THROMBOSIS

AND
DISORDERS
OF
HEMOSTASIS
 Hemostasis

 A series of reactions
designed for stoppage of
bleeding
 During hemostasis, three
phases occur in rapid
sequence
– Vascular spasms –
immediate vasoconstriction
in response to injury
– Platelet plug formation
– Coagulation (blood clotting)
 Blood Vessel Damage Vascular Phase
 Lasts about 30 mins
 Endothelial cells at the injury site
Smooth muscle in BV wall undergo changes:
contracts – Vascular Spasm – Contract and expose their
basement membrane to the
bloodstream
– Release chemical factors and local
BV diameter  hormones
 ADP, tissue factor, and
prostacyclin
– Endothelial cell membranes
become sticky
Blood loss slows
Platelet Plug Formation
 Platelets
 Platelets are Flattened disk-like cell fragments
that are about 1µm by 4µm diameter.
 Their granules contain serotonin, Ca 2+,
enzymes, ADP, and platelet-derived growth
factor (PDGF)
 Platelets function in the clotting mechanism by
forming a temporary plug that helps seal
breaks in blood vessels
 Platelets not involved in clotting are kept
inactive by NO and prostaglandin I2
Ultrastructure of platelets

Secretory granules
Diagrammatic Representation of the Platelet
 Platelet Plug Formation
 Platelets do not stick to each other or to the endothelial
lining of blood vessels
 Upon damage to blood vessel endothelium (which
exposes collagen) platelets:
– With the help of von Willebrand factor (VWF) adhere to
collagen
– Are stimulated by thromboxane A2
– Stick to exposed collagen fibers and form a platelet plug
– Release serotonin and ADP, which attract still more
platelets
 The platelet plug is limited to the immediate area of
injury by PGI2
 Coagulation

 A set of reactions in which blood is transformed

from a liquid to a gel
 Coagulation follows intrinsic and extrinsic
pathways
 The final three steps of this series of reactions are:
– Prothrombin activator is formed
– Prothrombin is converted into thrombin
– Thrombin catalyzes the joining of fibrinogen into a
fibrin mesh
BLOOD COAGULATION
Polymerisation of fibrinogen
ULTRASTRUCTURE OF THROMBUS
 Clot Retraction and Repair

 Clot retraction – stabilization of the clot by

squeezing serum from the fibrin strands
 Repair
– Platelet-derived growth factor (PDGF)
stimulates rebuilding of blood vessel wall
– Fibroblasts form a connective tissue patch
– Stimulated by vascular endothelial growth
factor (VEGF), endothelial cells multiply and
restore the endothelial lining
 Factors Limiting Clot Growth
or Formation
 Two homeostatic mechanisms prevent clots
from becoming large
– Swift removal of clotting factors
– Inhibition of activated clotting factors
Antithrombotic Attributes of Vascular
Endothelium
 Intrinsic Pathway of Blood Coagulation

 Clinical test to assess the functionality of this

pathway is the activated partial thromboplastin
time (aPTT)
– Kaolin and cephalin are added to the test plasma sample
– The normal range is ~30 – 50 seconds (varies slightly
depending on the laboratory)
– Prolongations in the aPTT are observed in deficiencies of
factor XII (or its cofactors), or factors XI, IX, VIII, X,
and V, or prothrombin (II).
– Used to test for common congenital hemophilias
(deficiencies in IX, VIII, or XI) and to monitor heparin
treatment
 Extrinsic Pathway of Blood Coagulation

 Clinical test to assess the functionality of this

pathway is the prothrombin time (PT)
– Lipidated tissue factor is added to test plasma sample
– The normal range is ~10-15 seconds (varies slightly
depending on the laboratory)
– Prolongations in the PT are observed in deficiencies of
factors VII, X, V, prothrombin (II), or fibrinogen (I).
– Used to test for the rare congenital deficiencies in these
factors: More often it is used to diagnose acquired
bleeding disorders resulting from vitamin K deficiency,
oral anticoagulants (e.g. warfarin), and liver disease
 Common Pathway of Blood Coagulation

 Tested clinically by the thrombin time (TT)

 In this test, thrombin is added to the plasma

– The normal range is ~10-15 seconds (varies slightly
depending on the laboratory)
– Prolongations in the TT are observed in congenital
fibrinogen deficiency or acquired fibrinogen deficiency
resulting from consumption of fibrinogen in DIC
(disseminated intravascular coagulation), or may occur
following treatment with fibrinolytic drugs
 NORMAL HAEMOSTATIC BALANCE

Naturally
occurring
Pro-coagulants anticoagulants
and platelets. and good
vascular flow

