From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., "Coagulation." In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.

Index of Tests

Protein S [CO004300]

Related Information

Abstract Protein S is a required cofactor for the anticoagulant activity of protein C. A hereditary deficiency of protein S leads to a hypercoagulable state with an increased risk for venous thrombosis. Protein S deficiencies are quantitative (type I) or qualitative (type II).

Patient Preparation Determine if patient is on oral anticoagulants or estrogen (eg, oral contraceptives, estrogen replacement) or if the patient is pregnant. Protein S levels are decreased by estrogen, pregnancy, and warfarin (Coumadin®).

Specimen Plasma

Container Blue top (sodium citrate) tube

Sampling Time Testing should be deferred until patients have not received Coumadin® for at least 10 days, because Coumadin® decreases protein S levels.

Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled. Deliver tubes immediately to the laboratory.

Storage Instructions Separate plasma from cells as soon as possible. Plasma may be stored on ice for up to 4 hours; otherwise, store frozen.

Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimens

Turnaround Time Several days (because testing is usually batched)

Special Instructions Elevated factor VIII (>200%) is a common cause of artifactually decreased protein S in PTT-based functional assays. It is recommended to measure factor VIII on the same specimen when the functional protein S is decreased by PTT-based methods, to determine if the decrease is due to elevated factor VIII.

Reference Interval Results are reported as a percent of the amount expected in normal plasma. By definition, the mean value in normal plasma is 100%. The reference range is approximately 70% to 140%; lower for women than for men.1,2 At birth, protein S (total antigen) levels are only 36% (range 12% to 60%) of adult normal values.3 Protein S rises into the adult reference range by age 6 months.

Use A functional assay should be performed first, because all subtypes of protein S deficiencies will be detected. The free antigen assay is needed only if the functional assay is decreased, and the total antigen assay is needed only if the free antigen is decreased, in order to determine the deficiency subtype. If the antigen assays are performed without the functional assay, patients with certain subtypes will not be detected (see Additional Information and the table).

Limitations Acquired protein S deficiencies are more common than hereditary deficiencies (see Additional Information).

Functional assays: Commonly encountered coagulation conditions interfere. For example, lupus anticoagulants can falsely increase the protein S test result. Elevations in factor VIII (>200%) can artifactually decrease PTT-based results; factor VIII elevations occur in patients with an acute phase reaction. In some assays, falsely low values have been reported in patients with the factor V Leiden.4,5 Assays that tolerate up to 1-2 units/mL heparin are available. The functional assay cannot be performed in patients on hirudin or argatroban anticoagulation.

Antigen assays: If not used in conjunction with a functional assay, patients with some subtypes will not be detected (see Additional Information and table).


Functional (activity) assays: Protein S is measured by its ability to serve as a cofactor required for activated protein C-mediated degradation of activated factors V and VIII, thereby prolonging a PTT- or PT-based clotting time.

Free antigen (immunoassay): Monoclonal antibodies specific for free (unbound) protein S are used in an enzyme-linked immunosorbent assay (ELISA). In an older assay, free protein S was determined by first treating specimens with polyethylene glycol (PEG), which precipitates bound protein S and leaves free protein S in the supernatant. In the new ELISA using monoclonal antibodies specific for free protein S, the PEG step is no longer necessary. Elimination of the PEG-precipitation step has significantly improved the accuracy of the test result.6

Total antigen (immunoassay): Measures total (free and bound) protein S by ELISA. An alternative method uses latex particles coated with antibodies directed against protein S. In the presence of protein S, the latex particles form aggregates that absorb light passing through the specimen. The amount of light absorbance is directly related to the amount of protein S in the specimen.7 A third method, rocket immunoelectrophoresis, is an older method that is still in use in some laboratories.8

Additional Information Protein S is a vitamin K dependent protein that is a required cofactor for activated protein C. Activated protein C, with protein S as a cofactor, acts as an anticoagulant by degrading activated factors V and VIII. Sixty percent of total protein S is bound to C4b-binding protein and is inactive. The remainder, called free protein S, is the functionally active form.

