coagulation
III. tertiary hemostasis-fibrinolysis

Phases cont’d
I. Primary hemostasis
 involves the blood vessels ( parietal or vascular
vasoconstriction phase and release of tissue or
exogenous factors)
 and the thrombocytes (platelate or endothelial –
thrombocyte phase, platelate aggregation and
release of platelate factors).
 After 3 to 5 minutes , blood flow is arrested with
the formation of a platelate plug.

Phases cont’d
II. Coagulation
 involves
plasma coagulation factors (plasma phase)
platelet factor 3
 provides for definitive hemostasis
 takes 5 to 10 minutes by formationof fibrin -
stable clot
 reinforces the platelet plug.
III Fibrinolysis
 essential final step in any hemostasis mechanism,
 enabling in 48 to 72 hours, and a
 return to normal by destroying fibrin and healing the
injured vessel.

I. Primary hemostasis
 Is a result of a three –way interaction between:
the vascular wall
the platelets
plasma glycoprotein
 Fibrenogen and
 vWF
 Triggered by
small injuries to blood vessels
the plasma glycoprotein factors desquamation /
damaging of epithelial cells in pinpricks

The vascular system
The Blood vessel wall
 first line of defense for normal hemostasis
 lined with endothelial cells and synthesize von
Willebrand factor (vWF) multimers
 vWF multimers are secreted into the circulation or
onto the collagen-containing subendothelium.
 Following endothelial cell damage and
subendothelial exposure, platelets bind to vWF
multimers and collagen to initiate hemostasis.
 which form a tight selective membrane that keeps
blood cells and plasma inside the vessel

The vascular system cont.d
 Endothelial cells also produce a fibrinolytic
activator
 protective function to prevent blockage of
blood vessels by clots
 Nerve and muscular tissue in the supporting
sub endothelial (under the endothelium) tissue
allow
 constriction of the vessel when injured
(muscle cells contract)

 The blood vessels allowing the passage of
gases
nutrients
selected cells to enter or leave the system
 In the normal state, the endothelial cells
produce a substance called prostacyclin &
other substances
inhibit platelet function (cause disaggregation of
platelets).

Vasoconstriction:
 Constriction of small vessels such as arterioles,
venules,
capillaries slows or stops blood flow
Serotonin
Thromboxane A2
 is a powerful vasoconstrictor and platelet
aggregating agent

Vasoconstriction &Blood Flow
Vasoconstriction creates stasis
facilitating PLT activation and
promoting secondary hemostasis.
Blood flow controls hemostasis by
diluting and removing activated
factors.
Graphic accessed at URL http://www.walgreens.com/library/contents.jsp?docid=8983&doctype=2, 2008.

 Slowed bleeding  more effective platelet contact
activation  adhesion of platelets to the exposed
subendothelial tissue
 wound sealed and vascular lumen narrows closes and
blood flow to the injured site minimized
Note: this effect is temporary lasting up to 20
seconds needs to be supplemented by platelets and
blood coagulation factors

Platelets
Platelets are
 anucleated,
 cytoplasmic fragments of the megakarocyte
mother cell
 4 stages of development:
 Megakaryoblast,
 Promegakarocyte,
 Megakaryocyte,
 Thrombocyte
 Each megakaryocyte produces 2000-4000
platelets

Platelets cont’d
 Young platelets are
larger & less dense than older platelets.
 also metabolically active and more effective in
hemostasis
Platelet turn over rate equals 35,000  4,300 per
L each day
Size 2-20 fL;
2-4 m in diameter, colorless,
with Wright’s stain they stain blue with pink
granules

Hema Chapter 24_Hemostasisnnnnnnnnnnnnnnnn (2).ppt

Platelets cont’d
 Life span ~9 days (7-10 days)
 Production regulated by thrombopoietin (plus others)
 Normally 2/3 of the platelets released from the bone
marrow stay in the circulation;
 the remaining sequestered in a splenic pool that is
freely exchangeable with circulating platelets

Platelets cont’d
Function of platelets
 Maintain the functional integrity of the endothelial
surface
 Initially arrest bleeding by
forming temporary hemostatic platelet plug
 Provide phospholipids (Platelet factor 3)
acts as a catalytic surface for initiation of the
coagulation process
 When there is an injury platelets undergo the
following actions:
 Adhesion
 Aggregation
 Shape change
 Secretions

Adhesion
 It is the binding of platelet to non platelet surface: sub
endothelial collagen
 involves changes from a disc shape to a slightly
broader, plate like form to increase surface area
 a number of plasma proteins are required for normal
platelet adhesion.
 fibronectin and von Willebrand factor (vWF
 vWF
 is the largest component of factor VIII
 secreted by platelets and by vascular endothelial cells.
 Thrombin also stimulates platelet adhesion

Adhesion cont’d
Collagen – vWF –Platelet
 Bridge physical distance between platelate and sub
endothelial collagen
 Increase bond that seal platelet to the vessel wall
 reversible

Release reaction(secretions)
 It is release of contents of the granules of platelet
Primarily ADP  stimulates aggregation
Cathecolamine (especially epinephrine) and
serotoni enhance vasoconstriction
Platelets contain 3 types of secretary granules:
Lysosome containing acid hydrolyses
 α-granules containing platelate specific proteins
(Plt factor 4, β- thromboglobulin, as well as other
proteins such as Platelet derived growth factor and
coagulation proteins found in plasma (fibrinogen &
von Willebrand’s factor)
-granules containing ATP, ADP, Calcium &
serotonin

Aggregation
Platelet-Platelet interactions
Triggered by ADP
Need FIBRINOGEN to bridge platelate-to-
platelate distance and encourage platelate plug
Platelate fill the open space to form a plug
Platelates shed membranes rich in phospholipid
(appearance of PLT factor 3 on the PLT
membrane)
this happens during PLT plug formation
serves as a catalytic site for the coagulation
proteins
helps initiation of the coagulation mechanism
 Aggregation is also a response to thrombin and
thromboxane

