Thrombotic Disorders
1
THROMBOTIC DISORDERS
A thrombotic disorder is an acquired or hereditary disorder predisposing to the
unnecessary formation of a thrombus (clot), which is the end result of activation of the
coagulation cascade. So in essence, a thrombotic disorder results either from the undue
activation or lack of inhibition of the coagulation cascade.
REGULATION OF THROMBOSIS:
Since thrombus formation starts from the formation of a platelet plug at the site of
vascular injury followed by deposition of fibrin as a result of activation of the coagulation
cascade, inhibitory factors exist at both levels that limit the formation of a thrombus
beyond what is physiologically required by the body.
INHIBITION OF PLATELETS:
As soon as an adequate platelet plug is formed, APD and thromboxane A2 are dissipated
in the circulation limiting the size of the thrombus physiologically required. In addition,
direct inhibition of the platelets by ADPase, prostacyclin, and nitric oxide is achieved.
INHIBITION OF COAGULATION CASCADE:
The control of the coagulation cascade occurs at multiple levels:
1. Inhibition of thrombin by a protein called anti-thrombin III (AT)
2. Inactivation of activated factor V (FVa) and VIII (FVIIIa) by activated protein C
(APC) and protein S inhibition pathway
3. Tissue factor pathway inhibition (TFPI)
4. Fibrinolysis
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Thrombotic Disorders
2
ANTITHROMBIN:
Antithrombin III (AT-III) is a plasma proteinase inhibitor synthesized in the liver that
inactivates thrombin and other enzymes responsible for the generation of thrombin. The
name antithrombin is somewhat misleading as it implies that this protein inhibits only
thrombin when in fact it inhibits many other activated coagulation proteins including
factors Xa, IXa, XIa, XIIa, and pre-kallikrein-HMWK in the intrinsic pathway and VIIaTF in the extrinsic pathway. This combination of being able to block thrombin-mediated
fibrin formation and inhibit enzymes in the pathways responsible for prothrombin
activation makes AT-III a powerful and important endogenous anticoagulant molecule
and explain why patients with even modest AT-III deficiencies may experience clinical
thrombosis.
[This picture is adapted from Hemostasis & Thrombosis by Colman et al. 4th edition. Lippincott-Williams & Wilkins]
Antithrombin III inhibits its target protein substrates by direct binding. This inhibition is
very efficient in the presence of cofactors such as heparin cofactor II (HcII) in blood.
Endothelial cell surface and subendothelial matrix contain heparan sulfate proteoglycans
(HSPGs) that bind to antithrombin III and form a complex on their surface. When after
vascular injury thrombin is formed AT-III in complex with HSPGs binds to thrombin and
other coagulation factors leading to their clearance. Circulating AT-III in complex with
HcII does the same job at the site of clot formation. Antithrombin III in complex with
thrombin and other activated coagulation factors is cleared from circulation by liver.
TISSUE FACTOR PATHWAY INHIBITOR (TFPI):
Tissue factor pathway inhibitor (TFPI) is a plasma proteinase inhibitor that regulates
tissue factor-mediated coagulation by producing factor Xa-dependent feedback inhibition
of factor VIIa-Tissue factor (VIIIa-TF) complex. The inhibition occurs in 2 steps: first,
TFPI binds to Xa and inactivates it then in the second step TFPI-Xa complex binds to
VIIa-TF complex and causes its inhibition resulting in the lack of activation of the
coagulation cascade by the extrinsic pathway.
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Thrombotic Disorders
3
ACTIVATED PROTEIN-C (APC) AND PROTEIN-S PATHWAY:
Two proteins called protein C and protein S, both synthesized in liver and vitamin Kdependent, are an important mechanism in the control of thrombus formation. Protein C,
a serum protease, binds to another protein called thrombomodulin (TM) in complex with
thrombin (T) and also binds to its receptor on the endothelial cell surface (EPCR) and
becomes activated. The activated protein C (APC) cleaves activated coagulation factors
V (FVa) and VIII (FVIIIa) with the help of its non-enzymatic cofactor, protein S. In fact,
protein S can independently cleave Va, VIIIa and also Xa. About 60% of the protein S is
inert and bound to C4BP, a component of the complement system and the remaining 40%
is free and is the active form. The cleavage of activated factor V and VIII to their
inactivated forms (Vi & VIIIi) takes place on the lipid bi-layer surface of platelets.