Hereditary protein S deficiency is present in 0.7% of the general population.9 It accounts for 2% of unselected patients with venous thrombosis and up to 7.6% of patients younger than 70 years of age with thrombosis.10,11 Many different mutations in the protein S gene are known to cause hereditary protein S deficiency.12 Individuals heterozygous for protein S deficiency have an increased risk for venous thrombosis, and the risk is further increased in the presence of a second risk factor.13 Heterozygotes generally have protein S levels between 20% to 65%.6,14 The age at onset of thrombosis is usually between 10-50 years in heterozygous individuals. Coumadin®-induced skin necrosis has been reported in protein S deficient patients who are started on Coumadin® without the addition of an immediate-acting anticoagulant (eg, heparin) until the Coumadin® levels are therapeutic. Homozygous deficiencies are rare, and are fatal if untreated. They present in the newborn period with severely decreased protein S, purpura fulminans, and disseminated intravascular coagulation (DIC).15

Decreased protein S can also arise from acquired conditions, such as:

* decreased hepatic synthesis from liver disease or L-asparaginase treatment

* synthesis of a dysfunctional protein due to vitamin K deficiency or warfarin (Coumadin®) use

* consumption from thrombosis, DIC or invasive procedures

* estrogen, including oral contraceptives, estrogen replacement therapy, or pregnancy (decreased protein S may persist for up to 2 months after delivery or estrogen discontinuation)

* acute phase reactions (due to elevated C4b-binding protein, which decreases free and consequently functional protein S)

May also become decreased in nephrotic syndrome, varicella infection16,17,18 or HIV infection.19 Acquired inhibitors (autoantibodies) to protein S have been reported,20 some of which arose in association with varicella infections. If an acquired cause is present, the test should be repeated once the condition is no longer present, if possible. Confirmation of a hereditary protein S deficiency may require documenting protein S deficiency in a relative.

In liver disease, protein S is occasionally normal despite decreased protein C and antithrombin (all three proteins are synthesized in the liver). It is speculated that this is because protein S is synthesized in endothelial cells and megakaryocytes in addition to the liver, whereas protein C and antithrombin are synthesized predominantly or exclusively in the liver.

Protein S deficiencies are quantitative (type I) or qualitative (type II). In type I deficiencies, normal protein S molecules are made, but in reduced quantity. In type II deficiencies, normal amounts of protein S are made, but the protein S is defective. Functional assays measure protein S function. The total antigen assay is an immunoassay that measures the total quantity of protein S, regardless of the quality of its function. Free antigen assays are immunoassays that measure only unbound (free) protein S, regardless of the quality of its function. Only free (unbound) protein S is active; protein S that is bound to its binding protein (C4b-binding protein) is inactive. Accordingly, type I deficiencies have decreased protein S in both functional and antigenic assays. Type II deficiencies have normal total antigen levels, with decreased functional protein S. A further type II subtype (known as type IIa or type III) is characterized by decreased functional and free antigen levels with normal total antigen levels (see table). This subtype may be due to mutations causing increased binding of protein S to C4b-binding protein. In summary, if only antigenic assays are performed, type II deficiencies will not be detected. Therefore, a functional assay should be used as the initial screening assay. If the result is decreased, a free antigen assay should be performed to determine the deficiency subtype. If the free antigen is decreased, a total antigen assay may be performed to further determine the deficiency subtype (see table).

Classification of Hereditary Protein S Deficiencies


Protein S

Free Protein S

Total Protein S

(Free Antigen Assay)

(Total Antigen Assay)





II (also called IIb)




III (also called IIa)





1. Leroy-Matheron C, Duchemin J, Levent M, et al, "Influence of the nt 2148 A to G Substitution (Pro 626 Dimorphism) in the PROS1 Gene on Circulation Free Protein S Levels in Healthy Volunteers - Reappraisal of Protein S Normal Ranges,"Thromb Haemost, 2000, 83(5):798-9.

2. Henkens CM, Bom VJ, van der Schaaf W, et al, "Plasma Levels of Protein S, Protein C and Factor X: Effects of Sex, Hormonal State and Age,"Thromb Haemost, 1995, 74(5):1271-5.