Aggregation cont’d
platelet aggregation

Aggregation cont’d
 Platelet membrane phospholipase is activated during
aggregation.
 This releases arachidonic acid from Platelet
phospholipids
 Local Platelet activation stimulates a similar parallel
series of reactions in the vascular endothelium &
produce Prostacyclin (opposes TXA2)
 Balance is established between production of TXA2
by Platelet and Prostacyclin by the vascular
endothelium
 Thus, PLTs can seal the exposed vessel wall without
forming a large plug that would block circulation
 Formation of definitive fibrin clot is necessary to
ensure the repair of a damaged vessel

Cont..
•Cyclicendoperoxides
Cyclo-oxigenase
Arachidonic acidinhibited
by asprin

2. Secondary hemostasis (coagulation)
 In the coagulation or plasma phase , blood changes
from the fluid state to the gelled state , a result of the
transformation of a soluble protein , fibrinogen , into
an insoluble protein , fibrin .
 This forms the network around which the clot will be
formed. This change in state corresponds to a
cascade of enzyme activity whose first step have the
function of amplifying the entire process of fibrin
formation.
 This cascade requires a large number of protein
factors , most of which are present as pro enzymes
and which are transformed by partial proteolysis to
active forms.
 Procoagulants are the enzymes ( zymogens),
substrates and Co - factors

Enzymes( Serine Proteases)
Hydrolyze peptide bonds
Synthesised as inactive zymogen
 –cleaved at sites by another proteases
Activation is localized ( at sites of injury)

Cont..
Factor I (Fibrinogen)
 Large, stable globulin protein (mol wt 341,000)
 Is the precursor of fibrin, which forms the
resulting clot
 When fibrinogen is exposed to thrombin, two
peptides split from the fibrinogen molecule,
leaving a fibrin monomer. The monomers
aggregate together to form the final polymerized
fibrin clot product
Factor II (Prothrombin)
 Is a stable protein (mol wt 63,000)
 In the presence of ionized calcium, prothrombin is
converted to thrombin by the enzymatic action of
thromboplastin from both extrinsic and intrinsic sources

Cont..
Factor IIa (Thrombin)
 Has a half-life of almost 3 days with 70%
consumption during clotting
 Thrombin (mol wt 40,000) is the activated form of
prothrombin, which is normally found as an inert
precursor in the circulation
 This proteolytic enzyme, which interacts with
fibrinogen, is also a potent platelet-aggregating
substance
 A unit of thrombin will coagulate 1 ml of a standard
fibrinogen solution in 15 sec at 28oC

Cont..
Tissue thromboplastin
 Tissue thromboplastin is the term given to any non-
plasma substance containing lipoprotein complex
from tissues. These tissues can be from the brain,
lung, vascular endothelium, liver, placenta, or
kidneys; these tissue types are capable of converting
prothrombin to thrombin
Ionized calcium (formerly factor IV)
 The term ionized calcium has replaced the term factor IV
 Necessary for the activation of thromboplastin, and for
conversion of prothrombin to thrombin. Ionized calcium is the
physiologically active form of calcium in the human body and
only small amounts are needed for blood coagulation. A
calcium deficiency would not be expressed as a coagulation
dysfunction, except in cases of massive transfusion

Cont..
Factor V (Proaccelerin)
 Factor V is an extremely labile globulin protein. It deteriorates
rapidly, having a half-life of 16 hours. Factor V is consumed
in the clotting process and is essential to the later stages of
thromboplastin formation
Factor VII (proconvertin)
 Factor VII, a beta globulin, is not an essential component of
the intrinsic thromboplastin-generating mechanism. It is not
destroyed or consumed in clotting and is found in both plasma
and serum, even in serum left at room temperature for up to 3
days. The action of factor VII acceleration of the production of
thrombin from prothrombin. This factor is reduced by vitamin
K antagonists.

Cont..
Factor VIII (Antihemophilic Factor)
 This factor is consumed during the clotting process and is not
found in serum. Factor VIII is extremely labile, with a 50%
loss within 12 hours at 4oC in vitro and a similar 50% loss in
vivo within 8-12 hours after transfusion.
 Factor VIII can be subdivided into various functional
components; a low molecular weight fraction consists of the
von Willebrand factor (vWF).
Factor IX (Plasma Thromboplastin component)
 Factor IX is a stable protein factor that is neither consumed
during clotting nor destroyed by aging at 4oC for 2 weeks.
 It is an essential component of the intrinsic thromboplastin
generating system, where it influences the amount rather than
the rate of thromboplastin formation.

Cont..
Factor X (Stuart Factor)
 This -globulin is a relatively stable factor that is not
consumed during clotting. Together with factor V, factor X in
the presence of calcium ion forms the final common pathway
through which the products of both the extrinsic and intrinsic
thromboplastin-generating systems merge to form the ultimate
thromboplastin that converts prothrombin to thrombin.
 The activity of factor X appears to be related to factor VII
Factor XI (Plasma Thromboplastin Antecedent)
 Factor XI, a -globulin, can be found in serum because it is
partially consumed during the clotting process.
 These factor is essential to the intrinsic thromboplastin-
generating mechanism.

