Protein S 

Updated: Jan 15, 2014
  • Author: Ashwin Pai, MBBS, MS (GenSurg), MRCS; Chief Editor: Eric B Staros, MD  more...
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Reference Range

Protein S, a vitamin K-dependent plasma glycoprotein, is synthesized in the endothelium. The 2 forms of protein S are a free form and a complex form. The free form of protein S is active. The bound form comprises 65% of the total protein S, and it is complexed to C4b-binding protein (C4bBP) and is inactive. The availability of C4bBP regulates the proportion of the free and bound forms of protein S. Synthesis of protein S occurs in the liver, endothelial cells, and megakaryocytes. The half-life of protein S is 42 hours. [6]

The reference ranges are as follows:

  • Males - Greater than 73 U/dL
  • Females - Greater than 63 U/dL

Protein S levels are low at birth and do not reach adult values until approximately age 6 months. Patient age, sex, health history, the method used for the test, and many other factors can affect laboratory test results, possibly causing results to vary. [1]

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Collection and Panels

Test preparation

Protein C and protein S tests should not be ordered for at least 10 days after an episode or while a person is on oral anticoagulant therapy. First, the patient is treated for venous thromboembolism (VTE); the patient is put on a limited course of anticoagulant therapy (often about 3-6 months). After the VTE is adequately treated, protein C and free protein S functional and activity tests are ordered. Other tests for hypercoagulability are also performed to help determine the cause of the thrombus and to help evaluate the patient’s risk for recurrence.

The protein C and free protein S functional and activity tests measure the amount of each protein and evaluate whether they are performing their proper function in the body.

The blood sample is collected from a vein, and a bandage, cotton ball, or gauze is placed on the needle insertion site, with pressure applied to the area. The patient should be instructed to avoid strenuous exercise immediately after blood is drawn. If any pain or redness, swelling, or discharge occurs at the puncture site, the patient should consult a healthcare worker. Hematoma formation, bruising, or infection may occur but are rare.

Method

Protein S assays can be either functional or immunological. Techniques for measuring protein S antigen include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays. The most commonly used technique is ELISA, for which a number of commercial kits are available. With either method, it should detect protein S regardless of the level of C4bBP.

For detecting free protein S, the C4bBP-bound protein S is removed from the assay by a pre-assay step involving precipitation of the C4bBP-bound protein S with polyethylene glycol and then using centrifugation to remove the precipitate. The resultant protein S plasma sample is free, unbound protein S. Another method is to direct monoclonal antibody against protein S epitopes that are not accessible in the bound form; these residues are involved in the binding to C4bBP.

Functional assays

Functional assays measure only free (physiologically active) protein S. Functional assays are usually based on either the prothrombin time (PT) or the activated partial thromboplastin time (aPTT).

  • With aPTT-based functional protein S assays, platelet-poor plasma is incubated at 37°C with the following: protein S–deficient plasma, phospholipid, a contact activator (eg, Kaolin), and either an excess of activated protein C or an activator of protein C .Typically, the incubation period is 1-4 minutes, after which calcium is added to initiate clotting. The time elapsed to clot formation is noted, and the protein S level is then determined based on comparison to a reference curve.
  • For PT-based functional protein S assays, the activated protein C cofactor activity of protein S is measured based on the degree of prolongation of a PT-based clotting time. Plasma depleted of protein S is mixed with the test plasma sample and Protac (a protein C activator)–activated protein C, tissue factor, phospholipid, and calcium ions. The extent of duration of the clotting time is proportional to the concentration of protein S in the plasma sample. [2]

Immunological assays

Immunological assays measure total protein S, both free protein S and bound protein S. In rare circumstances, the level of immunological protein S is normal but some functional abnormality exists (ie, a true type 2 deficiency).

Immunological assays are ELISAs and latex particle-based agglutination assays.

ELISAs are used more commonly than latex particle-based agglutination assays. Rabbit or goat antihuman protein S antibodies are used to coat the wells of a microtiter plate. The wells are incubated with plasma. After washing, diluted antiprotein S conjugated to horseradish peroxidase (HRP) is added. The quantity of HRP-bound antibody is assessed using a third incubation with an HRP-specific substrate. The degree of absorbance is measured. A curve is charted using a known concentration of protein S.

For latex particle-based agglutination assays, purified C4BP is adsorbed onto the first latex reagent, which is then incubated with the patient’s plasma sample. Free protein S is then adsorbed on the C4BP latex particle, and an agglutination reaction with the second latex reagent sensitizes monoclonal antibodies against human protein S. The free protein S concentration in the test sample is directly proportional to the degree of agglutination.