[This picture is adapted from BLOOD: Principles & Practice of Hematology by Handin et al. Lippincott Press]
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Thrombotic Disorders
4
CLASSIFICATION OF THROMBOTIC DISORDERS:
A hypercoagulable state is suspected in a patient with a documented thrombotic disorder
with the following presentations:
 Age below 40
 Venous thrombosis in unusual sites such as
(a) Cerebral veins and sinuses
(b) Retinal vein thrombosis
(c) Renal vein thrombosis
(d) Venous thrombosis in portal venous system including mesenteric veins,
splenic veins, portal vein, hepatic vein
 Recurrent thrombosis
 Family history of thrombosis
 Repeated thrombosis despite anticoagulation therapy
 Recurrent fetal loss; HELLP syndrome
 Apparently unprovoked thrombosis
Hypercoagulable states can be divided into two general categories:
1. Inherited disoders
(i)
Antithrombin III deficiency or dysfunction
(ii)
Protein C deficiency
(iii) Protein S deficiency
(iv)
Resistance to activated protein C by factor V (factor V Leiden)
(v)
Prothrombin polymorphism (G20210A)
(vi)
Heparin cofactor II deficiency
(vii) Dysfibrinogenemia –such as fibrinogen Marburg, fibrinogen Paris V
(viii) Hyperhomocysteinemia – associated with arterial thrombosis
2. Acquired disorders and hypercoagulable states
(i)
Antiphospholipid syndromes – discussed later
(ii)
Paroxysmal Nocturnal Hemoglobinuria (PNH) – cerebral vein
thrombosis is a feature
(iii) Myeloproliferative disorders – increased risk for portal venous system
thrombosis
(iv)
Acute promyelocytic leukemia – cells release procoagulant proteins
(v)
Mucin-secreting adenocarcinomas –
(vi)
Nephrotic syndrome – increased renal loss of anticoagulant proteins
(vii) Hyperviscosity syndromes – Waldenstrom’s macroglobulinemia
Hyperlipidemia – in particular with type II hyperbetalipoproteinemia
(viii) Pregnancy & high estrogen states – complex mechanisms
(ix)
Immobilization – venous stasis predisposing to formation of thrombus
(x)
Heparin induced thrombocytopenia syndrome – discussed elsewhere
(xi)
Thrombotic thrombocytopenic purpura – discussed elsewhere
(xii) Disseminated intravascular coagulation (DIC) – discussed elsewhere
(xiii) Intravascular devices
(xiv) A variety of other conditions (Behcet’s syndrome, ulcerative colitis)
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Thrombotic Disorders
5
INHERITED AND ACQUIRED THROMBOTIC DISORDERS
Acquired disorders are more commonly associated with venous thromboembolism
(VTE). Nevertheless, hereditary predisposition is increasingly being identified in, at least,
some of the patients thought to have VTE as a result of an acquired disorder. Also,
spontaneous VTE in the absence of an identifiable acquired disorder is an area of intense
research because in a large proportion of individuals with an arterial or venous
thrombosis no known genetic predisposition to VTE could be demonstrated.
HEREDITARY RISK FACTORS OF THROMBOPHILIA:
ACTIVATED PROTEIN C (APC) RESISTENCE / FACTOR VLEIDEN MUTATION:
This is the most common of all known risk factors for hereditary venous
thrombophilia. As you learned previously that activated protein C (APC) is required for
inactivation of activated coagulation factors V (FVa) and factor VIII (FVIIIa) such that
resistance of factor V to cleavage by protein C results in an undue prolonged activation of
factor V favoring a thrombophilic state. This resistance is most commonly caused by a
single base substitution at position 1691 (G to A) which results in the formation of a
mutated factor V called factor VLeiden (First described in Leiden, Netherland) in which
the normal Arginine amino acid at position 506 is replaced by Glutamine (Arg506 to
Gln). This mutated factor VLeiden is resistant to cleavage by APC. Resistance of factor V
to APC has also been seen with other mutations in factor V (Factor VCambridge). The
normal activation and inactivation of factor V is shown below:
Fig. The top part shows a factor V molecule that is cleaved at three different sites (709, 1018 & 1545) by
thrombin or activated factor X (Xa) for full cofactor activity (Va). After cleavage heavy and light chains
are held together by a metal ions (Me). After serving as an activated cofactor, the activated factor V (Va) is
physiologically shut down by cleavage by protein C at site 306 & 506 releasing inactivated factor V (iVa).