Cont..
Factor XII (Hageman factor)
 Factor XII is a stable factor that is not consumed during the
coagulation process.
 Adsorption of factor XII and kininogen (with bound
prekallikrein and factor XI) to negatively charged surfaces
such as glass or subendothelium (Collagen) exposed by blood
vessel injury initiates the intrinsic coagulation pathway.
 Surface absorption alters and partially activates factor XII to
factor XIIa by exposing an active enzyme (protease) site.
Because of a feedback mechanism, Kallikrien (activated
Fletcher factor) cleaves partially activated factor XIIa
molecules adsorbed onto the subendothelium to produce a
more kinetically effective form of XIIa.
Factor XIII (Fibrin-Stabilizing Factor)
 Fibrin-stabilizing factor in the presence of ionized calcium
produces a stabilized fibrin clot

Cont..
 The clotting mechanism responsible for the formation of fibrin
involves a cascade of reactions in which inactive enzymes
(zymogens) are activated, and the activated enzymes in turn
activate other inactive enzymes.
 Plasma coagulation factors have various names but an
internationally standardized nomenclature system is using
Roman numeral designations. A lower case “a” indicates the
active factor (e.g. factor IXa)
 Roman numerals indicate inactive forms as they exist in the
plasma except factors III & IV.
 They reflect order of discovery but not the sequence of
reaction in the coagulation system

Cont..
Can be grouped as
 Fibrinogen group
 Thrombin sensitive
 I, V, VIII,XIII
Prothrombin group
- Vitamin-K dependent
- II,VII,IX and X
Contact group
- XI, XII, PK, HMWK

 Additional components:
Ca 2+, v W F, Phospholipid
Coagulation reaction occur on the surface of
platelate phospholipid ( Platelate Factor 3) or
endothelial cell membrane (phospholipid)
Not in fluid phase
Phospholipid is an assembly molecule

Cont..
 Serine protease bind negatively charged
phoapholipid surfacethrough positively
charged Ca 2+
Ca involved in most reaction
Ca bridge platelate factor with phospholipid,
Phospholipid and Ca 2+ :
 used to overcome the influence of Inhibitory
factors on plasma factors
prevent diffusion of plasma factor in to the
systemic circulation

Cont..
 vWF is a large glycoprotein
Platelate adhesion
Transports procoagulant F VIII
Synthesised in megakaryocytes and
endothelial cells

Vitamin K in blood coagulation
 Coagulation factors II, VII, IX and X as well as
protein C and protein S are dependent on vitamin K
for their normal function
 •
Vit K is found:

Cont..
 They are synthesised in an inactive form that cannot
bind Ca2+ only after posttranslational modification
by γ carboxylation of glutamic acid residues can they
bind

Cont..
 In vit K deficiency ( in the presence of vit k
antagonists:warfarin) there is no γ carboxylation →
noncarboxylated forms of above proteins released
into circulation
 These proteins cannot bind Ca2+ ions and thus
cannot bind phospholipid surfaces, and hence cant
participate in the coagulation reaction

Intrinsic system
 The initial reaction in this system is
conversion of inactive factor XII to XIIa
 This activation is catalysed by high-
molecular weight kininogen (HMW
kininogen) and kallikrein
 Can be brought about in vitro by exposing
the blood to electro-negatively charged
wettable surfaces such as glass & collagen
fibers
 Activation in vivo occurs when blood is
exposed to collagen fibers underlying the
endothelium in the vessels

Cont..
 Contact factors do not have an in vivo procoagulant
function. But responds to the negatively charged
surfaces: non siliconized glass ( in a test tubes)
 In vivo activated by
Valve prostheses
Artificial implants in surgery
Exposure to foreign substances: sub endothelial
collagen

Cont..
 Active factor XII then activates factor XI
 Active factor XI activates factor IX
 Activated factor IX forms a complex with factor VIII
leading to activation of factor X; factor VIII itself
needs activation by thrombin inorder to participate in
the activation of factor X
 Phospholipids (PF-3) from aggregated PLTs & Ca++
are necessary for full activation of factor X

Cont..
 Although intrinsic pathway is more complex &
slower, it accounts for the majority of the coagulation
activity in vivo
 Factor XII, Prekallikrein, HMW kininogen are
referred to as the contact proteins, because their
activation occurs on contact with an abnormal surface
or (glass or kaolin)

Cont..
 THROMBIN
1) cleaves Fibrinopeptides A and B from
Fibrinogen
2)Amplifies co agulation mechanism- activates co
factors V and VIII and F XI
3)activates F XIII
Because of Multiple autocatalytic functions
THROMBIN is considered the most important
protease of the coagulation pathway

Extrinsic system (measured by PT)
 Coagulation is triggered by the exposure of tissue
thromboplastin to plasma protein ( F-V II)
-Tissue thromboplastin is not circulating in the
blood but released when cells are damaged e.g.
intravascular hemolysis, spontaneous abortion,
traumatic head injury
 Concentrated sources of tissue thromboplastin
(factor III) are:
-RBC membranes,Platelets,Brain
tissue,Placenta,Lung tissue

Cont..
 The extrinsic system activates factor X rapidly since
there are fewer reaction steps involved. Tissue factor
forms a complex with factor VIIa and activates factor
X to Xa

Cont..
 Interdependence between the extrinsic and intrinsic pathways
has been demonstrated; the tissue factor-Factor VIIa complex
can activate factor IX, providing a mechanism for bypassing
the initial steps of the intrinsic pathway
 Role of contact factors is questionable, because patients
lacking the contact factors (factor XII, PK, HMWK) do not
have bleeding problems whereas patients who have
deficiencies of factors VIII, IX, or X bleed.

Common pathway
 Activated factor X, in association with
cofactor on phospholipid surface and
calcium,converts prothrombin to
thrombin
 Thrombin converts fibrinogen to fibrin

Cont..
Currently proposed model is as follows:
 Following injury
 Tissue factor is expressed
 Complex formation with factor VIIa activates factors
IX and X
 FXa binds FVa on Phospholipid surface in the presence of
Ca 2+
 This Xa-Va complex activates Prothrombin to Thrombin
 THROMBIN cleaves Fibrinopeptides A and B from plasma
FIBRINOGEN causing the formation of Fibrin monomer (
soluble) then Fibrin polymer,which stabilized by the cross
–linking action of F XIII to form Insoluble fibrin

Cont..
 When small amounts of Xa are produced, tissue factor
pathway inhibitor inhibits subsequent tissue factor activity
(I.e., extrinsic pathway)
 Thrombin generated by the initial tissue factor activates factor
XI to initiate the intrinsic coagulation and additional thrombin
formation
 Thrombin generation is amplified by thrombin feedback
activation of factors V and VIII

