Inherited Thrombophilias

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