[This picture is adapted from Williams Hematology by Beutler et al. 6th edition. McGraw Hill Press]
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Thrombotic Disorders
6
ATTRIBUTES OF FACTOR VLEIDEN MUTATION:
Occurrence
General population
4-7% (Caucasians)
Cause of First DVT
In 20% cases
Thrombophilic families
40% incidence
Genetic Basis
Inheritance
Mutation
Autosomal dominant
Exon 10 (G to A at position 1691)
Physiologic Basis
Derangement
Basis
Lack of inactivation of activated factor V
Resistance of factor Va to cleavage by APC
Risk for thrombosis
Homozygous
Heterozygous
Relative risk of VTE increased to 80-fold
Relative risk of VTE increased to 7-fold
Laboratory testing
Assay
Functional assay for activated protein C
PCR assay for factor VLeiden mutation detection
PROTHROMBIN GENE MUTATION (G20210A):
A specific mutation in the gene for prothrombin, which results in the replacement of
Guanine (G) by Adenine (A) at position 20210 results in elevated levels of prothrombin.
This is thought to create a hypercoagulable state by providing more substrate and hence
more thrombin generation once the coagulation cascade is physiologically activated. It is
speculated that subclinical physiologic activation of coagulation at local sites of minor
injury in individuals with this mutation results in formation of unusually larger and/or
frequent clot formation that would not have occurred in individuals without this mutation.
ATTRIBUTES OF PROTHROMBIN GENE MUTATION (G20210A):
Occurrence
General population
1-2% heterozygosity (Caucasians)
Cause of First DVT
In 6% cases
Thrombophilic families
18% incidence
Genetic Basis
Inheritance
Mutation
Autosomal dominant
G to A at site 20210 in the 3`untranslated region
Physiologic Basis
Derangement
Basis
Increase thrombin formation
Elevated prothrombin (factor II) in plasma
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Thrombotic Disorders
7
Risk for thrombosis
Homozygous
Heterozygous
Increased risk of VTE & also for arterial thrombosis
Relative risk of VTE increased to 3-fold
Laboratory testing
Assay
PCR assay for prothrombin 20210 mutation
PROTEIN C DEFICIENCY:
As we learned previously that under physiologic conditions, protein C is converted to
its activated form (APC) by thrombin:thrombomodulin complex on the endothelial cell
surface after which APC inactivates factors Va and VIIIa. Lack of protein C function
could be result of a quantitative or a qualitative defect.
Type 1: Quantitative defect; (1a) Partial deficiency, (1b) Total deficiency
Type 2: Qualitative defects
ATTRIBUTES OF PROTEIN C DEFICIENCY:
Occurrence
General population
0.2% heterozygous (Caucasians)
Cause of First DVT
In 3% cases
Thrombophilic families
6% incidence
Genetic Basis
Inheritance
Mutation
Autosomal recessive
Many (>150 mutation)
Physiologic Basis
Derangement
Basis
Unregulated coagulation, decreased fibrinolysis
Decreased inactivation of Va and VIIIa
Risk for thrombosis
Homozygous
Heterozygous
Neonatal purpura fulminans, cerebral vein thrombus
Relative risk of VTE increased to 7-fold
Laboratory testing
Assay
Functional assay detecting decreased anticoagulant
activity or antigen level
PROTEIN S DEFICIENCY:
Protein S serves as a cofactor for protein C in the cleavage of factor Va and VIIIa and
hence its deficiency either quantitative or qualitative also predisposes for thrombosis.