3. Andrew M, Paes B, Milner R, et al, "Development of the Human Coagulation System in the Full-Term Infant,"Blood, 1987, 70(1):165-72.

4. D'Angelo SV, Mazzola G, Valle PD, et al, "Variable Interference of Activated Protein C Resistance in the Measurement of Protein S Activity by Commercial Assays,"Thromb Res, 1995, 77(4):375-8.

5. Faioni EM, Boyer-Neumann C, Franchi F, et al, "Another Protein S Functional Assay Is Sensitive to Resistance to Activated Protein C,"Thromb Haemost, 1994, 72:648.

6. Aillaud MF, Pouymayou K, Brunet D, et al, "New Direct Assay of Free Protein S Antigen Applied to Diagnosis of Protein S Deficiency,"Thromb Haemost, 1996, 75(2):283-5.

7. Laroche P, Plassart V, and Amiral J, "Rapid Quantitative Latex Immunoassays for Diagnosis of Thrombotic Disorders,"Thromb Haemost, 1989, 62:379.

8. Edson JR, Vogt JM, and Huesman DA, "Laboratory Diagnosis of Inherited Protein S Deficiency,"Am J Clin Pathol, 1990, 94(2):176-86.

9. Rodeghiero F and Tosetto A, "The Epidemiology of Inherited Thrombophilia: The VITA Project,"Thromb Haemost, 1997, 78(1):636-40.

10. Heijboer H, Brandjes DPM, Buller HR, et al, "Deficiencies of Coagulation-Inhibiting and Fibrinolytic Proteins in Outpatients With Deep-Vein Thrombosis,"N Engl J Med, 1990, 323:1512-6.

11. Melissari E, Monte G, Lindo VS, et al, "Congenital Thrombophilia Among Patients With Venous Thromboembolism,"Blood Coagul Fibrinolysis, 1992, 3(6):749-58.

12. Borgel D, Grandrille S, and Aiach M, "Protein S Deficiency,"Thromb Haemost, 1997, 78(1):351-6.

13. Simioni P, Sanson BJ, Prandoni P, et al, "Incidence of Venous Thromboembolism in Families With Inherited Thrombophilia,"Thromb Haemost, 1999, 81(2):198-202.

14. Finazzi G and Barbui T, "Different Incidence of Venous Thrombosis in Patients With Inherited Deficiencies of Antithrombin III, Protein C and Protein S,"Thromb Haemost, 1994, 71:15-8.

15. Pegelow CH, Ledford M, Young J, et al, "Severe Protein S Deficiency in a Newborn,"Pediatrics, 1992, 89:674-6.

16. Nguyen P, Reynaud J, Pouzol P, et al, "Varicella and Thrombotic Complications Associated With Transient Protein C and Protein S Deficiencies in Children,"Eur J Pediatr, 1994, 153:646-9.

17. Manco-Johnson MJ, Nuss R, Key N, et al, "Lupus Anticoagulant and Protein S Deficiency in Children With Postvaricella Purpura Fulminans or Thrombosis,"J Pediatr, 1996, 128(3):319-23.

18. Peyvandi F, Faioni E, Alessandro Moroni G, et al, "Autoimmune Protein S Deficiency and Deep Vein Thrombosis After Chickenpox,"Thromb Haemost, 1996, 75(1):212-3.

19. Stahl CP, Wideman CS, Spira TJ, et al, "Protein S Deficiency in Men With Long-Term Human Immunodeficiency Virus Infection,"Blood, 1993, 81(7):1801-7.

20. Sorice M, Arcieri P, Griggi T, et al, "Inhibition of Protein S by Autoantibodies in Patients With Acquired Protein S Deficiency,"Thromb Haemost, 1996, 75(4):555-9.


Makris M, Leach M, Beauchamp NJ, et al, "Genetic Analysis, Phenotypic Diagnosis, and Risk of Venous Thrombosis in Families With Inherited Deficiencies of Protein S,"Blood, 2000, 95(6):1935-41.

Van Cott EM and Laposata M, "Laboratory Evaluation of Hypercoagulable States,"Hematol Oncol Clin North Am, 1998, 12(6):1141-66.


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