Cont..
 Factor XII initiation is important when artificial
surfaces are present, but not for in vivo coagulation
 So the current model explains why patients with
deficiencies of factors VIII, IX, or XI bleed and why
patients with contact factors deficiency do not

Fibrinolysis
 Lysis or dissolution of the clot (by the fibrinolytic system)
 Necessary for tissue repair to proceed and for normal
circulation to resume
 For hemostasis to be effective, normal balance must exist
between clot formation and removal
 Plasmin is the active component of the fibrinolytic system.
 It lyses fibrin and fibrinogen, with the production of fibrin degradation
products

Cont..
 In summary, following a vascular injury (injury of smaller
vessels such as arteriole, venule, or capillary):
 Initially, rapid vasoconstriction reduces blood flow and
promotes contact activation of platelets and coagulation factors
 In the second phase, platelets adhere immediately to the
exposed sub endothelial connective tissue, particularly
collagen. The aggregated platelets enhance sustained
vasoconstriction by releasing thromboxane A2 and vasoactive
amines, including serotonin and epinephrine
 In the third phase, coagulation is initiated through both
intrinsic and extrinsic systems

Cont..
 Finally, fibrinolysis occurs following the release of tissue
plasminogen activators from the vascular wall. Fibrinolytic
removal of excess hemostatic material is necessary to
reestablish vascular integrity.
 Once tissue repairing is taking place, the clot dissolves
gradually and the particulate matter is phagocytized by the
mononuclear phagocytic system

Cont..
Normal control of the clotting process
 Equally important are mechanisms that prevent
inappropriate activation of the cascade. Natural or
innate inhibitors and anticoagulants circulate in the
plasma, limiting the initiation and extent of fibrin
formation.
 There are several protective mechanisms against
thrombosis; the most important are:
 Removal of activated clotting factors by blood flow
 Inactivation of clotting factors by circulating inhibitors.
The natural anticoagulant system in vivo includes:

 The extent of the clot should be confined to the immediate
surrounding area of the vascular lesion.
 Inhibitors to the fibrinogen activators are present in the blood
to help control fibrinolysis

PHYSIOLOGICAL COAGULATION INHIBITORS
1. Antithrombin III
-Is a serine protease inhibitor (SERPIN)
- a glycoprotein of hepatic origin most powerful
physiological coagulation inhibitors
- markedly inhibiting thrombin (FIIa)
- to a lesser degree on factors Xa, IXa, XIa, XIIa
and kallicrein
 Requiers Heparin for effective anticoagulant activity

Proposed Mechanism of AT III-
Heparin System
Heparin
Thrombin Antithrombin
III
Lysine
sites
Serine site
Arginine
site
H
Th
H
AT III
AT III
Th

Cont..
2. Protein C-Protein S
Protein C : vitamin K-dependent
- synthesized in the liver
 activated by thrombin in the presence of Ca 2+ and
a cofactor located on the surface of endothelial cells,
thrombomodulin.
 Protein Ca (activated Protein C) inactivates the major
proteins, factors Va and VIIIa.
 It requires as a co factor a phospholipids surface, Ca
2+ and is greatly enhanced by a plasma protein
,Protein S

Cont..
Protein S is
-Vitamin K- dependent factor
-synthesized in the liver
 -present in two forms in the plasma, a circulating form, and a
form bound to the fourth component of complement (C4b)
binding protein
 -only circulating Protein S is active as a cofactor of activated
Protein C
 -Protein Ca activity is regulated by inhibitor ( PCI)
 Nb (Protein C is also active during fibrinolysis by neutralizing
PAI

Cont..
3. Heparin cofactor II (HC II)
 - a glycoprotein synthesized in the liver
 -unlike AT III, HC II is a very specific inhibitor and
only neutralizes thrombin efficiently
 - this action is accelerated by heparin and dermatan -
sulphate

Cont..
4) Tissue factor pathway inhibitor (TFPI)
- is emerging as the most important regulatory
mechanism in vivo coagulation
A) synthesised by endothelial cells and circulates in
plasma bound to low density lipoproteins
B) also present in platelets and bound to heparan
sulphate at the endothelial surface
C) TFPI inhibits coagulation by binding to factor Xa
and TF:VIIa complex and inhibiting their proteolytic
activity

FIBRINOLYSIS
 Fibrinolysis is the physiological process whereby
fibrin is broken down by a specific enzyme,
plasmin.
 When blood coagulation is activated ,it maintains
hemostatic balance by dissolving the fibrin deposits
which could occur spontaneously in the circulation
and re open thrombosed blood vessel
 Plasmin is a result of activation of plasminogen.The
activation process can have several pathways:

Cont..
(i) - tissue plasminogen activator (t-PA)
- is synthesized in the endothelial cells and related
in large quantities by various stimuli : venous stasis,
acidosis, stress, physical exercise,
- t-PA has a great affinity to fibrin which it binds to
rapidly ,thus enabling plasminogen to be transformed
in to plasmin at the fibrin clot, carrying out
fibrinolysis in situ.
-The t –PA bound to the fibrin clot is protected by the
action of its inhibitors
t-PA is found in fairly large quantities in organs such
as the uterus, the prostate and the lungs

Cont..
 (ii)-the urokinase –
This activator has been detected in plasma and
circulates in a zymogen form
 D –Dimers are specific products of fibrin degradation
(4 main products called X, Y, D, E fragments).
 These fragments act as strong anticoagulants
 as its specificity is low , plasmin can attack other
substrates such as anti hemophilic factor A ( F VIII),
PRO ACCELERIN ( FV), etc

