An Introduction to Protein S and Free Protein S

An Introduction to Protein S and Free Protein S:

Pathophysiology of protein S in vivo and the clinical significance of laboratory testing.

Protein S and Protein C are both vitamin K dependant potentiators of fibrinolysis and anticoagulation.

Congenital protein S deficiency is an autosomal dominant disorder that is present in 2% to 6% of patients with venous thrombosis, accounting for a 10 fold increase in incidence compared to a normal population.

Protein S is available in two forms, free and bound. Free protein S is active and promotes the thrombolytic and anticoagulant effects described above. Bound protein S does not exhibit any activity due to inhibition by C4b binding protein. Therefore the activity of protein S is dependent on the availability of unbound active protein S, which is typically between 30-40% of the total detectable protein S.

Protein S and Protein C are both vitamin K dependant potentiators of fibrinolysis and anticoagulation. Protein C is activated in response to thrombin bound thrombomodulin, therefore providing a homeostatic feedback mechanism by which clots are broken down. Activated protein C destroys Factors V & VIII therefore reducing further thrombin generation, and promotes fibrinolysis via the inactivation of Plasminogen activator inhibitor-1 (PAI-1).

Protein S is primarily a cofactor for protein C, optimising its effect by binding activated protein C to phopholipid surfaces and enhancing the inactivation of factors Va & VIIIa. Protein S also exerts an anticoagulant effect independent of protein C, via factor Va binding and inhibition of the prothrombinase complex.

Congenital protein S deficiency is an autosomal dominant disorder that is present in 2% to 6% of patients with venous thrombosis, accounting for a 10 fold increase in incidence compared to the normal population. In addition they may also experience recurrent miscarriage, complications of pregnancy and are at higher risk for arterial thrombosis.

Protein S is available in two forms, free and bound. Free protein S is active and promotes the fibrinolytic and anticoagulant effects described above. Bound protein S does not exhibit any activity due to inhibition by C4b binding protein. Therefore the activity of protein S is dependent on the availability of unbound active protein S, which is typically between 30-40% of the total detectable protein S.

This difference is important when evaluating laboratory results as the reference ranges are different and abnormalities are clinically distinct.

Three different classes of protein S deficiency have been described by the ISTH:

  • Type I deficiency is characterised by a decrease in the total protein S antigen and free protein S antigen together (quantitative deficiency of protein S)
  •  Type II deficiency is characterised by normal total and free antigen levels but reduced protein S activity (functional deficiency of protein S)
  •  Type III deficiency is characterised by low free protein S levels, whereas the total plasma concentration of protein S is normal (Functional alteration to C4b)
     

 Type

Free protein S

Total protein S

Protein S activity

 I

Low

Low

Low

 II

Normal

Normal

Low

 III

Low

Normal

Low

An increase in total protein S and a decrease in free protein S is often associated with acute or chronic inflammation. Both C4b binding protein and protein S are upregulated during inflammation, resulting in an acquired deficiency. An increase in total protein S is observed due to an increase in production with a reduction in free protein S as a result of increased C4b binding.

Total protein S is quantitated in the lab using ELISA. A monoclonal antibody with a binding epitope that does not discriminate between bound and unbound protein S is used to accurately quantitate the total protein S level.

Protein S activity is perhaps the most widely used method when screening for protein S deficiency as this technique is clot based and suitable for the vast majority of routine coagulation analysers. In this assay, dilutions of normal plasma are mixed with Protein S depleted plasma. The mixed plasma is then activated by reagents which contain activated protein C, other human plasma proteins and phospholipids. After activation, clotting is initiated by the addition of calcium chloride. Human activated protein C and other plasma proteins are added at such quantities, so as to normalise any deficiencies in the patient sample, preventing false positive or negative results for Protein S functionality. A prolonged clotting time is therefore directly proportional to the concentration of the Protein S in the patient plasma.

Free protein S quantitation has historically been determined using ELISA based methods with monoclonal antibody specific to the C4b binding site of protein S. These are protracted techniques compared to recent advances in the market. Helena Biosciences have developed an immunoterbidometric assay capable of the accurate quantitation of free protein S. Two distinct monoclonal antibodies with specificity to the C4b binding site have been conjugated to polystyrene micro-particles. These particles agglutinate in the presence of free protein S and the subsequent reduction in absorbance is inversely proportional to the level of free protein S. This test can be ran on any analyser capable of measuring absorbance between 400-600nm allowing for easy integration into a laboratories portfolio.

The use of protein S activity and free protein S assays has been demonstrated to increase the diagnostic accuracy when investigating protein S deficient patients. Helena Biosciences can provide robust, accurate and sensitive methods for quantitation of protein S and free protein S.  

References

  1. Heeb MJ, Griffin JH. (2002) Activated protein C-dependent and –Independent anticoagulation activities of protein S have different structural requirements. Blood cells, molecules and disease. 29(2):190-199
  2. Comp PC, Forristall J, West CD,  Trapp RG (1990) Free Protein S Levels Are Elevated in Familial C4b-Binding Protein Deficiency. Blood. 76(12): 2527-252
  3. Kovac MK, Lalic-Cosic SZ, Dmitrovic JM, Djordjevic VJ, Radojkovic DP. (2015) Thrombin generation, D-dimer and protein S in uncomplicated pregnancy. Clinical Chemistry and Laboratory Medicine. 2015 May 6. pii: /j/cclm.ahead-of-print/cclm-2014-1030/cclm-2014-1030.xml. doi: 10.1515/cclm-2014-1030. [Epub ahead of print]
  4. Borgel D, Gandrille S, Aiach M (1997) Protein S deficiency. Thrombosis and Haemostasis. 78(1):351-356

More Information

Activated Protein C-Dependent and –Independent Anticoagulant Activities of Protein S Have Different Structural Requirements (sciencedirect.com)

Free protein S levels are elevated in familial C4b-binding protein deficiency (bloodjournal.org)

Thrombin generation, D-dimer and protein S in uncomplicated pregnancy (ncbi.nlm.nih.gov)

Protein S deficiency (ncbi.nlm.nih.gov)

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