Heritable Thrombophilias (BSH 2010, BSH 2022)

Key Messages

MEGA Study – testing for heritable thrombophilias in unselected patients presenting with VTE is not indicated and does not reduce, or predict, rates of recurrence.

The decision to test should be based on whether the result will change management. For the majority of patients, including pregnancy, this is not the case.

**Selected patients you might test**

In selected patients, testing may indicate a risk of recurrence following completion of anticoagulation, where you might otherwise have decided to stop treatment.

Examples

<40 y.o. & 2 or more symptomatic VTE episodes in family members
Children with pupura fulminans (Prot C and S)
Select asymptomatic first-degree relatives of people with Prot C, Prot S or AT def where the results may influence the management and life choices of the individual.
Pregnant women at risk of thrombosis, where a positive result will change their risk group
- E.g. An asymptomatic pregnant woman with a FH of VTE in a 1st degree relative (would change their score from 1 to 2 or 4 (depending which thrombophilia)

**Who not to test**

Example scenarios where testing is not routinely recommended:

First-degree relatives of people with a history of VTE
Unusual site VTE (e.g. Upper limb, intra-abodminal vein)
Retinal vein thrombosis
Pregnant women at risk of thrombosis who are already in a high-risk group
Arterial thrombosis, including stroke
Paediatric stroke
Recurrent miscarriage / adverse pregnancy outcomes (see bottom of page / 2022 guideline for details)
Prior to assisted conception

COCP / HRT and family history of VTE

Essentially, advise use of alternative contraception. Do not test for thrombophilias.
Specifics
- 1st degree relative with VTE who has not been tested – Use alternative. Do not test
- 1st degree relative with VTE who has tested negative – Use alternative. Do not test
- 1st degree relative with VTE who has tested positive – Use alternative. Testing patient may lend weight to your counselling but a negative test does not exclude the inherent increased risk of VTE with hormonal medications.

Overview of risks with heritable thrombophilias

VTE risk in 30-year-old, healthy individual is 1 in 10,000

VTE risk in pregnancy is 1 in 1,000 (and rises further in the 6 weeks post-partum)

Relative Risk increase for a 1st VTE:

Factor V Leiden (FVR506Q) Homozygous 10-80x

Heterozy. Antithrombin deficiency 10-20 fold

Heterozy. Protein C deficiency 5-7 fold

Heterozy. Protein S deficiency 5-7 fold

Factor V Leiden (FVR506Q) Heterozygous 5 fold

Prothrombin (F2G20210A) 3 fold

(N.B. Many other genes have been reported to increase risk of VTE - e.g. MTHFR, SERPINE1 (gene for PAI-1) & FXIII - but the association is either unconvincing or the effect too weak to alter patient management. Testing is not recommended.

using coag assays for heritable thrombophilias whilst on anticoagulants

DOAC Remove now available. Used to treat samples before testing to remove drug from the plasma

Consider alternative assays, e.g. my local lab uses Taipan snake venom to test for lupus in warfarinised samples

Factor V Leiden Mutation (FV R506Q)

95% of cases of activated protein C resistance are due to the Factor V Leiden mutation

Mutation results in the loss of the APC cleavage site on FVa (APC also inactivates FVIIIa)

Found in 5% of healthy European controls

In combination with Prothrombin mutation accounts for 65% of cases of heritable thrombophilia

APC Resistance Assay

(APTT + Protein C) / APTT = APC Sensitivity Ratio
Normally, adding protein C into the APTT should prolong the clotting time
Therefore, a normal APC sensitivity ratio is >2.2
Normal ratio >2.2, Heterozygotes ~1.7, Homozygotes ~1.2
This test can be ‘normalised’ against a reference plasma pool
Limitations
- Requires a normal baseline APTT
- There is considerable overlap between healthy individuals and heterozygotes
- Low protein S will also skew the ratio (see list below)

Chromogenic assay

Based on capacity for APC to limit the generation of Xa by inactivating VIIIa

Genetic Testing

Test for gene mutation if positive or reason to suspect false negative (see Prot S below)

Prothrombin Gene Mutation (F2 G20210A)

Gain of function mutation

Missense mutation resulting in increased prothrombin levels (30% rise hetero, 70% if homo)

Increased prothrombin levels assoc. with increased thrombin generation —> increased risk VTE

Found in 1-2% of healthy European controls, 6% in patients w/ thrombosis, 18% pts with unexplained VTE

In combination with FV Leiden accounts for 65% of cases of heritable thrombophilia

Particular combo of mutation plus use of COCP —> odds ratio 150 for cerebral vein thrombosis

PCR-based diagnostics

Antithrombin Deficiency

Antithrombin (AT) inhibits IIa (thrombin), Xa and less so IXa and XIa

Synthesised in the liver & circulates single chain protein with a half-life of three days

