A Practical Guide to Haemostasis

Resistance to Activated Protein C [APC-R] Assays


Resistance to Activated Protein C [APC-R] was first reported in 1995 and in approximately 95% of cases is due to the Factor V Leiden [FVL] mutation – a G1691A missense mutation within the F5 gene resulting in the replacement of the Arginine 506 residue by a Glutamine [R506Q] and the abolition of an Activated Protein C [APC] inactivation cleavage site in Factor Va. This is illustrated diagrammatically below:
[APC - Activated Protein C; A1-A3, B & C1-C2 represent the domains of Factor V].

Va Cleavage sites

Principles & Method

A number of tests have been developed to screen for APC resistance [APC-R]:

Method Explanation
APTT The original test for APC resistance [APC-R] involved measuring the APTT of a plasma sample with or without the addition of exogenous APC. In a plasma sample without APC-R, the addition of APC inactivates Factor Va and Factor VIIIa and so prolongs the clotting time of the APTT. In contrast in a sample with the Factor V Leiden mutation, the prolongation in the clotting time is less.

A ratio is derived from: [APTT+APC]/[APTT-APC]

A limitation of this test is that it requires a normal APTT in the patient and so cannot be used in cases in which there is a prolongation of the APTT e.g. patients on oral anticoagulants or with a Lupus Anticoagulant. In addition deficiencies of Factor VIII, IX, XI or XII that prolong the APTT may generate false positive APC-R results.

Polybrene may be included in some assays. Polybrene can neutralise Heparin [<1 IU/mL] and therefore removes this anticoagulant from the assay.

Individuals without the FVL mutation generally have a ratio of >2.0 and individuals who are heterozygous for the FVL mutation have a ratio <2. However, there is considerable overlap between healthy individuals and heterozygotes.

This test is also useful for detecting acquired APC-R and which represents an independent risk factor for venous thrombosis. In addition the test will detect other causes of APC-R arising from mutations within the F5 gene other than the FV Leiden mutation.
Normalised APTT The normalised APTT was established to try and improve the discrimination between FVL heterozygotes and individuals without the FVL mutation i.e. a normal Factor V.
The normalised ratio is derived by first dividing the APTT in the presence of APC by the APTT in the absence of APC [as with the original APTT screening test above.] This ratio is then normalized against a reference pool to obtain an APC sensitivity ratio.

[APTT+APC]/[APTT-APC] of patient sample
[APTT+APC]/[APTT-APC] of a normal reference plasma pool

80% of patients with an APC sensitivity ratio <0.84 and 100% of patients with an APC sensitivity ratio <0.70 were heterozygous or homozygous respectively for the FVL mutation.

It is important to remember that the presence in the normal plasma pool of a mutant FV [i.e. someone who is heterozygous for the FVL mutation] may reduce the APC ratio of the 'normal' plasma pool.
Modified APTT with Pre-dilution in Factor V-deficient plasma
Pefakit® APC-R Factor V Leiden screen The PefakitŪ APC-R Factor V Leiden screen is a plasma-based functional clotting assay and differs from other functional APC resistance tests by using a specific FV activator isolated from a snake venom (RVV-V from Daboia russelli).
The test is performed with and without the addition of APC and involves a pre-dilution [1 +4] of patient plasma with Factor V-deficient plasma.

Coagulation is triggered by the addition of a Factor V dependent Prothrombin activator - [Noscarin isolated from the venom of a snake Notechis scutatus scutatus] - in the absence of calcium. The time for clot formation is recorded and the ratio:
(Clotting Time + APC/Clotting Time - APC] determined. 

If the FVa molecules have been eliminated during the incubation step, the velocity of Prothrombin activation by the FV-dependent Noscarin is slow and therefore, the clotting time is prolonged.

The reagents employed in this test contain polybrene which makes it insensitive to Heparin up to a concentration of <2 IU/ml (UFH and LMWH) or Pentasaccharide levels <2 µg/ml.

The test is insensitive to the presence of the Direct Xa Inhibitors because the snake venoms bypass Factor Xa.  In addition, the test is independent of phospholipid and therefore, the test is unaffected by the presence of a Lupus Anticoagulant.
Chromogenic Factor X Assay The chromogenic assay for detecting APC resistance is based on the capacity of APC to limit the generation of Factor Xa by inactivating Factor Vllla in plasma. The ratio of the Factor Xa amidolytic activity in a sample without APC to its Factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. 
Russell Viper Venom Assay This test is based on the dilute Russell Viper Venom Time (dRVVT). The dRVVT is prolonged when a plasma sample is pre-incubated with a diluted snake venom isolated from Agkistrodon contortrix contortrix which activates Protein C.
The test result is expressed as the ratio between the dRVVT with and without addition of the venom.

The reagents contain a high phospholipid concentration which reduces their sensitivity to Lupus Anticoagulants and in addition a heparin neutraliser.
Pre-dilution of the test plasma sample in Factor V deficient plasma allows the distinction between controls and patients with deficiencies of Protein C, Protein S, Antithrombin and the Factor V Leiden mutation.


The interpretation of the test is very much dependent upon the specific test. Variables that have been shown to affect the original APTT-based test include:
   - Pregnancy
   - In association with oral contraceptive use
   - The presence of a Lupus Anticoagulant
   - Elevated Factor VIII level
   - Elevated Factor II [Prothrombin] level
   - Reduced Protein S
   - Anti-Protein C antibodies

Reference Ranges

These very much depend upon the specific test. A test ideally should be able to differentiate normal individuals without APC-R from individuals who are heterozygous or homozygous for the FVL mutation.

What Test Next

Individuals with a low APC ratio usually go on to have a screen of their F5 gene for the Factor V Leiden mutation. APC resistance independent of the FV Leiden mutation appear to a risk Factor for VTE. Some F5 mutations associated with APC-R but which are not due to the FVL Arg506Gln mutation have also been reported:

Protein Mutation Location
Factor V Leiden Arg506Gln Exon 10
Factor V Cambridge Arg306Thr Exon 7
Factor V Hong Kong Arg306Gly Exon 7
Factor V Nara Trp1920Arg Exon 20
Factor V Liverpool Ile359Thr Exon 8
Factor V Bonn Ala540Val Exon 10