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Interpretation

Elevated protein S levels are not usually associated with medical problems and are not clinically significant. Normal protein C and protein S antigen activity and concentrations usually indicate adequate clotting regulation.

A low level of protein S activity can cause excessive or inappropriate blood clotting. If the protein is not functioning properly (ie, normal protein levels but improper function), insufficient regulation of the coagulation process ensues, which can result in an increased risk of clot development and vein blockage. The severity of the risk is dependent on the magnitude of the deficiency and/or the degree of dysfunction of the protein.

Table. Three Types of Protein S Deficiency Distinguished on the Basis of Laboratory Test Results (Open Table in a new window)

Deficiency Type Functional Protein S Free Protein S Antigen Total Protein S Antigen
1 Decreased Decreased Decreased
2 Decreased Normal Normal
3 Decreased Decreased Normal

Because the neonate’s protein S levels are usually not detectable at presentation, neonatal homozygous type I protein S deficiency is usually easy to diagnose. However, because the neonatal reference range is very wide, repeat testing at 6 months may be required for heterozygous or type II or III deficiency. Alternatively, evaluations for other vitamin K–dependent coagulation proteins can be performed for comparison or parental levels of protein S can be measured.

For the rare type II deficiency, a functional assay for protein S deficiency may be helpful if the index of suspicion is high, results from other testing are normal, possible interfering factors can be ruled out, and the assay is reliable.

Acquired deficiencies

Decreased protein S concentrations can result from inadequate production or an increase the body’s usage. Owing to the fact that protein S is produced in the liver and is vitamin K dependent, reduced levels of protein S can occur in liver disease, vitamin K deficiency, or anticoagulant therapy that opposes vitamin K. Conditions that cause simultaneous clotting and bleeding throughout the body, such as disseminated intravascular coagulation (DIC), exhaust clotting factors (including protein C and protein S), at higher rates, thereby causing decreased concentrations in the blood. [3]

Severe inflammatory infections, renal disease, cancers, HIV disease, pregnancy, oral contraceptive pill usage, status immediately post thrombotic episode, and warfarin or heparin anticoagulant therapy may result in decreased concentrations of protein S. Decreased production or increased usage of protein S occur in these conditions. While typically mild and self-limited, they can sometimes be severe and may be acute, chronic, or progressive. [4]

Sickle cell disease may cause decreased protein S levels, reportedly due to absorption of protein S by the sickle cell. However, the protein S level do not fall precipitously during a sickle cell crisis. [5]

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Background

Protein S, a vitamin K-dependent plasma glycoprotein, is synthesized in the endothelium. The 2 forms of protein S are a free form and a complex form. The free form of protein S is active. The bound form comprises 65% of the total protein S, and it is complexed to C4b-binding protein (C4bBP) and is inactive. The availability of C4bBP regulates the proportion of the free and bound forms of protein S. Synthesis of protein S occurs in the liver, endothelial cells, and megakaryocytes. The half-life of protein S is 42 hours. [6]

Protein S plays a significant role in the anticoagulation cascade, where it functions as a cofactor to the serine protease activated protein C in the inactivation of factors Va and VIIIa. Only the free form of protein S is active. [5] Additionally, through direct binding to factors Va, Xa, and VIII, protein S exerts activated protein C–independent anticoagulant activity.

When is it ordered?

The protein S test is usually performed as part of the evaluation to detect a possible hypercoagulable clotting disorder. It can also be performed to help diagnose the cause of deep venous thrombosis (DVT) or VTE, especially if it has occurred in a relatively young patient (< 50 y) or if it occurred in an unusual location (eg, veins leading to the liver or kidney, blood vessels of the brain [cerebral]).

Test results elucidate the function (activity) or quantity (antigen) of protein S. Functional tests for protein C and protein S, as well as other tests for hypercoagulability, are usually ordered to screen for sufficient, normal factor activity. Results determine the quantity of protein S antigen and free, or occasionally total, protein S antigen in order to assess if the decreased production is due to an inherited or acquired condition. The type of deficiency can also be classified. Shortages due to the rare inherited genetic conditions can be further defined as homozygous or heterozygous based on the quantity of protein C or protein S available and the degree of activity.

Protein S testing can also be ordered if a newborn is suspected of having a severe clotting disorder, such as DIC or purpura fulminans.

If test results show decreased activity or a decreased quantity of protein S, the test should be repeated before a diagnosis is made. If an acquired deficiency is identified, protein C or protein S concentrations may be monitored occasionally as the underlying condition progresses or resolves.

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