PHYSIOLOGICAL INHIBITOR OF THE FIBRYNOLYTIC
SYSTEM
 As in coagulation, fibrynolysis is confined to
the clot surface and controlled by inhibitors
Their target is :
-either the activation system
The strongest inhibitor is the Plasminogen
Activator Inhibitor (PAI).
PAI inhibits t-PA and u-PA
 or plasmin
whose principal inhibitor is alpha 2- antiplasmin

Bleeding disorder
 1. Bleeding and Coagulation disorders
 Vascular defects
 Platelet defects
 Coagulation factors
 Vascular defects
 Inability to contract after injury
Causes include:-
 Ascorbic acid (vit C) deficiency
 Inflammation
 Certain toxins
 Aging and
 Congenital defects e.g. hereditary hemorrhagic
telangiectasia
 In these conditions, bleeding in to the skin produces
ecchymoses called “vascular purpura”

Platelet defects
 Quantitative and qualitative defect
 Thrombocytopenia
Decreased production
 Hypoplasia
 Marrow replacement by tumor or malignant cells
 Immune damage from toxins, drugs, bacterial & viral
infections
 Idiopathic (ITP)
Ineffective maturation  e.g. in megaloblastic
anemia
Increased destruction or utilization
 Autoimmune antibodies
 DIC

 Pooling of platelets by the spleen (without destroying
PLTs)
 The spleen is responsible for destruction, anti-platelet
antibody production and pooling
 Disorders of PLT function (qualitative defect)
 Acquired or inherited
 Acquired Platelet disorders
 Many drugs e.g. asprin, other non-steroidal anti-
inflammatory drugs
 Hereditory Platelet disorders
 Thrombasthenia  defect in primary platelet aggregation

Coagulation factor defect and inhibitors
 Coagulation factor defect or abnormal function
Factor deficiencies
 The most important congenital deficiency is
Factor VIII deficiency:
 Called Hemphilia A
 Graded as severe, moderate, and mild depending on the
coagulant activity of Factor VIII
 Inherited as sex-linked recessive manner and occurs
exclusively in males

 von Willebrand’s disease:
 Due to deficiency of vWF
 Characterized by defects in platelet adhesion
 Factor IX deficiency:
 Called Hemophilia B
 Sex linked recessive
 Occurs less frequently and milder in its clinical
presentation than factor VIII deficiency (Hemopilia A)

Abnormal coagulation factors function
 Abnormality in function is seen in vit K deficiency. The
binding of Ca++ to factors II, VII, IX, X is required for
normal clotting. Without the attached Ca++, these fators will
not bind to phospholipids and rate of factor activation will
be sharply decreased. To bind Ca++ they need
gamacarboxylation with vit K (to make II, VII, IX & X
functional)
 Vit K dependant factors are: Factors II, VII, IX, X, Protein
C & protein S

Consumption of coagulation factors
 e.g. DIC  acquired coagulation defect secondary to
other pathologic processes which results in
accelerated consumption of platelets and several
coagulation factors, particularly fibrinogen
 Inhibitors of coagulation
 e.g Lupus anticoagulant in patients with SLE and other
related disorders. Lupus anticoagulant interferes with the
phospholipid portion of Factor Xa-V-Ca++-Plt
phospholipids complex.

 Disturbance of the balance between promotors and inhibitors
of coagulation due to:
 Various disorders including bacterial, viral, rickettsial,
infections
 Complication of pregnancy
 AML
 Tissue damage (shock, heat strock, burns)
 Hemolytic transfusion reactions
 Venome snake bites

2. Laboaratory investigation of bleeding
and coagulation disorders
 Bleeding Time
 Coagulation time
 Platelet count
 Clot retraction time
 Prothrombin time (PT)
 Accctivated Partial Thromboplastin Time (APTT)
 The Thrombin time
 Fibrinogen quantitative assays
 specific factor assays are commonly used to assess
coagulation factors.
 preoperative screening tests usually include a bleeding
time, platelet count, PT & APTT

Bleeding time:
 the time required for a small standardized wound cut to
stop bleeding
 a measure of vascular integrity and platelet function
 prolonged in:
shortage of platelets (Plt<50,000
cells/L)(Thrombocytopenia)
inadequate function of platelets
 von Willebrand’s disease
poor retractability of capillaries (e.g. scurvy-vit C
deficiency)
deficiency of plasma factors

Different methods:
 Duke method & Ivy method (common ones), Mielke (9 mm
long, 1mm deep), Template, Simplate (5mm long, 1mm deep)
 Duke method: oldest method, which is performed by
puncturing the ear lobe using a sterile lancet, recording the
time, blotting the blood every 30 seconds until bleeding
ceases, and recording the time. Blotting is done without
allowing the filter paper to touch the wound

Cont..
 Time between the puncture and the cessation of
bleeding is the bleeding time
 NR = 1-3 min (3-6 min boarder line)
 Drawback: impossible to standardize the depth of the
incision; as a result not recommended

Cont..
 Ivy method
A blood pressure cuff is placed on the patient’s arm above the
elbow, inflated & maintained at a constant pressure (40
mmHg) throughout the procedure. This is to standardize the
pressure in the vascular system. Two or three standardized
(3 mm) punctures of the forearm are made by holding the
skin tightly i.e. by grasping the underside of the arm firmly.
The length of time required for bleeding to stop is recorded.
Report the average of the two results including method and
normal values

Cont..
 Note: wait for 30 sec after applying the sphygmomanometer
and inflating it to 40 mm Hg to allow the capillary filling to
equilibrate. Select an area in the lower arm 3-finger width
below the bending in the elbow, with no hair and superficial
veins.
 NR= 1-7 min; 7-11 min boarder line
 In general, if bleeding continues for more than 15 min,
discontinue the procedure and report as >15 min.
 It is recommended to repeat the procedure on the other arm if
bleeding time < 1min and >7 or 15 min, except in excessively
prolonged tests.

