Heritable Thrombophilias (BSH 2010)

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.



  • <40 y.o. & 2 or more symptomatic VTE episodes in family members

  • Children with pupura fulminans (Prot C and S)

  • 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)

  • ?Cerebral vein thrombosis – testing has uncertain predictive value

  • ?Intra-abdominal vein thrombosis – testing has uncertain predictive value


Who not to test



  • Upper limb DVT

  • Retinal vein thrombosis

  • Pregnant women at risk of thrombosis who are already in a high-risk group

  • Arterial thrombosis

  • Paediatric stroke

  • 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


Relative Risk increase:

Factor V Leiden (FVR506Q) Homozygous              10-80x

Antithrombin deficiency                                            10-20x

Protein C deficiency                                                  10x

Protein S deficiency                                                  10x

Factor V Leiden (FVR506Q) Heterozygous              3-5x

Prothrombin (F2G20210A)                                        3-5x



Do not use coag assays for heritable thrombophilias whilst on anticoagulants



Factor V Leiden Mutation (FVR506Q)


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

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

APC resistance causes an odds ratio >2 for increased risk of thrombosis


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


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


Prothrombin Gene Mutation (F2G20210A)


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 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

Levels fall in sepsis and with asparaginase treatment in ALL.


Quantitative (Type 1) and Qualitative (Type 2) variants exist (more details on pract haemostasis)


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


Principle of test

  • 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.


Next test – sequencing of the antithrombin gene (SERPINC1)


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.


Principles for lab tests:


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


Causes of Acquired Prot C Deficiency

  • Acute phase reactant (dramatic fall in meningococcal sepsis)

  • DIC

  • Liver disease

  • VKA antagonists

  • Sickle Cell Disease

  • Chicken pox (Rx: PLEX)


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)


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


Causes of Acquired Protein S Deficiency (& therefore cause of false positives in APC assay)

  • Pregnancy

  • COCP

  • Warfarin

  • Acute VTE

  • Liver disease

  • Chicken pox

  • HIV (Purpura fulminans)

  • Sickle Cell Disease