inherited thrombophilias
TRANSCRIPT
Inherited Risk Factors for Venous Thromboembolism
22/11/12
Introduction• Inherited thrombophilia is a genetic tendency
to venous thromboembolism• Combination of factors are usually present in
the development of thrombosisAcquired
prothrombotic stimulus
One or more prothrombotic
mutationsThrombosis
Inherited Thrombophilias
PrevalencePrevalence of Defects in Patients with
Venous ThrombosisActivated protein C resistance (factor V
Leiden)12–40%
Prothrombin gene mutation (G to A transition at position 20210 in the 3’-
untranslated region)
6–18%
Deficiencies of antithrombin III, protein C, protein S
5–15%
Antiphospholipid antibody syndrome 5–10%
CAVEAT: All data for caucasians.
Inherited Thrombophilias
• Commonly grouped according to nature of defect
1. Deficiencies or qualitative abnormalities of inhibitors of activated coagulation factors
2. Impaired clot lysis3. Metabolic defects4. Abnormalities of coagulation factors or
cofactors
Disorder Inheritance Prevalence
Deficiency or qualitative abnormalities of inhibitors of activated coagulation factors
AT deficiency AD 1-2%
TM deficiency AD 1-5%
Protein C deficiency AD 1-5%
Protein S deficiency AD 1-5%
APC resistance, Factor V Leiden AD 20-30%
Impaired Clot lysis
Dysfibrinogenemia AD 1-2%
Plasminogen deficiency AD,AR 1-2%
TPA deficiency AD Unknown
Excess PAI-1 activity AD unknown
Metabolic defect
Hyperhomocysteinemia Not known 10-25%
CBS,MTHFR deficiency AR ~1 in 300000 LB
Coagulation factor abnormality
Prothrombin gene mutation AD 5-10%
Elevated Factor VIII levels Not known 20-25%
Elevated Factor IX, X, XI levels Not known ~10%
The Leiden Thrombophilia Study
Incidence of Inherited Thrombophilias in a Western Indian Population
Unknown45%
Protein C10%
Protein S6%
Antithrombin3%
Factor V Leiden
3%
MTHFR15%
ACLA10% LA
8%
Incidence in 432 patients
Ghosh K et al Clin Appl Thromb Hemost 2001 7: 158
Incidence of Inherited Thrombophilias in a Northern Indian Population
• 431 patients • Low levels of protein C were detected in 21.1% – 11.3% were attributed to acquired factors.
• Protein S deficiency was found in 19.0% – 10.4% cases were associated with acquired risk factors.
• Antithrombin deficiency was detected in 6.4% of patients– 4.8% were secondary to acquired factors.
• Activated protein C resistance (APC-R) was present in 12.5% cases.
Bhattacharya M et al, Ind J Path Micro.2003, 46(4):621-624
APC resistance and Factor V Leiden
• 1st described in 1993 by Dahlback et al in a group of
individuals with unexplained venous
thromboembolism whose plasmas exhibited a poor
response to activated protein C (APC) in an activated
partial thromboplastin time assay
• Heterozygosity for the factor V Leiden mutation
accounts for 90-95% cases
Physiology of APC
resistance
Position 506 replacement of arginine by glutamine
Decreased inactivation of Factor Va and persistent coagulation activity
Dual defect leading to hypercoagulable state
• Decreased anticoagulation – Factor V cleaved at position 506 is also thought to
be a cofactor along with protein S, in supporting the role of activated protein C in the degradation of factor VIIIa (in the tenase complex) as well as factor Va (in the prothrombinase complex).