Cont..
Sources of error
 No bleeding because of too gentle an incision
 Severe bleeding: superficial veins have probably been cut
 If filter paper touches the wound, a platelet aggregate might be
removed resulting in prolonged bleeding
 Decrease bleeding time:
 Failure to cleanse the area
 Making superficial puncture

Cont..
 Increase bleeding time:
 Puncturing a red flushed area
 Too deep puncture
 Applying pressure to the punctured area

Cont..
Coagulation time
 The time required for a measured amount of blood to clot
under certain specified conditions
 Capillary blood methods
 Slide method (NR 2-6 min)
 Capillary tube (NR 2-6 min)
 Dale & Laidlaw (NR 1-3 min)
 Venous blood methods
 Lee & Wite method (NR 5-15 min)
 Howell method (NR 10-30 min)
 Silicone tube method (NR 20-60 min)
 Note: the report should always include method and normal
range

Cont..
Slide method
 Puncturing the finger, recording the time, placing 3 drops of
blood on a glass slide, drawing the point of the needle or lancet
through the drops until fibrin threads appear & recording the
time
Capillary tube method
 Puncturing, recording the time, filling a capillary tube with
blood, allowing 3 min to ellapse, breaking the capillary tubes
at half min intervals until a span of fibrin is seen and recording
the time

Cont..
Dale & laidlaw
 Puncturing, recording the time, allowing the blood to flow into
a capillary tube, which contains a lead bead, immersing the
capillary tube in 37oC water, tilting the tube until the lead
bead is held firmly by fibrin threads, & recording the time
Lee & White
 Drawing blood from a vein & noting the time the blood enters
the syringe, or vacutainer tube, transferring the blood to 3 test
tubes, tilting each test tube one after the other until coagulation
takes place, & recording the time

Cont..
Howel method
 Coating a syringe with petroleum, drawing blood from a vein,
recording the time the blood enters the syringe, transferring the
blood to a test tube, tilting the tube until coagulation takes
place, & recording the time
Silicone tube method
 Coating a syringe with silicone to decrease the contact of
blood with glass (because contact with glass promotes
coagulation), drawing blood from a vein & noting the time the
blood enters the siliconized syringe, transferring the blood to 2
siliconized test tubes, placing the tubes on 37 oC water bath,
tilting the tubes until coagulation takes place & recording the
time
 The Lee & White method is the most widely used method.

Cont..
Lee & White method
 In the past, it was used as a screening test to measure all
intrinsic coagulation system & to monitor heparin therapy.
However,
 it is time consuming,
 sensitive to only severe factor deficiencies &
 insensitive to high doses of heparin, and has poor reproducibility.
Therefore, of limited use in today’s laboratory
Procedure
 Label 3 clean 13 x 100 mm test tubes
 Draw 4 ml of blood, start the stopwatch as soon as blood
enters the syringe
 Remove the needle & gently transfer about 1 ml of blood to
each of the 3 test tubes (excessive agitation will hasten
coagulation)

Cont..
 Place the 3 test tubes in a 37oC water bath
 Allow 4-5 min to elapse & gently tilt test tube #1 to a 45 angle
every 30 sec until the blood is completely clotted
 Repeat the process with the 2nd and 3rd test tubes & record
the time
 Coagulation time is the time elapsed between the withdrawal
of blood and completion of coagulation in test tube #3. Note: 3
test tubes are used because each successive tube is subjected to
less tilting, & therefore, less agitation of the blood, and
consequently a more accurate coagulation time. Since agitation
and handling speed up coagulation, the coagulation time of test
tube #3 is the reported result.

Cont..
Sources of error
 Coagulation hastened:
 Dirty test tubes
 Poor venipuncture can introduce tissue thromboplastin
 Excessive agitation during transfer
 Coagulation retarded:
 Temperature < 35 oC & > 45oC

Cont..
 Diameter of the test tube; the smaller the diameter, the more
rapid the clot formation is because the amount of foreign
surface area (glass) to the amount of blood is increased.
Therefore, all test tubes should be of the same diameter.
 Increased volume of blood per tube
 At completion of the Lee & White clotting time, it is suggested
that 1 test tube remain in the 37oC water bath to be checked
after 2 & 4 hours for clot retraction. Also the same tube may
be allowed to remain in the water bath over night & checked
the next day for clot lysis
 NR 5-15 min

Cont..
 Standardization of Clotting time can be achieved
when:
 The amount of blood is consistent
 Process occurs at specified temperature and
 Clotting occurs in the same physical environment

Cont.
Platelet count (Thrombocyte count)
 Also involved in clot retraction
 Thrombocytosis
 In Polycythemia Vera: over active bone marrow resulting
in increased production of WBC, RBC & Plt
 Idiopathic thrombocythemia (increased platelet number)
 CML
 Following splenectomy
 Sickle cell anemia: hyperactive bone marrow to produce
more RBC, but increased WBC & Plts as well

Cont..
Thrombocytopenia
 Thrombocytopenia purpura (Idiopathic or secondary
to other diseases)
 Aplastic anemia: characterized by pancytopenia
 Acute leukemia: decrease in Plt and RBC production
 Pernicious anemia: decreased production due to
deficiency of vit B12
 Sometimes following chemotherapy and radiation
therapy
 Platelet count may be below or above NR values in
certain normal conditions and activities
 e.g. below normal: before menstruation
 above normal: at high altitude and after severe exercise

Cont..
 Platelets are difficult to count, small, disintegrate easily, and
are hard to distinguish from dirt, readily adhere to each other
(aggregation) and also become easily attached to any foreign
body (adhesiveness).
 Platelet counts from finger or heel prick blood is less
satisfactory and significantly lower than platelet counts
obtained from venous blood. Significant number may probably
be lost at the puncture site. When obtaining capillary blood, it
is important that the platelet count should be obtained first