Thrombosis Bleeding
 Hemostasis Disorders:
Thrombus – a clot that develops and persists in
an unbroken blood vessel
− Thrombi can block circulation, resulting in tissue death
− Coronary thrombosis – thrombus in blood vessel of the
heart

Embolus – a thrombus freely floating in the

blood stream
−Pulmonary emboli can impair the ability of the body to
obtain oxygen
−Cerebral emboli can cause strokes
Thrombi & Emboli
 A thrombus is formed when
platelets begin to stick to the wall
of an intact blood vessel
– Platelets are often attracted to
arteriosclerotic plaques – where Above: Normal artery
endothelial and smooth muscle cells
Below: Same artery with a
contain lots of lipids. large thrombus (arrow)

 If the clot (thrombus) breaks off

and begins to drift in the
bloodstream, it is called an
embolus.
 VENOUS THROMBOSIS

 Swollen painful leg

Dr. Rudolph Virchow
1821-1902
 Endothelial Injury:
 Especially important in thrombus formation
in the heart and arterial circulation
 Causes of endothelial
injury:
 Myocardial infarction –formation of the thrombus
within the cardiac chambers
 Ulceration or rupture of plaque in atherosclerotic
arteries
 Traumatic injury
 Inflammation of the artery wall – vasculitis
(bacterial or autoimmunological)
 Cigarette smoke
 Hypercholesterolemia, homocystynuria
Rupture of plaque in
atherosclerotic artery
Alterations in Normal Blood Flow:
 Laminar flow
– Streamlined
– Outermost layer moving
slowest and center moving
fastest
– Cells do not contact with
endothelium

 Turbulent flow
– Interrupted
– Rate of flow exceeds
critical velocity
– Fluid passes a constriction,
sharp turn, rough surface
 Alterations in Normal Blood
Flow:
 Transformation from laminar blood flow to
turbulent flow contributes to formation of
countercurents and local pockets of stasis
 Disruption of laminar flow brings platelets
into contact with endothelium and damages
endothelial cells
 Alterations in Normal Blood
Flow:
 Artherosclerotic vascular constrictions are
common sources of turbulent flow and
thrombosis
 Aneurysms –abnormal aortic and arterial
dilations causes local stasis and turbulence
 Conjunction of atrial fibrillation and atrial
dilation results in profound stasis and
thrombus development.
left atrial appendicular thrombus
 Inherited hypercoagulability
states
 Antithrombin deficiency
 Abnormalities in protein C and protein S system
- protein C deficiency
- protein S deficiency
- abnormal thrombomodulin
● Elevated level of prothrombin in plasma
 Resistance to activated protein C (FV Leiden, FV Cambridge)
Inherited Activated Protein C Resistance
“Factor V Leiden”

• Point mutation in the Factor V gene - Factor V

Leiden

• Prevalence in Western Populations ~2-5%

• Rare in Eastern/Oriental populations

• Autosomal dominant trait

• Responsible for ~60% of familial thrombosis

 Factor V Leiden
Factor Va

Arg 306 Arg 506 Arg 1765

Arginine Glutamine
CGA CAA
Result: Factor Va resistant to APC cleavage

Coagulation cofactor activity continues

 Prothrombin Gene Mutation

• Second most common hereditary risk

factor for venous thrombosis

• Present in 2% of Caucasian population

• Caused by a point mutation (G20210A) in

the prothrombin gene
 Prothrombin G20210A Mutation

 A G-to-A substitution in nucleotide position 20210 is

responsible for a factor II polymorphism
 The mutation causes a 30% increase in prothrombin
levels.
 Antithrombin III Deficiency

 Inhibits coagulation by irreversibly binding the

thrombogenic proteins thrombin (IIa), IXa, Xa, XIa
and XIIa
 Antithrombin’s binding reaction is amplified 1000-
fold by heparin, which binds to antithrombin to cause
a conformational change which more avidly binds
thrombin and the other serine proteases
 Protein C Deficiency
 Protein C is a vitamin K dependent glycoprotein produced in the
liver
 In the activation of protein C, thrombin binds to thrombomodulin,
a structural protein on the endothelial cell surface
 This complex then converts protein C to activated protein C
(APC), which degrades factors Va and VIIIa, limiting thrombin
production
 For protein C to bind, cleave and degrade factors Va and VIIIa,
protein S must be available
 Protein C deficiency, whether inherited or acquired, may cause
thrombosis when levels drop to 50% or below
 Protein C deficiency also occurs with surgery, trauma, pregnancy,
OCP, liver or renal failure, DIC,or warfarin
 Antiphospholipid Antibody
Syndrome
 Antiphospholipid syndrome (APS) is a disorder that
manifests clinically as recurrent venous or arterial
thrombosis and/or fetal loss.
 Characteristic laboratory abnormalities in APS include
persistently elevated levels of antibodies directed against
membrane anionic phospholipids (ie, anticardiolipin [aCL]
antibody, antiphosphatidylserine) or their associated
plasma proteins, predominantly beta-2 glycoprotein I
(apolipoprotein H); or evidence of a circulating
anticoagulant.
Antiphospholipid Antibody
Syndrome
 Antiphospholipid antibodies associated with vaso-
occlusive events without any underlying disease
process is termed the primary antiphospholipid
antibody syndrome (PAPS).