– Lack of this cleavage product anticoagulant activity of APC
Genetics• Heterozygosity for factor
V Leiden– Caucasians — 5.3 %– Hispanic Americans — 2.2
%– Native Americans — 1.2 %– African Americans — 1.2 %– Asian Americans — 0.45 %
• Homozygosity for factor V Leiden – ~1% of patients
• Other Defects• Mutations at the Arg306
residue – Arg306 with threonine
(factor V Cambridge)– Arg306 with glycine– Factor V Liverpool
(Ile359Thr)• HR2 haplotype
– Described in 1996– Group of 6 polymorphisms
assoc with FV activity and APC resistance
Other genetic influences• Blood Group – Non-O blood group risk of
developing VTE in both heterozygotes and homozygotes for factor V Leiden by two- to four-fold
• Protein Z mutations• SNPs in Factor V gene may decreased
normal factor V levels in heterozygotesBlood Transfus. 2012 Oct11:1-5.
Clinical Manifestations• Venous thrombosis affecting
multiple sites• Most commonly DVT of the
lower limbs– 10 -26% of such patients have this
mutation• Risk of recurrent DVT may also
be increased• Cerebral Venous Thrombosis
and Abdominal vein thrombosis including the Budd Chiari Syndrome
• Important interaction with other acquired risk factors e.g. OCP use, Obesity, Smoking
Factors Risk Incidence(per 100 person-years)
Normal - 0.008
Prothrombin gene mutation
2.8x 0.022
OCP 4x 0.03
FVL heterozygotes
7x 0.057
OCP + FVL 35x 0.285
FVL Homozygotes
80x 0.5-1
Prothrombin gene mutation
• Vitamin K dependent factor produced by the liver
• GA transition at nucleotide 20210 in the 3'
untranslated region of the prothrombin gene
increased risk of thrombophilia
• Heterozygous carriers have 30 percent higher
plasma prothrombin levels than normal
Blood. 1996;88(10):3698.
Prevalence
• Prevalence highly variable• White population heterozygous for the allele
varies from 0.7-6.5 %• Extremely rare in the nonwhite (black or
Asian) population• Mutation probably occurred after the
divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations
• May be co-inherited with the FVL mutation
Risk Of Thrombosis• Isolated mutations may not
confer as much thrombotic risk as previously thought
• Combination with other mutations increases the risk
• Double heterozygotes 2.2 % with VTE
• 12 %heterozygous for FVL• 23 % heterozygous for the
prothrombin gene mutation
Odds ratios for the risk of VTE
Prothrombin gene mutation heterozygotes
3.8
Factor V Leiden
heterozygotes
4.9
Both mutations (ie,
double heterozygotes)
20.0
Other prothrombin gene mutations
• Prothrombin C20209T
• Prothrombin A19911G
• Prothrombin Yukuhashi
Antithrombin Deficiency
• Formerly called AT III
• Also known as heparin cofactor I
• Natural anticoagulant, a vitamin K-independent
glycoprotein
• Major inhibitor of thrombin and other coagulation
serine proteases (ie, a serpin), including factors Xa
and IXa
Pathophysiology
• Exists in an active monomer and an inactive "latent" form
• Two active functional sites: the reactive center, Arg393-Ser394; and the heparin binding site
• Progressive antithrombin activity – AT slowly inactivates thrombin in the absence of heparin
• Heparin cofactor activity – In the presence of heparin, thrombin or factor Xa is rapidly inactivated by AT
Genetics of AT deficiency
• First identified heritable thrombophilia• Gene localised to chromosome 1q (SERPINC1)
Seven exons and six introns• Inheritance is usually AD, with variable
penetrance• Two types– Type I – Reduced synthesis of biologically normal
molecules. Proportionately reduced functional and antigenic activity
– Type II – Markedly reduced functional activity but normal immunologic activity
Type II Deficiency
• Prevalence ~0.5-1 % patients with a first
thrombotic event
• 3 subtypes
– Heparin binding site defect
– Thrombin binding site defect
– Pleiotropic defects
Clinical Manifestations
• ~60 % develop recurrent thrombosis• Thrombotic episodes start to occur with some
frequency after puberty, with a peak between 15 and 35 years of age
• Initial thrombotic event occurs spontaneously in ~42 %
• Heparin resistance may be seen in some individuals
Protein C Deficiency
• Vitamin K-dependent protein synthesized in the liver
• Gene for protein C is located on chromosome 2q13-14.