Platelet counts
 Blood is mixed with a diluent (1% ammonium oxalate) that
causes hemolysis of red cells.
 A hemocytometer is filled with the diluted sample and
platelets are counted under the microscope
 Note: Amonium oxalate should be stored in refrigerator and always be
filtered just before use to remove crystals and other debris, which may be
mistaken for platelets.
Platelet estimates (for QC purpose)
 In normal blood smear there are 8-20 Plts per field in the thin
area (4-8 Plts/100 RBC; 30 Plts for every 500 RBC)
 One method of estimating platelets is to determine the average
number of platelets per field, using 10 different fields, taking
the average and multiply the result by 20,000 (comment as
NR, decreased or increased)
 Never report an estimate; this is just for QC purpose

Procedure
 Venous EDTA blood recommended, not > 1 hr old because platelets
disintegrate easily
 Pipet 0.38 ml of diluting fluid, Mix the blood
 Add 20 L blood and wash out pipet by drawing up the diluting fluid and
expelling into the tube a few times
 Mix at least 10 min to ensure proper mixing and RBC lysis
 Blood diluted with 1% ammonium oxalate is stable for 8 hrs
Technical tips
 The hemocytometer should be clean (ethyl alcohol & lint free cloth
recommended)
 Shake gently, at least 2 min
 Allow 10-20 min for platelets to settle
 Count platelets in the central squares; there should be even distribution of
cells in the counting chamber (there should be no platelet clump)
 Calculate the number of platelets and report with normal values

Cont..
 Sources of error
 Capillary blood: platelets adhere to the wound; squeezing with
heavy pressure may cause disintegration
 Ammonium oxalate shoud be refrigerated and must be discarded if
there is evidence of bacterial contamination
 Presence of clumps: test should be repeated. Clumps are usually
due to inadequate mixing or poor technique in obtaining blood
sample
 Unfiltered diluting fluid: debris and dirt may be mistaken for
platelets
 Dirty tubes, pipets, and counting chamber: Plts will stick to the dirt
and falsely lower Plt count
 For QC purpose charge both sides of the hemocytometer
 Results should be double checked by examination of Plts on a
Wright stained blood smear. If the count does not agree with
the estimate, it should be repeated

Cont..
Clot retraction Time
 When blood coagulation is complete, the clot normally
undergoes contraction, where serum is expressed from the clot,
and the clot becomes denser (firm).
 Thrombosthenin, released by platelets is responsible for clot
retraction.
 The time is affected by quantitative and qualitative defects in
platelets.
 When the red cell count is high, degree of retraction decreased
because of large volume of RBC in the clot, and vise versa.
 There are different methods. Some inspect the clot after 1, 2, 4
and 24 hours
 NR 2-4 hrs; Poor 4-24 hrs; >24 hrs reported as none

Cont..
Prothrombin time (PT)
 PT is the time required for plasma to clot after an optimal
amount of tissue thromboplastin and calcium chloride have
been added to trigger the coagulation process.
 It evaluates the generation of thrombin and the formation of
fibrin via the extrinsic and common pathways

Cont..
Diagnostic significance
 It measures the functional activity of factors VII, X, and V,
and factor II or I.
 Especially useful for initiation and monitoring of oral
anticoagulant therapy to adjust the dose.
 Therefore, extreme care is needed. e.g. Warfarin is a vitamin K
antagonist and interferes with the production of vit K dependent factors
(factor II, VII, IX, and X) and Protein C & protein S. Protein C and S
are natural anticoagulants.
 To diagnose deficiencies in the coagulation factors of the
extrinsic system
 Useful for checking the synthesis performance of the liver in
hepatic disease

Cont.
Principle:
 The coagulation process is triggered by incubation of plasma
with the optimal amount of thromboplastin and calcium, and
the time required for the formation of a fibrin clot is measured
in seconds.
Prolonged in:
 Deficiency of one or more coagulation factors in the extrinsic
pathway: i.e., factors VII, X, V, and II or I
 Vit K deficiency
 Certain liver diseases
 Circulating anticoagulants
 Anticoagulant therapy (e.g. Coumarin)
 DIC (disseminated intravascular coagulation)

Cont..
Quick’s one stage Prothrombin Time
 Specimen required: 9 parts blood + 1 part 3.8% sodium
citrate. Avoid formation of foam. The sample should be
centrifuged at 3000 rpm for 15 min as soon as possible with
the plasma removed from the erythrocytes. Plasma may be
stored several hours at 2-6oC prior to testing

Cont..
Quality control
 Control and patient plasma should be run in duplicate and the
two results averaged to obtain the final value.
 Duplicates should agree within 2 sec.
 Report both patient and contol value; for patients on
anticoagulant therapy, the control value is twice the normal.
Normal values range from 10-13 sec. If the PT of the control
plasma doesn’t lie within the specified values provided by the
manufacturers, it indicates failure in equipment, reagent or
techniques used and the test must be repeated

Cont..
Most common sources of error
 The 9:1 ratio of blood to sodium citrate should be precise
 Failure to follow directions in the manufacturers instruction
(package insert) strictly while preparing patient plasma,
control plasma, reconstituting reagents
 Use of dirty or wet test tubes, pipets etc to perform the test
 Test must be performed within 4 hrs of specimen collection
(within 2 hrs is best)
 Mistakes in pipetting
 Hemolysis
 Timing, incubation temperature, contact activation influence
the test

Cont..
International Normalized Ratio (INR)
 INR values preferable to the PT because different
thromboplastin reagents have different sensitivities to warfarin
induced changes in levels of clotting factors
 The INR corrects most of reagent differences, expressed as ISI
 ISI is the international sensitivity index of the thromboplastin
reagent; it is a correction factor assigned by the manufacturer

Cont..
 ISI values reported by manufacturers vary depending on the
instrument used to perform the PT
 The PT, utilized to adjust the dose of oral anticoagulation,
should be reported according to the INR and not the PT ratio
or PT in seconds
 The INR is essentaially a “corrected” PT
 INR = PT patient ISI
PT normal

Cont..
Activated Partial Thromboplastin Time
(APTT)
 Major screening test for coagulation disorders in
the intrinsic system
 Especially for sensitive detection of fatctors VIII and
IX and the contact factors (except for platelets and
factor XIII)
 Also a method of choice for monitoring heparin
therapy

Cont..
Test principle:
 The plasma after centrifugation contains all intrinsic
coagulation factors except Ca and platelets.
 Calcium and a substitute for Platelet factor III,
(partial thromboplastin-Cephalin) and an activator
such as kaolin, to ensure maximal activation, are
added to the plasma. The time required for the plasma
to clot is the APTT. The activator is added to ensure
maximal activation.

