Transcript INHERITED THROMBOPHILIA

PATHOPHYSIOLOGY OF THROMBOSIS
“Virchow’s Triad”
1. Injury to blood vessels
Trauma, atherosclerosis, surgery
2. Stasis of blood
Immobility, venous incompetence, heart failure
3. Increased coagulability of blood “thrombophilia”
Various inherited and acquired conditions

In general, vessel injury is the most important
contributing factor to arterial thrombosis (heart
attack, stroke) while stasis and increased
coagulability are more important in venous
thrombosis
INHERITED THROMBOPHILIA
• Venous >> arterial thrombosis
• Most venous thrombi in legs
– Occasionally mesenteric, portal, cerebral,retinal veins
• Prevalence of thrombosis varies between families
• Thrombotic problems may begin in 20s and 30s –
rarely in childhood
• About half of thrombotic episodes occur in
association with other identifiable risk factors
(pregnancy, oral contraceptives, surgery, etc)
DEEP VENOUS THROMBOSIS
PULMONARY EMBOLISM
Arrow points to large
clot in pulmonary
artery
Clot dissolved after
administration of
fibrinolytic drug
THERE ARE MANY POTENTIAL GENETIC CAUSES
OF THROMBOPHILIA
“If something can go wrong, it will”
(Murphy)
THERE ARE FIVE KNOWN CAUSES OF
INHERITED THROMBOPHILIA
Defects in physiologic anticoagulant pathways
1.
2.
3.
4.
Antithrombin deficiency
Protein C deficiency
Protein S deficiency
Factor V Leiden
Increased production of procoagulant
5. Prothrombin G20210A gene mutation
QUANTITATIVE VS QUALITATIVE DEFICIENCY OF
CLOTTING PROTEINS
• Quantitative deficiency: decreased protein
production (gene deletion, nonsense mutation,
etc)
– Both antigen and activity low
– “Type I” deficiency
• Qualitative deficiency: normal protein production,
decreased activity (missense mutation)
– Antigen normal, activity low
– “Type II” deficiency
ANTITHROMBIN
AKA “ANTITHROMBIN III”
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Serine protease inhibitor
Made in liver
20 mg/dl plasma concentration
Inhibits thrombin, Xa, other clotting
enzymes
• Activity enhanced by heparin and heparinlike molecules on endothelium
THE ANTITHROMBIN SYSTEM
Serine protease mechanism
ANTITHROMBIN-HEPARIN
INHIBITORS OF MULTIPLE STEPS IN THE CLOTTING CASCADE
XIIa
TF
VII(a)
Inhibits all serine protease
clotting factors except VIIa
XIa
VIIIa
IXa
ANTITHROMBIN
HEPARIN
Xa
IIa
Va
INHERITED ANTITHROMBIN DEFICIENCY
• Prevalence: 0.2-0.4% of population; 2-5% of
inherited thrombophilia
• Dominant inheritance with variable
penetrance
– No homozygotes known (lethal?)
ANTITHROMBIN ASSAYS
• Patient plasma + heparin + thrombin
– Thrombin activity measured with chromogenic
substrate
– Measure decay of thrombin activity with time
• Detects both quantitative and qualitative
deficiency
• Other serine protease inhibitors in plasma may
contribute to measured activity causing decreased
sensitivity
• Alternative assay uses factor Xa rather than
thrombin, greater specificity and sensitivity
THE PROTEIN C SYSTEM
• PROTEIN C
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–
–
Proenzyme precursor of serine protease
Made in liver, vitamin K-dependent
0.4 mg/dl in blood
When activated by thrombin, degrades Va and VIIIa
• PROTEIN S
–
–
–
–
No intrinsic enzymatic activity
Made in liver, endothelium, vitamin K-dependent
Bound/inactive and free/active forms in plasma
Cofactor for protein C
• THROMBOMODULIN
– Endothelial cell surface component
– Binds thrombin
– TM-bound thrombin activates protein C
THE PROTEIN C SYSTEM
Vi
VIIIi
Va
VIIIa
APC
+
PS
IIa
PC
PC
IIa
IIa
TM
EC
TM
EC
PROTEIN C DEFICENCY
• Dominant form: 30-60% of normal protein C
activity in blood
– Found in about 5% of inherited thrombophilia
– Both quantitative and qualitative deficiency can occur
• Recessive form: < 10% of normal protein C activity
– Parents (heterozygous) have about 50% of normal level,
asymptomatic
– Rare affected individuals (homozygous) have severe
thrombotic tendency that may begin in infancy
• Biologic basis for dominant vs recessive forms
unknown
DOMINANT INHERITANCE OF PROTEIN C DEFICIENCY
32
30
24
22
Protein C
deficient
19
30
26
21
Protein C
normal
22
18
17
13
8
History of
thrombosis
RECESSIVE INHERITANCE OF PROTEIN C DEFICIENCY
HOMOZYGOUS PROTEIN C DEFICIENCY
CAUSES NEONATAL PURPURA FULMINANS
PROTEIN C LEVELS DROP FASTER THAN LEVELS
OF OTHER VITAMIN K-DEPENDENT PROTEINS
DURING WARFARIN TREATMENT
Prothrombin
Protein C
WARFARIN-INDUCED SKIN NECROSIS IN A
PROTEIN C-DEFICIENT PATIENT
PROTEIN C ASSAYS
• Immunologic
– Detects only quantitative deficiency
• Functional, chromogenic substrate
– Snake venom enzyme activates protein C in test plasma
– Activated protein C cleaves chromogenic substrate
– Detects quantitative, most qualitative deficiency
• Functional, clotting time-based
– Detects any deficiency
– Not useful in patients taking warfarin
PROTEIN S
Crossed immunoelectrophoresis showing bound and free forms
