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]
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
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®).
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,
Causes for Rejection Specimen received more than 4 hours
after collection, tubes not filled, clotted specimens
Turnaround Time Several days (because testing is usually
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
* decreased hepatic synthesis from liver disease or L-asparaginase
* 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
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,
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,
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,
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,
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,
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.