Type 1
Type 2
Type 3
Protein S antigen
Protein S activity
Total
Free
Low
Low
Low
Normal
Normal
Low
Normal
Low
Low
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Thrombotic Disorders
8
ATTRIBUTES OF PROTEIN S DEFICIENCY:
Occurrence
General population
Uncertain
Cause of First DVT
In 1-2% cases
Thrombophilic families
6% incidence
Genetic Basis
Inheritance
Mutation
Autosomal dominant
Many (>150 mutation)
Physiologic Basis
Derangement
Basis
Reduced protein C function
Decreased cofactor activity for protein C
Risk for thrombosis
Homozygous
Heterozygous
Neonatal purpura fulminans, cerebral vein thrombus
Relative risk of VTE increased to 6-fold
Laboratory testing
Assay
Functional assay, decreased “free” protein S activity
ANTITHROMBIN III DEFICIENCY:
Antithrombin III (ATIII) is a major inhibitor of proteinase coagulation factors.
Type 1:Quantitative defect
Type 2:Qualitative defect (Thrombin binding site defects)
Type 3:Qualitative defect (Heparin binding site defects)
ATTRIBUTES OF ANTITHROMBIN III DEFICIENCY:
Occurrence
General population
0.02%
Cause of First DVT
In ~1% cases
Thrombophilic families
4% incidence
Genetic Basis
Inheritance
Mutation
Autosomal dominant
Many (~200 mutation)
Physiologic Basis
Derangement
Basis
Reduced antithrombin III activity
Reduced inhibition of thrombin & other proteinases
Risk for thrombosis
Homozygous
Heterozygous
Laboratory testing
Assay
Lethal in utero
Relative risk of VTE increased to 5-fold
Functional assay for decreased activity
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Thrombotic Disorders
9
ACQUIRED RISK FACTORS OF THROMBOPHILIA
ANTIPHOSPHOLIPID SYNDROME (APLS):
Antiphospholipid syndrome is a clinicopathologic entity characterized by both venous
and arterial thromboembolism, including thrombotic strokes, and recurrent fetal loss,
and prolongation of in vitro phospholipid based coagulation tests such as aPTT, PT, and
dilute Russell Viper Venom Time (dRVVT). It is perhaps the most common acquired risk
factor for unprovoked thrombosis. Antiphospholipid antibodies (APLA) include a broad
family of autoantibodies that include
(a) Lupus anticoagulant (LA) – so named because it was first identified in
patients with systemic lupus erythromatosus (SLE); the term anticoagulant is a
misnomer because they cause thrombosis instead of bleeding.
(b) Anticardiolipin antibodies (ACA) – so named because it was first shown to
react with cadiolipin from bovine heart in the test reagent.
These APLAs are immunoglobulins (IgG, IgM, IgA and mixtures) that occur in <5% of
normal adult individuals, in a proportion of patients with certain infections and with a
higher frequency in patients with autoimmune disorders.
FEATURES OF ANTIPHOSPHOLIPID SYNDROME:
A) Clinical features:
1. Venous and arterial thromboembolism
2. Unexplained pregnancy losses
3. Thrombocytopenia
4. Stroke
5. Coronary artery disease
6. Livedo reticularis skin lesions, necrotizing skin vasculitis
7. Microangiopathy
8. Other features
B) Laboratory criteria: either 1 or 2 or both
1. Anticardiolipin antibodies identified on two or more occasions at least 6
week apart as measured by enzyme linked immunosorbant assay (ELISA)
2. Lupus anticoagulant antibodies detected on two or more occasions at least 6
week apart in the following manner
(a) Prolongation of aPTT, PT, kaolin clotting time, dRVVT
(b) Failure to correct the prolonged coagulation time by mixing studies
(c) Correction of coagulation time by the addition of excess phospholipid in
the test reagent
(d) Exclusion of other coagulopathies
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Thrombotic Disorders
10
THROMBOPHILIC PREDISPOSITION:
There are many hereditary predispositions, any one or several of which can be
complicated by an acquired illness, the combination of which could amplify the
thrombotic risk. The thrombotic risk of a given hereditary predisposition or an acquired
illness varies considerably, such that a low-risk condition alone is unlikely to cause a
clinical venous thromboembolic disease (VTED). A combination of two genetic factors
or a genetic factor plus an acquired factor could amplify the risk to a modest or high
level. Some hereditary and acquired disorders carry more risk than others.