• aPC inactivates coagulation factors Va and VIIIa• Inhibitory effect is enhanced by Protein S• Multiple cytoprotective effects, including anti-
inflammatory activities and protection of endothelial barrier function
Types
• AD inheritance• Type I – More common• Most affected patients are heterozygous• Plasma protein C concentration ~50 percent of
normal in both immunologic and functional assays
• Type II — Normal plasma protein C antigen levels with decreased functional activity
Clinical Manifestations
• Venous thromboembolism in heterozygous and rare homozygous or doubly heterozygous teenagers or adults
• Neonatal purpura fulminans in homozygous or doubly heterozygous newborns
• Warfarin-induced skin necrosis in certain heterozygous teenagers or adults
Protein S Deficiency
• Cofactor of the protein C system• Also serves as a cofactor for protein C
enhancement of fibrinolysis• Synthesized by both hepatocytes and
megakaryocytes and circulates in two forms• 40-50 % as free form, and the remainder
bound to the complement component, C4b-binding protein (C4b-BP)
Genetics of Protein S Deficiency
• Two homologous genes on chromosome 3• PROS1 – 80 kb, 15 exons• PROS2 – pseudogene• Congenital PS deficiency, 1st reported in 1984• Heterozygotes – Thromboembolic
complications• Homozygotes – Neonatal purpura fulminans
Types
• Type I – Classic type of protein S deficiency– Associated with ~50 % of the normal total S
antigen level– More marked reductions in free protein S antigen
and protein S functional activity• Type II – Normal total and free protein S levels
but diminished functional activity• Type III – Total protein S antigen measurements in
the normal range and selectively reduced levels of free protein S and protein S functional activity to <40 %
Clinical Manifestations
• Similar to that of antithrombin or protein C
deficiency
• Age at first thrombotic event was 28 years with a
range between 15 and 68 years
• 56 % episodes are apparently spontaneous
• Free Protein S levels correlate better with risk of
thrombosis
Hyperhomocysteinemia
Prothrombotic properties of homocysteine
• Attenuation of endothelial cell tissue plasminogen activator binding sites
• Activation of factor VIIa and V• Inhibition of protein C and heparin sulfate• Increased fibrinopeptide A and prothrombin
fragments 1 and 2• Increased blood viscosity• Decreased endothelial antithrombotic activity
due to changes in thrombomodulin function
Genetics of Hyperhomocysteinemia
• Most commonly results from production of a
thermolabile variant of methylene
tetrahydrofolate reductase with reduced
enzymatic activity (T mutation)
Clinical Manifestations
• Implicated in atherosclerotic disease and arterial thrombosis
• Venous thrombosis also increased (odds ratio of 2.5-2.95)
• May also be a risk factor for recurrent venous thrombosis
• Patients with other inherited thrombophilias may be at even higher risk (10-50 times)
Elevated Coagulation Factor Levels
• Elevated levels of factors VIII, IX, X and XI have been associated with risk of thrombosis
• fVIII > 150 IU/dL were associated with a 4.8 fold risk of thrombosis
• ~20-25 % patients with venous thrombosis had an elevated fVIII level
• Likelihood of recurrent venous thrombosis was 37 % at 2 years
• Overall risk of recurrence was ~ 7x that of the control population
Koster T , et al. Lancet. 1995 Jan 21;345(8943):152-5.
Elevated Coagulation Factor Levels
• 20 % of patients with venous thrombosis had fIX levels >129 U/dL
• Risk of thrombosis was 2-3x higher than controls• Highest risk was seen in postmenopausal females
who did NOT use OCPs (12x)• Elevated fX levels may also confer an increased risk
of thrombosis in women who do not use OCPs• Data is limited
Joost et al N Engl J Med 2000;342:696-701.