Cont.
 When an activator is not added the test is called PTT and the
amount of time required for normal plasma to clot is prolonged
 Normal plasma should be run each time a new reagent is
opened
 The APTT assay reflects the activity of prekallikrein, HMWK,
and factors XII, XI, IX, VIII, X, V, II, and I
 APTT may be prolonged due to a factor decrease or presence
of circulating anticoagulants.
 The normal APTT is less than 35 seconds depending on the
activator used

Interpretation
The common causes of a prolonged APTT are as
follows:
 Disseminated intravascular coagulation
 Liver disease
 Massive transfusion with plasma-depleted red blood
cells
 Administration of or contamination with heparin or
other anticoagulants
 A circulating anticoagulant (inhibitor)
 Deficiency of a coagulation factor other than factor
VII

Prolonged APTT + Prolonged PT:
 Vitamin K deficiency
 Liver disease due to:
-Malabsorption of vitamin K
-Decreased synthesis of clotting factors
- An acquired dysfibrinogenemia due to changes in the
sialic acid content of the fibrinogen.
 Combined deficiency of clotting factors e.g. Factors V
and VIII
 DIC - due to the consumption of clotting factors
 Massive blood transfusion leading to a dilutional
coagulopathy

Cont..
Thrombin Time
 Determines the rate of thrombin induced cleavage of
fibrinogen to fibrin monomers and the subsequent
polymerization of fibrin polymers
 NR=<20 sec
 Prolonged
 When fibrinogen concentration is <100 mg/dL(
Hypofibrinogenemia)
 Dysfibrynogenemia
 Afibrinogenemia: DIC,Liver disease
 Thrombin inhibitors or substances that interfere with fibrin
formation (e.g heparin, fibrin degradation products)

Cont..
Fibrinogen levels
 Useful to detect deficiencies of fibrinogen and alterations in
the conversion of fibrinogen to fibrin
 NR= 200-400mg/Dl
 May be decreased in liver disease or due to consumption of
fibrinogen when there is accelerated intravascular clotting
 mainly affected by the concentration of Fibrinogen and the
FDP level
 Elevated ;-in infection, Inflammation, Traumatic injury

Platelet
Count
PT APTT TT Fibrinogen Interpretation
N N N N N Normal profile
Factor XIII deficiency
Mild VWD
Qualitative platelet disorder
Connective tissue problem e.g. Ehlers Danlos
Mild coagulation factor deficiency
N ↑ N N N FVII deficiency
N N ↑ N N FVIII, FIX, FXI or FXII deficiency
VWD - if the FVIII level is reduced
Lupus anticoagulant [Occasionally a very strong lupus anticoagulant or a lupus
anticoagulant that has anti-prothrombin activity can result in a prolonged PT.
Similarly a lupus anticoagulant can also be associated with thrombocytopaenia.]
Other contact factor deficiency
N ↑ ↑ N N Common pathway' deficiency i.e. FII, FV or FX deficiency
Multiple clotting factor deficiencies e.g. combined FV and FVIII deficiency
Warfarin or vitamin K antagonist
Vitamin K deficiency or a mutation within one of the genes encoding key
enzymes involved in VK metabolism.
Occasionally a very strong Lupus anticoagulant can cause these findings but it is
unusual to see a prolongation of the PT with a LA due to the high concentration
of PL used in the PT test.
↓ ↑ ↑ ↑ ↓ DIC
Massive transfusion
Liver disease
↓ N N N N Primary platelet problem e.g. ITP.
The Mean Platelet Volume [MPV] can be helpful in establishing the causes of
thrombocytopaenia.
A raised MPV is often associated with increased peripheral destruction e.g. ITP
- whereas a reduced MPV is often seen in association with bone marrow failure.
Changes in the MPV are also seen in patients with various inherited platelet
disorders e.g. Wiskott Aldrich syndrome

 35-year-old woman needs to have an ovarian cyst removed.
Her mother has a history of bleeding after tooth extraction.
The physician needs to determine if there is a bleeding
disorder. The coagulation test results are as follows:
 PT 20.5 seconds (Reference range, 10.5 to 13.3)
 aPTT 32.1 seconds (Reference range, 28.7 to 35.5)
 Platelets 320,000/mm3 (Reference range, 150,000 to 400,000/mm3)
 Bleeding time 11 minutes
What is impaired factor?
A. V
B. VII
C. VIII
D. IX

 A 52-year-old woman needs to have tooth extraction. She had no
pregnancy or delivery history. Her sister has a history of bleeding after
minor surgery. The physician needs to determine if there is a bleeding
disorder. The coagulation test results are as follows:
 PT 11.0 seconds
 aPTT 30.1 seconds
 Platelets 320,000/m m3
 Bleeding time 13 minutes (Reference, 8 minutes)
 What is the most significant abnormal result in the coagulation panel?
A. PT
B. aPTT
C. PLT
D. BT

Hema Chapter 24_Hemostasisnnnnnnnnnnnnnnnn (2).ppt

More Related Content

Similar to Hema Chapter 24_Hemostasisnnnnnnnnnnnnnnnn (2).ppt (20)

More from ErmiyasBeletew (16)

Recently uploaded (20)

Hema Chapter 24_Hemostasisnnnnnnnnnnnnnnnn (2).ppt

Editor's Notes