RISK
Low
HEREDITARY
ACQUIRED
Heterozygous factor VLeiden
General surgery
Heterozygous prothrombin 20210 Oral contraceptive/Pregnancy
mutation
Immobilization
Sickle cell anemia
Elevated factor VIII levels
Moderate
Heterozygous protein C deficiency
Heterozygous protein S deficiency
Heterozygous AT-III deficiency
High
Homozygous factor VLeiden
Surgery for total hip or
Homozygous prothrombin 20210 replacement
mutation
Hip fracture
Acute promyelocytic leukemia
Surgery for malignancy
Sepsis
Antiphospholipid syndromes
Myeloproliferative syndromes
Paroxysmal nocturnal hemoglobinuria
knee
Excessive Homozygous protein C deficiency
Mucin secreting adenocarcinomas
Homozygous protein S deficiency
Homozygous AT-III deficiency
Double heterozygous for protein C,
protein S and AT-III deficiency.
Heterozygosity for factor VLeiden
mutation + heterozygosity for
protein C, S or AT-III deficiency
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Thrombotic Disorders
11
LABORATORY TESTING:
In a patient with clinical diagnosis of thrombosis, following approach may be taken. In
addition prothrombin gene mutation (G20210A) and fibrinogen levels are also
recommended. Laboratory testing is best performed several weeks after completion of a
course of oral anticoagulants in patients with thrombosis, to avoid confounding effects of
acute thrombosis or heparin or warfarin therapy on the assay results. With the exception
of protein C and protein S assays, all other testing can be performed in patients taking
oral anticoagulants.
[This picture is adapted from Dacie & Lewis Practical Haematology by Lewis et al. 9th ed. Churchill-Livingstone]
FBC
MSU
FOB
CXR
PNH
MPD
VTE
APCR
FVL
LAC
ACLA
AT
FVIII
Lp (a)
TG
HDL
LDL
Full blood count (complete blood count, CBC)
Midstream urine specimen for evaluation of nephritic syndrome
Fecal occult blood for gastrointestinal cancers
Chest X-ray for lung cancers
Paroxysmal nocturnal hemoglobinuria
Myeloproliferative disorders
Venous thromboembolism
Activated protein C resistance assay
Fcator V Leiden assay
Lupus anticoagulant
Anticardiolipin assay
Antithrombin III
Factor VIII assay
Lipoprotein little a
Triglycerides
High density lipoprotein
Low density lipoprotein
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Thrombotic Disorders
12
INDICATIONS FOR LABORATORY TESTINGS:
A laboratory testing for thrombophilia should be performed if the results of testing could
make a difference in the clinical care of the patient or family members. These conditions
include:
 Changes in the duration or intensity of oral anticoagulation
 Administration of specific therapy (antithrombin concentrates, vitamins for
homocyteinemia)
 More intense prophylaxis for high-risk situations (e.g. surgery, acute illness)
 Prolonged immobilization (transcontinental flights) with a family history of
thrombosis
 Better accuracy in estimates of the future risk of thrombosis in clinical settings
(surgery and pregnancy)
 Counseling of women as to the risks of oral contraceptives, pregnancy or
hormone replacement therapy
 Study of family members at risk of thrombosis
FUNCTIONAL ASSAYS:
(a) Activated protein C resistance
(b) Protein C levels
(c) Protein S levels
(d) Antithrombin III levels
(e) Factor VIII levels
PLASMA LEVELS:
(a) Plasma homocystein levels
(b) Plasma fibrinogen levels
MOLECULAR TESTS (POLYMERASE CHAIN REACTION):
(a) Factor VLeiden mutation
(b) Prothrombin gene mutation (G20210A)
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