Protein S

Overview of Protein S in Hemostasis

Definition: Protein S is a vitamin K-dependent glycoprotein synthesized in the liver that functions as a cofactor for activated protein C (APC) in the inactivation of Factors Va and VIIIa, key components of the coagulation cascade
Significance: Protein S, along with Protein C, acts as a natural anticoagulant, helping to regulate thrombin generation and prevent excessive clot formation. Protein S deficiency is a well-established risk factor for venous thromboembolism (VTE)
Two Forms of Protein S:
- Free Protein S: The active form that functions as a cofactor for APC
- Bound Protein S: Bound to C4b-binding protein (C4BP), an acute-phase reactant. This bound form is inactive and does not function as a cofactor for APC

Assays for Protein S

Protein S Activity Assay:
- Principle: Measures the functional activity of protein S, i.e., its ability to act as a cofactor for APC in the inactivation of Factor Va
- Methods:
  - Activated Partial Thromboplastin Time (aPTT)-Based Assay:
    - A modified aPTT assay is used, where the aPTT is performed in the presence and absence of APC
    - The degree to which APC prolongs the aPTT is proportional to the amount of functional Protein S present
  - Chromogenic Assay:
    - A chromogenic substrate is used to measure the activity of APC in the presence of Protein S
- Interpretation:
  - Decreased Protein S Activity: Suggests Protein S deficiency
Total Protein S Antigen Assay:
- Principle: Measures the total amount of Protein S protein present, regardless of whether it is in the free or bound form
- Methods:
  - Immunoturbidimetry: Antibodies specific to protein S are added to the sample, and the turbidity of the solution is measured
  - Nephelometry: Measures the light scattered by the immune complexes
  - ELISA (Enzyme-Linked Immunosorbent Assay): Uses enzyme-labeled antibodies to detect protein S
- Interpretation:
  - Decreased Total Protein S Antigen: Suggests a quantitative deficiency of Protein S
Free Protein S Antigen Assay:
- Principle: Measures the amount of Protein S that is not bound to C4b-binding protein (C4BP). Only free Protein S has anticoagulant activity
- Methods:
  - Immunometric Assay with Precipitation: Free Protein S is captured by an antibody, and then total protein S is measured after dissociation
- Interpretation:
  - Decreased Free Protein S Antigen: Suggests a deficiency in the active, free form of Protein S

Interpreting Protein S Assay Results

The interpretation of Protein S assay results can be complex due to the different types of Protein S deficiency and the influence of C4b-binding protein (C4BP) levels

Types of Protein S Deficiency (based on laboratory findings):
- Type I: (Quantitative Deficiency)
  - Decreased Total Protein S Antigen
  - Decreased Free Protein S Antigen
  - Decreased Protein S Activity
- Type II: (Qualitative Deficiency)
  - Normal Total Protein S Antigen
  - Normal Free Protein S Antigen
  - Decreased Protein S Activity
- Type III: (Low Free Protein S)
  - Normal Total Protein S Antigen
  - Decreased Free Protein S Antigen
  - Decreased Protein S Activity
Important Considerations:
- Pregnancy: Pregnancy is associated with decreased Protein S levels, making interpretation difficult
- Oral Contraceptives and Hormone Replacement Therapy: Can decrease Protein S levels
- Acute Thrombosis: Acute thrombotic events can consume Protein S
- Inflammation: C4b-binding protein (C4BP) is an acute-phase reactant. Increased C4BP can bind more Protein S, leading to decreased free Protein S levels
Recommendations: * Confirm the results by repeating the testing when the patient is not acutely ill or pregnant, and not taking oral birth control. * Check C4b-binding protein level to confirm that there is not an increased level lowering free protein S

Factors Affecting Protein S Assay Results

Pre-Analytical Variables:
- Improper Specimen Collection: Traumatic venipuncture, underfilled collection tube, or clots in the sample
- Delayed Testing: Protein S activity and antigen levels can decrease with prolonged storage
- Improper Storage: Samples should be stored properly to maintain protein S activity
Analytical Variables:
- Reagent Quality: Use fresh, properly stored reagents
- Instrument Calibration: Ensure proper calibration of the instrument
- Interfering Substances: Heparin, lupus anticoagulants, and other substances can interfere with some assays
Patient-Related Variables:
- Anticoagulant Therapy: Warfarin can affect Protein S levels
- Pregnancy: Protein S levels decrease during pregnancy
- Liver Disease: Impairs the synthesis of Protein S
- Nephrotic Syndrome: Protein S loss in the urine

Troubleshooting Erroneous Results

If the Protein S results are unexpected or inconsistent with the patient’s clinical presentation:
- Check the sample for clots or hemolysis
- Verify the quality control data
- Repeat the test on a fresh sample, ensuring proper collection and handling
- Consider the possibility of interfering substances or medications
- Review the patient’s medical history and medication list
- Repeat the test during a period of stability (e.g., after pregnancy or discontinuing warfarin)

Reflex Testing

If the initial Protein S activity or antigen level is low, further testing may be performed to:
- Rule Out Acquired Causes of Protein S Deficiency:
  - Check liver function tests
  - Evaluate for nephrotic syndrome (urine protein)
  - Rule out DIC
- Assess C4b-Binding Protein (C4BP) Level: Elevated C4BP can lead to decreased free protein S
- Perform Genetic Testing: To identify specific PROS1 gene mutations (rarely done)

Key Terms

Protein S: A vitamin K-dependent anticoagulant protein that acts as a cofactor for activated protein C
Thrombophilia (Hypercoagulable State): An increased tendency to develop blood clots
Vitamin K-Dependent Factors: Coagulation factors (II, VII, IX, X) and anticoagulant proteins (C and S) that require vitamin K for their synthesis
Free Protein S: The active form of protein S that is not bound to C4b-binding protein
Total Protein S: The total amount of protein S, including both free and bound forms
C4b-Binding Protein (C4BP): An acute-phase reactant that binds to protein S, reducing the amount of free protein S
VTE (Venous Thromboembolism): A condition that includes deep vein thrombosis (DVT) and pulmonary embolism (PE)

Protein C

Molecular