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Protein S or Free Protein S, which assay is the right one

Date : 24 Sep, 2018

Protein S is a vitamin K–dependent anticoagulant protein that was first discovered in Seattle, Washington in 1979. It was arbitrarily named after that city. The major function of Protein S as a cofactor is to facilitate the action of activated Protein C (APC) on its substrates, activated factor V (FVa) and activated factor VIII (FVIIIa). Protein S deficiencies are associated with thrombosis. This deficiency may be hereditary or acquired; the latter is usually due to hepatic disease or a vitamin K deficiency. Protein S deficiency usually manifests clinically as Venous Thromboembolism (VTE).

Any association of Protein S deficiency with arterial thrombosis appears coincidental or weak at best. Arterial thrombosis is not evident with other hereditary anticoagulant abnormalities (eg. protein C or antithrombin III deficiency, factor V Leiden gene mutation). Protein S and C levels are lower in Sickle Cell Anemia and decrease significantly further during a crisis. [1]

Hereditary Protein S deficiency is an autosomal dominant trait. Thrombosis is observed in both heterozygous and homozygous genetic deficiencies of Protein S. This deficiency is diagnosed using laboratory tests for the Protein S antigen and by using other tests for functional Protein S activity (based on clotting assays). Management takes place in the event of acute VTE. Prophylaxis may be used in selected patients with asymptomatic carrier states without a thrombotic event.

Protein S functions predominantly as a non-enzymatic cofactor for the action of another anticoagulant protein, Activated Protein C (APC). This activity occurs via a coordinated system of proteins, termed the Protein C system. The image below shows a simplified outline of the function of Protein S in the Protein C system.

Types of Protein S

In blood plasma, Protein S exists in both a ‘bound’ and a ‘free’ state. A portion of Protein S is non-covalently bound with high affinity to the complement regulatory protein C4b-Binding Protein (C4BP).

In healthy individuals, approximately 30-40% of total Protein S is in the free state. Only free Protein S is capable of acting as a cofactor in the Protein C system.

Importance of Protein S Testing

This distinction between ‘free’ and ‘total’ Protein S levels is important and gives rise to the current terminology regarding the deficiency states.

Types of Deficiency Free Protein S % Activity (Functional) Total Protein S % Activity (Functional) Protein S Antigenic
Type I Low Low  
Type II   Low Normal
Type III Low Normal  

Clinical studies reveal that Type 1 and Type III are the most common defeciencies. Type II is quite rare comparatively.

Age affects total Protein S but not free Protein S levels. Generally, the total Protein S level, increases in people over 50 years of age.

Families with the same recognized genetic defect in Protein S can have both Type I and Type III deficiencies. When families with the same genetic Type I defect are surveyed, older individuals even with deficiency in Protein S have an increase in total Protein S and now appear to have Type III deficiency.

Acquired Protein S Deficiency

Acquired conditions associated with decreased Protein S levels include the following:

  • Oral contraceptive use
  • Warfarin anticoagulant use
  • Vitamin K deficiency
  • Chronic liver disease
  • Certain viral infections (eg. HIV, varicella)
  • Systemic lupus erythematosus
  • Myeloproliferative disorders

In addition, Protein S levels decrease in pregnancy and can fall into the abnormal-low laboratory range. These low levels of Protein S in pregnancy do not cause thrombosis by themselves. Another seldom recognized cause for acquired Protein S deficiency is Sickle Cell Anemia.

Diagnostic Considerations

Screening for inherited thrombophilia is appropriate in cases of the following:

  • VTE without obvious cause in patients younger than 45 to 50 years
  • VTE in patients with a family history of thrombosis
  • Recurrent VTE
  • Thrombosis at an unusual location
  • VTE during pregnancy, use of oral contraceptives, or hormone replacement therapy 

Thrombophilia testing should be performed in younger VTE patients without known acquired risk factors. Testing for Protein C, Protein S and prothrombin gene mutations should be followed by additional molecular assessment in patients with suspicious findings.

Challenges in Diagnostic Testing

Total Protein S Activity is a clotting test and can be done on most coagulation analyzers. Free Protein S is an immunoturbidimetry test and is possible on some coagulation systems. 

Protein S Antigenic test is predominantly an ELISA test and is cumbersome.

Clotting kits have very low reconstitution stability, the calibration and tests must be performed within two hours of sample collection.

Protein S is a cofactor; hence it is highly unstable, limiting the diagnostic window for testing.


Total Protein S and Free Protein S are tests to distinguish the Protein S deficiency types. Hence, both the tests are a must.

The Erba Coagulation range has advanced analyzers and reagents. Marketed through Transasia Bio-Medicals Ltd., Erba’s Protein S detection kit helps in the quantitative measurement of Protein S assay.