Dysfibrinogenemias
• Qualitative abnormalities of fibrinogen • Inheritance – AD • Heterogenous group of disorders– Bleeding diathesis– Venous or arterial thromboembolism
• Possible mechanism– Defect in release of fibrinogen cleavage– Abnormalities in the binding of thrombin to fibrin
Defective Fibrinolysis
• Plasminogen deficiency– Chromogenic assay
• tPA deficiency– Chromogenic assay– Euglobulin lysis time– ELISA
• Increased PAI-1 levels– ELISA
Screening in Asymptomatic populations
• Unselected population-based screening is not recommended because of
1. The low frequency of the symptomatic condition in the general population
2. The low penetrance of the symptomatic condition among carriers of the most common thrombophilic conditions (eg, factor V Leiden and prothrombin G20210A mutations), AND
3. The lack of a safe, cost-effective, long-term method of prophylaxis if an abnormality is found.
Benefits of Thrombophilia Testing
• Prediction of VT recurrence risk• Improving patient understanding of
thrombosis
• Family testing
Screening for APC resistance
• Principle – APC inhibits factor Va induced prolongation of clotting time
• 1st generation APC resistance assays – aPTT based assays– aPTT is performed in the presence and absence of a
standardized amount of APC, and the two clotting times are converted to an APC ratio
– Ratio is compared to the normal range – Advantage – Simple to perform– Disadvantages – Needs careful standardisation,
determination of normal range. Cannot be used in patients on anticoagulants
Screening for APC resistance
• 2nd generation assays – Highly sensitive and specific– Patient plasma is diluted in a sufficient volume of
factor V-deficient plasma– Either an aPTT-based assay or a tissue factor-
dependent factor V assay is then performed– Can be performed in patients on anticoagulants or
with deranged aPTT• Genetic Testing for FVL
Genetic Testing
• DNA test – for Arg506Gln mutation
• RFLP based PCR test: exon 10 of FV – Uses restriction enzyme MnlI to digest a 220 bp
amplified fragment of patient DNA.
116 37 67
Normal FV – restriction by Mnl1 enzyme
153 67
Mutant FV – restriction by Mnl1 enzyme
220bp
Heterozygote – 4 bands
Normal – 3 bands
Homozygote – 2 bands
Screening for AT Deficiency
• Type I deficiency can be detected by immunoassays that provide quantification
• Type II defects require functional assays– Progressive AT activity assay– AT-heparin cofactor assay – Measures the ability of
heparin to bind to lysyl residues on AT and catalyze the neutralization of coagulation enzymes such as thrombin and factor Xa
• Either a thrombin inhibition assay or a factor Xa inhibition assay depending upon the enzyme that is used.
AT Heparin Cofactor Assay
Antithrombin + heparin
[Antithrombin:Heparin] + thrombin (excess)
[Antithrombin:Heparin:Thrombin] + thrombin (residual)
[Peptide:pNA] Peptide + pNA
Timing of screening for AT Deficiency• AT-heparin cofactor assay should be performed
when the patient is off heparin• Heparin can lower AT levels by ~ 30 %• Oral anticoagulants rarely raise plasma AT
concentrations into the normal range• Acute thrombosis can also lower the
concentration of AT• Optimal time to testing – 2 weeks after
completing the initial 3-6 month course of oral anticoagulant therapy
Screening for Protein C Deficiency
• Antigen estimation – Electroimmunoassay, ELISA, and RIA
• Functional assays – Protein C is activated using thrombin or the thrombin-thrombomodulin complex
• Enzyme activity is assessed using either a chromogenic substrate or by measuring its anticoagulant activity in a factor Xa one-stage clotting assay
Screening for Protein S Deficiency
• Most difficult of the hereditary thrombophilias to document with certainty
• Levels of total or free PS antigen <60-65 IU/dL are considered to be in the deficient range
• Functional assays are not specific as they are also sensitive to the defect characterized by APC resistance
Timing of screening for Protein C and Protein S Deficiency
• Erroneous diagnoses can be made due to the influence of acute thrombosis, comorbid illness, or anticoagulant therapy