Bound
(inactive)
Free
(active)
PROTEIN S DEFICIENCY
•
Dominant inheritance, prevalence unknown
Found in about 5% of inherited thrombophilia
•
Three patterns of deficiency
1. Reduced (30-60%) total protein S antigen with
proportionate reduction in free protein S
2. Reduced free protein S with normal total protein S
antigen
3. Reduced protein S activity with normal total and free
protein S antigen
PROTEIN S ASSAYS
• Total protein S (immunologic)
– Detects only type 1 deficiency
• Free protein S (immunologic)
– Detects type 1 and type 2 deficiency
• Protein S activity
– Theoretically should detect any deficiency
– Some assays give false positive result in
patients with activated protein C resistance due
to factor V Leiden
PROTEIN C AND S
Acquired deficiency states
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Warfarin treatment
Vitamin K deficiency
Liver disease
Newborn
DIC (protein C)
Inflammation (free protein S)
Pregnancy (protein S)
Oral contraceptive use (protein S)
MEASURING PROTEIN C AND S IN
WARFARIN-TREATED PATIENTS
• Problem: warfarin causes decreased protein C
and protein S level
• Solution: compare levels of these proteins to
another vitamin K-dependent protein (factor X)
• Low ratio of protein C or S to factor X suggests
underlying deficiency state
• Requires steady state warfarin treatment (same
dose for at least a week)
• Only applicable to antigen measurements
Factor V Leiden
• Missense mutation changes amino acid 506 of
factor V from arginine to glycine
• Mutation is at preferred protein C cleavage site,
slows inactivation of factor Va by protein C
• Factor Va procoagulant activity not affected
• Single mutation responsible for almost all
cases
• Very common (up to 5% of population
heterozygous)
• Accounts for up to 50% of inherited
thrombophilia
Vi
VIIIi
Va
VIIIa
APC
+
PS
IIa
PC
PC
IIa
IIa
TM
EC
TM
EC
MODIFIED FUNCTIONAL ASSAY FOR FVL
1.
Mix patient plasma with factor V deficient plasma (1:4)
2.
Plasma mixture
aPTT
3.
Mixture + APC
aPTT
4.
aPTT with APC
APC ratio =
aPTT without APC
THE FACTOR V LEIDEN MUTATION
DNA TESTING FOR FACTOR V LEIDEN
FVL DNA AMPLIFIED BY PCR, DIGESTED WITH RESTRICTION ENZYME
NORMAL HETEROZYGOUS HOMOZYGOUS
PROTHROMBIN G20210A GENE MUTATION
• Mutation in 3' untranslated (non-coding) part of
prothrombin gene
• No effect on prothrombin structure or function
• Heterozygotes have 5-10% higher plasma levels of
prothrombin
• Heterozygotes have 2-3 fold risk of venous
thromboembolism
 Risk in homozygotes uncertain
• About 1-2% of population heterozygous; 5-7% of
young patients with DVT/PE
• Diagnosis: DNA testing
COMPARISON OF INHERITED THROMBOPHILIAS
Phenotype
Number of
genotypes
Approx prevalence
in thrombophilia
Approx relative
risk of thrombosis
Antithrombin
deficiency
Many
5% or less
Up to 10 (varies
with mutation)
Protein C
deficiency
Many
5%
Up to 10 (varies
with mutation)
Protein S
deficiency
Many
5% or less
Up to 10 (varies
with mutation)
Factor V Leiden
One
40-50%
3-7
Prothrombin
G201210A
One
5-10%
2-3
INHERITED THROMBOPHILIA: GENE DOSE
Relative risk of thrombosis in heterozygous
and homozygous factor V Leiden
Genotype
Relative Risk
Normal
1
Heterozygous
7
Homozygous
80
Rosendaal et al, Blood 1995;85:1504
INHERITED THROMBOPHILIA: GENE
INTERACTIONS
Co-inheritance of protein C deficiency and
factor V Leiden within a family
Gene Mutation
Thrombosis
present (%)
Thrombosis
absent (%)
Protein C and
Factor V
16 (73)
6 (27)
Protein C
5 (31)
11 (69)
Factor V
2 (13)
11 (87)
None
0
11 (100)
Koeleman et al, Blood 1994;84:1031
RISK OF VENOUS THROMBOSIS
Factor V Leiden plus oral contraceptive
RISK FACTOR
RELATIVE RISK OF
THROMBOSIS
Oral contraceptive
4
Factor V Leiden
8
Both
35
Vandenbroucke et al, Lancet 1994;344:1453
INHERITED THROMBOPHILIA IS A RISK FACTOR,
NOT A DISEASE
Thrombophilia is a weak
(not statistically
significant) predictor of
recurrence in patients
with venous thrombosis
RISK OF VENOUS THROMBOSIS IN AFFECTED VS UNAFFECTED
RELATIVES OF THROMBOPHILIC PATIENTS
Unaffected (no defect)
PT
FVL
PC
AT
PS
FVL, PT mutation:
Most carriers remain
asymptomatic
PC, PS, AT deficiency:
Higher chance of
thrombosis, but many
carriers asymptomatic
Blood 2009;113:5314
TESTING FOR INHERITED THROMBOPHILIA
When is it indicated?
• Young patient
• Family history
• Thrombosis in absence of known risk factors
• Warfarin-induced skin necrosis (protein C)
• Neonatal purpura fulminans (protein C, S)
DIAGNOSIS OF INHERITED THROMBOPHILIA
What's next?
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Rapid, cheap (?) screening for large numbers of
mutations and polymorphisms using DNA chip
technology
More accurate diagnosis of inherited antithrombin,
protein C, protein S deficiency
Discovery of many new genetic conditions that
affect thrombotic risk
More information than we know what to do with