 The presence of antiphospholipid antibodies and a

vaso-occlusive event superimposed on an
underlying disease, such as SLE or malignancy, is
a secondary antiphospholipid antibody syndrome.
 APS Pathophysiology
Possible mechanisms by which antiphospholipid antibodies may induce
thrombotic events include the following:
 Antiphospholipid antibodies may combine with platelet membrane
phospholipids, resulting in increased platelet adhesion and aggregation.
 Antiphospholipid antibodies may combine with the endothelial cell
membrane phospholipids and induce endothelial cell damage, impaired
prostacyclin production, increased platelet adhesion, and aggregation. 
 Antiphospholipid antibodies can stimulate production of antibodies against
coagulation factors, including prothrombin, protein C, protein S, and
annexins
 b2-GPI and oxidized low-density lipoprotein (oxLDL) complexes may be
bound up by antiphospholipid antibodies, cleared by macrophages, and, thus,
promote accelerated development of atherosclerosis in autoimmune patients.
 BLEEDING DISORDERS or
HEMORRHAGIC DIATHESES
Excessive bleeding may result from:
 Increased fragility of blood vessels
 Platelet deficiency or dysfunction
 Derangements in coagulation mechanism
 Combintion of more than one reasons
 Bleeding disorders caused by
vessel wall abnormalities
 Relatively common
 Usually induce small hemorrhages in the
skin or mucous membranes (particularly the
gingivae)
 Tests of coagulation (platelet count,
bleeding time, PT, PTT) are usually normal
 Bleeding disorders caused by
vessel wall abnormalities
 Induced by infection – vasculitis caused by
diplococcus menigitidis (meningococceal
septicemia)
 Result of deposition of immune complexes in the
vessel walls (Schonlein –Henoch purpura,
disorders induced by drugs)
 Induced by impaired formation of collagenous
support of vessels walls (Ehlers-Danlos syndrome,
excessive corticosteroids levels, vitamin C
deficiency)
 Ehlers-Danlos Syndrome
 Ehlers-Danlos syndrome (EDS) is the name
given to a group of more than 10 different
inherited disorders;
 all involve a genetic defect in collagen and
connective-tissue synthesis and structure.
 Ehlers-Danlos syndrome can affect the skin,
joints, and blood vessels.
 Is characterised by joint hypermobility,
cutaneous fragility, and hyperextensibility
 Ehlers-Danlos Syndrome
 The collagen defect has been identified in only 6 of the 11
types of Ehlers-Danlos syndrome.
 Type IV is characterized by a decreased amount of type III
collagen.
 Types V and VI are characterized by deficiencies in
hydroxylase and lysyl oxidase,
 Type VII has an amino-terminal procollagen peptidase
deficiency.
 Type IX has abnormal copper metabolism.
 Type X has nonfunctioning plasma fibronectin.
 Henoch-Schonlein Purpura
 Henoch-Schoenlein purpura is an acute immunoglobulin A (IgA)–
mediated vasculitis that primarily affects children.
 The dominant clinical features of Henoch-Schoenlein purpura
include cutaneous purpura, arthritis, abdominal pain, GI bleeding,
orchitis, and nephritis.
 Is characterised by increased serum IgA concentrations, IgA-
containing circulating immune complexes, and IgA deposition in
vessel walls and renal mesangium.
 IgA aggregates or IgA complexes with complement deposited in
target organs, resulting in elaboration of inflammatory mediators,
including vascular prostaglandins such as prostacyclin, may play a
central role in the pathogenesis of Henoch-Schoenlein purpura
vasculitis.
 Bleeding disorders related to
reduced platelet number
 Condition known as Trombocytopenia
 Platelet count normally range between 150,000
and 300,000/mm3
 Spontaneous bleeding does not occur until the
platelents count fall below 30,000/mm3
 Characterized by bleeding from small vessels of
the skin and mucous membranes (petechie)
 Prolonged bleeding time
 Normal PT and PTT
 Common causes of
Trombocytopenia:
 Decreased production of platelets
 Decreased platelet survival
 Sequestration of platelets
 Dilutional trombocytopenia
Decreased production of platelets