• Warfarin therapy reduces functional and, to a lesser extent, immunologic measurements of protein C,S
• Testing is best performed at least 2 weeks after the completion of therapy
Management issues in Inherited Thrombophilias
• Standard therapy with Heparin or LMWH f/b warfarin except in the following situations
1. AT deficiency – May have heparin resistance
2. Protein C deficiency
3. Heterozygous protein C deficiency and a
history of warfarin-induced skin necrosis
Management of AT Deficiency
• May be resistant to heparin in large doses• Patients may have– Unusually severe thrombosis– Recurrent thrombosis despite adequate
anticoagulation– Difficulty achieving adequate anticoagulation
• AT concentrate may be used in these situations• Dose = [desired AT level (%) - baseline level (%) ]
x weight (kg) ÷ 1.4• Plasma levels should be kept >80 %
Protein C Deficiency and Warfarin Induced Skin Necrosis
• Seen in heterozygotes who are given large doses of warfarin without heparin coverage
• Pathogenesis– Protein C has a shorter t1/2
than other vitamin K-dependent proteins
– Protein C levels decrease rapidly once warfarin therapy has been initiated
– Net procoagulant state induces microvascular thrombosis affecting dermal vessels
Management of Protein C deficiency
• Routine screening is not recommended in all cases as– The frequency of asymptomatic hereditary protein C
deficiency is relatively high (eg, one in 200 in a report of healthy blood donors)
– The occurrence of warfarin induced skin necrosis among patients with protein C deficiency is infrequent
– There is difficulty in making a rapid and definitive laboratory diagnosis of the protein C deficiency state
Management of Protein C deficiency
• Warfarin should only be started under cover of heparin
• Initial dose of warfarin should be low• In patients with heteroqygous Protein C
deficiency or a history of skin necrosis– Protein C concentrate of FFP should be used along
with heparin and warfarin until a stable level of anticoagulation is achieved
Screening and Management of Hyperhomocysteinemia
• Benefit more definite for arterial thrombosis and atherosclerotic disease
• Screening for MTHFR mutations is not cost effective
• Treatment (in the absence of thrombosis)– Folic acid@ 1 mg/day (may be to 5 mg/day)– vitamin B6@ 10 mg/day– vitamin B12@ 0.4 mg/day
• Doses of vitamin B6 up to 50 mg/day were used in secondary prevention No benefit
den Heijer M et al Blood. 2007;109(1):139
Duration of anticoagulation
• Indefinite therapy is recommended for patients with high risk disease including– Two or more spontaneous thromboses or one
spontaneous thrombosis in the case of antithrombin deficiency or the antiphospholipid syndrome
Bauer KA Ann Intern Med. 2001;135(5):367
Duration of anticoagulation
– One spontaneous life-threatening thrombosis (eg, near-fatal pulmonary embolism; cerebral, mesenteric, or portal vein thrombosis)
– One spontaneous thrombosis at an unusual site (eg, mesenteric or cerebral vein)
– One spontaneous thrombosis in the presence of more than a single genetic defect predisposing to a thromboembolic event (eg, combined heterozygosity for protein S deficiency, protein C deficiency, or antithrombin deficiency)
Bauer KA Ann Intern Med. 2001;135(5):367
High Risk Situations
• Surgery• Trauma• Medical Illness• Pregnancy and Puerperium• Immobilization
• Thromboprophylaxis should be given in all these situations
Summary
• Inherited thrombophilia is a genetic tendency to venous thromboembolism
• FVL is the most common mutation• Unselected screening is not recommended• Screening of asymptomatic patients is
recommended in the presence of a strong family history of VTE
• Certain patients may require prophylaxis
Summary• Prophylactic therapy may be considered in– Presence of homozygosity and/or multiple thrombophilic
defects– Protein C deficiency and the use of vitamin K antagonists– Antithrombin deficiency in the pregnant woman– Contemplated use of oral contraceptives– Pregnancy and obstetric complications
• Special therapy may be needed in AT deficiency and Protein C deficiency
• Indefinite therapy may be needed in high risk situations