Normal plasma level is 150 ug/ml

Quantitative (Type 1) and Qualitative (Type 2) variants exist (more details on pract haemostasis and in the 2022 guideline)

Found in 0.02-0.2% of normal population, 1-2% of patients with VTE

The risk of a 1st VTE is 15-fold in heterozygous deficiency vs general population

Testing (only after 3 months AC for acute thrombosis)

Functional assays based on IIa and Xa inhibition in presence of heparin.
The anti-Xa or anti-IIa effect of AT can be measured by clotting or chromogenic assay
Chromogenic is more convenient and more commonly used.
Genetic testing – sequencing of the antithrombin gene (SERPINC1). Available as part of a thrombophilia panel from Genomics England.

Causes of Acquired Antithrombin Deficiency

Pregnancy
Liver disease
DIC / Severe sepsis
Nephrotic syndrome
Acute VTE
Heparin therapy
L-asparaginase therapy

Additional factors affecting Antithrombin assays

Physiological low levels in neonates
Artefactual rise in clotting-based assays if DOAC present

Protein C Deficiency (Autosomal Dominant)

Protein C is a Vit-K dependent anticoagulant, with a half-life of 6 hours

It is converted by thrombin into activated protein C (APC).

APC in combo with Protein S degrades Va and VIIIa

APC also binds to Plasminogen Activator Inhibitor Type 1 (PAI-1) —> enhanced fibrinolysis

APC is inhibited by Protein C Inhibitor (PCI or PAI-3)

Normal plasma level 65-135 IU/dL

Homozygous deficiency – Rare, presents in newborns with purpura fulminans (fatal if not Rx’d)

Heterozygous deficiency – 0.2% of pop, 3% of unselected patients with VTE. Skin purpura at the start of warfarin therapy.

Testing (only after 3 months AC for acute thrombosis)

Chromogenic assay
- Preferred test.
- Protein C is activated by Protac venom and level determined from the rate of colour change due to cleavage of a chromogenic substrate.
- False negative: Mutation in GLA domain of protein C will give normal chromogenic assay (Clot based assay will still be abnormal. GLA domains present on all Vit K factors)
ELISA – Quantitative, not functional
Clot-based functional APTT assay – time to clot after addition of a protein C activator
Genetic testing - Available as part of a thrombophilia panel from Genomics England.

Causes of Acquired Prot C Deficiency

Acute phase reactant (dramatic fall in meningococcal sepsis)
DIC / Severe sepsis
Liver disease
VKA antagonists / Vit K deficiency
Chicken pox (Rx: PLEX)

Additional factors affecting Prot C assays

Physiological low levels in neonates and children
Artefactual rise in clotting-based assays if DOAC or heparin present
Artefactual fall in clotting-based assays if Factor V Leiden present

Protein S Deficiency (Autosomal Dominant)

Protein S acts as a co-factor to Protein C in the inactivation of Va and VIIIa

It also acts independently on Va, Xa and VIII.

Synthesised in the liver, endothelial cells and megakaryocytes, with a half-life of 42 hours.

Homozygous deficiency – Rare, presents in newborns with purpura fulminans (fatal if not Rx’d)

Testing (only after 3 months AC for acute thrombosis)

ELISA – Quantitative
Latex Agglutination – Quantitative. C4b binding protein fixed to latex --> binds free protein S from patient’s plasma --> addition of 2nd latex reagent coated with anti-Protein S Ab --> agglutination
APTT or PT-based functional assays
Genetic testing - Available as part of a thrombophilia panel from Genomics England.

Causes of Acquired Protein S Deficiency

Pregnancy
COCP
VKA Antagonists / Vit K deficiency
Acute VTE
Liver disease
Chicken pox
HIV (Purpura fulminans)
Sickle Cell Disease
Nephrotic syndrome
DIC / Severe sepsis

Additional factors affecting Prot S assays

Physiological low levels in neonates
Artefactual rise in clotting-based assays if DOAC or heparin present
Artefactual fall in clotting-based assays if Factor V Leiden present

What about procoagulant factor levels?

Discussed in the 2022 guideline

Raised levels of Factors II, VIII, IX, X, XI and FGN found to have an association with thrombosis in the MEGA study but there are too many confounding acquired factors to draw conclusions about the significance of heritable elevations in procoagulant factors. Testing is not recommended.

heritable thrombophilias and pregnancy loss

Several studies have failed to show a benefit for the use of anticoagulation to reduce miscarriage in women with inherited thrombophilia.

ALIFE2 2022 - 326 women with inherited thrombophilia and 2+ pregnancy losses. Low dose LMWH from 7wks gestation. Primary outcome: Live births. LMWH vs standard of care. No difference in live birth rate.