 Aplastic anemia
 Neoplasmatic infiltration of the bone
marrow (leukemia, metastases)
 B12 or folic acid deficiency (impaired
synthesis of DNA)
 Decreased platelet survival

 Platelet destruction is caused by circulating

platelet auto-antibody
 Idiopathic trombocytopenic purpura (ITP)
 Drug induced thrombocytopenia
 HIV-associated thrombocytopenia
 Mechanical injury to platelets
(microangiopathic conditions)
 Idiopathic Thrombocytopenic Purpura
 Idiopathic thrombocytopenic purpura (ITP), also known as
primary immune thrombocytopenic purpura and autoimmune
thrombocytopenic purpura, is defined as isolated
thrombocytopenia with normal bone marrow and the absence of
other causes of thrombocytopenia.

 ITP is primarily a disease of increased peripheral platelet

destruction, with most patients having antibodies to specific
platelet membrane glycoproteins.

 Acute ITP often follows an acute infection and has a spontaneous

resolution within 2 months. Chronic ITP persists longer than 6
months without a specific cause.
Heparin induced thrombocytopenia
•Antibody against PF4-heparin
complex causes inactivation
and destruction of platelets
•Autoantibody binds platelets
to each other via interaction of
PF4 receptor and Fc-receptors
•Autoantibody IgG may bind to
endothelial cell bound heparin-
PF4 complex and cause
vascular damage
•Vascular damage and platelet
activation may provoke
thrombus formation
 Bleeding disorders related
to defective platelet
functions

 Defects of adhesion
 Defects of aggregation
 Disorders of platelet secretion
Bernard – Soulier syndrome:

•Autosomal recessive
condition
•Deficiency of platelet
membrane – bound
glycoprotein complex Ib
(receptor for von Willebrand
complex)
•Results in defective platelets
adhesion
Glanzmann’s thrombastenia
 Autosomal recesive
trait
 Deficiency of
glycoprotein IIb –IIIa
 Bleeding due to
defective platelet
aggregation
 Types of bleeding related to
derangements in coagulation mechanism

 Hemophilia A (factor VIII deficiency)

 Hemophilia B (factor IX deficiency)

 Hemophilia C – mild type, (factor XI deficiency)

 von Willebrand Disease (vWD)

 Other
 Hemophilias A and B

 Hemophilias A and B are cause by deficiencies in factors VIII

or IX, respectively

 Hemophilia A affects: 20.6 per 100,000 males

– Severe: 50-60%

 Hemophilia B affects: 5.3 per 100,000 males

– Severe: 44%

 Inherited as a recessive X-linked trait (Mom would be an

unaffected carrier)

 ~30 % of cases of hemophilia are new mutations

 VIII concentration and symptoms
Concentration,  g/l Symptoms

100 – 50

50 - 30 Intensive and long lasting

bleeding after big trauma
30 – 5 Long bleeding after surgery and
minor trauma
5–1 Intensive/long bleeding after
minor trauma
<1 Spontaneous bleeding
Types of bleeds in hemophilia

 Joint bleeding - hemarthrosis

 Muscle hemorrhage
 Soft tissue
 Life threatening-bleeding
 Other
 Hemophilic Arthropathy
 As blood is catabolized, it is absorbed by
synovium
 Iron is toxic to cells – synovial cells
disintegrate releasing lysosomes which
destroy cartilage and inflame synovium
 Hypertrophic, hypervascular synovium
 Chondrocytes also affected
 FIBROSIS
Acute and Chronic Hemophilic Arthropathy
 Von Willebrand’s Disease
 Most common inherited disorder
 Usually autosomal dominant
 Caused by mutation in vWF gene
 Split into several subgroups
 May be quantitative or qualitative defect
Role of vWF
 Multimeric glycoprotein
secreted by vascular
endothelium and
megakaryocytes
 Carries and stabilises
Factor VIII in plasma
 Mediates adhesion of
platelets to damaged
endothelium
 Laboratory Diagnosis
 APTT may be prolonged, PT normal
 Bleeding time may be prolonged
 F VIII and vWF levels usually reduced
 Defective Platelet Aggregation
 Types of
von Willebrand Disease
 Type I: quantitative - normal multimers but decreased amounts
 Type 2: qualitative abnormality in VWF
– 2A: absence of (the hemostatically most effective) large multimers
of VWF
– 2B: the large multimers of VWF are abnormal, having a
heightened affinity for platelets
 Type 3: severe form of VWD- extremely low levels of VWF and
FVIII