Protein C

Overview of Protein C Assays

Definition: Laboratory tests used to measure the activity and/or concentration of Protein C, a vitamin K-dependent anticoagulant protein, in plasma
Clinical Significance:
- Diagnosis of Inherited Protein C Deficiency: Helps identify individuals with a genetic predisposition to thrombosis
- Evaluation of Thrombotic Events: Used to investigate patients with unexplained venous thromboembolism (VTE) or arterial thrombosis
- Assessment of Warfarin-Induced Skin Necrosis: Helps identify patients at risk of developing this rare but serious complication when starting warfarin therapy
- Differentiation of Thrombophilia Risk Factors: Used to determine if protein C deficiency is contributing to an increased risk of thrombosis
Types of Assays:
- Protein C Activity Assay (Functional Assay)
- Protein C Antigen Assay (Immunological Assay)

Protein C: Background and Function

Protein C:
- A vitamin K-dependent serine protease synthesized in the liver
- Circulates in the blood in an inactive form
Activation of Protein C:
- Thrombin binds to thrombomodulin, an endothelial cell receptor
- The thrombin-thrombomodulin complex activates protein C to Activated Protein C (APC)
Anticoagulant Function of APC:
- Activated Protein C (APC), along with its cofactor Protein S, inactivates Factors Va and VIIIa, inhibiting thrombin generation
- APC also promotes fibrinolysis by inhibiting plasminogen activator inhibitor-1 (PAI-1)

Types of Protein C Deficiency

Type I (Quantitative Deficiency):
- Reduced production of Protein C, resulting in decreased levels of both Protein C activity and antigen
- Caused by mutations that affect protein synthesis or secretion
Type II (Qualitative Deficiency):
- Normal levels of Protein C protein, but the Protein C molecule is dysfunctional
- Normal Protein C antigen level but decreased Protein C activity
- Caused by mutations that affect the catalytic activity or cofactor binding of Protein C

Protein C Activity Assay

Principle:
- Measures the functional activity of protein C in plasma
- Based on the ability of activated protein C (APC) to prolong the clotting time in a modified aPTT assay
- Patient plasma is mixed with a reagent containing thrombin and thrombomodulin to generate APC
- The ability of the generated APC to prolong the clotting time is measured
Methods:
- Clotting-Based Assay:
  - APC Resistance Assay: Measures the ability of APC to prolong the aPTT in the presence of Factor V Leiden. This is actually a test for Factor V Leiden, not Protein C. It’s included because Protein C and Factor V interact, but the test itself detects Factor V Leiden.
  - Protein C Clotting Assay: Measures the ability of generated protein C to prolong the clotting time in a modified aPTT assay.
  - These are now rarely used and have generally been replaced by chromogenic assays
- Chromogenic Assay:
  - A synthetic chromogenic substrate that is cleaved by APC
  - The amount of colored product released is measured spectrophotometrically
  - The intensity of the color is proportional to the APC activity
Procedure:
- Follow the manufacturer’s instructions for the specific assay being used
- Typically involves:
  1. Preparing the patient plasma sample
  2. Adding the reagents to activate protein C
  3. Measuring the chromogenic activity to determine Protein C’s function
Reporting:
- Reported as a percentage of normal or in IU/dL
- Reference Range: Varies depending on the laboratory and the assay used (e.g., 70-130%)
Interpretation:
- Decreased Protein C Activity:
  - Suggests Protein C deficiency
  - Levels below the reference range indicate there is not a normal amount of Protein C

Protein C Antigen Assay

Principle:
- Measures the amount of Protein C protein in the plasma, regardless of its function
- Based on immunochemical methods that use antibodies specific for Protein C
Methods:
- Enzyme-Linked Immunosorbent Assay (ELISA):
  - Microplate wells are coated with antibodies against protein C
  - The sample is added to the wells, and any protein C present binds to the antibodies
  - A secondary antibody conjugated to an enzyme is added, which binds to the protein C-antibody complex
  - A substrate for the enzyme is added, and the resulting color change is measured spectrophotometrically
- Immunoturbidimetric Assay:
  - Antibodies against protein C are added to the sample
  - As the antibodies bind to protein C, immune complexes form
  - The turbidity of the solution is measured, which is proportional to the protein C concentration
Procedure:
- Follow the manufacturer’s instructions for the specific assay being used
- Typically involves:
  1. Preparing the patient plasma sample
  2. Incubating the sample with the antibody reagent
  3. Measuring the absorbance or turbidity to determine Protein C levels
Reporting:
- Reported as a concentration (e.g., mg/dL or IU/mL)
- Reference Range: Varies depending on the laboratory and the assay used (e.g., 70-130%)
Interpretation:
- Decreased Protein C Antigen: Suggests protein C deficiency
  - Helps distinguish between type I (quantitative) and type II (qualitative) protein C deficiency
    - Type I: Decreased antigen and activity
    - Type II: Normal antigen, decreased activity

Interpreting Protein C Results

Decreased Protein C Activity and Antigen: Suggests a quantitative deficiency (Type I)
Decreased Protein C Activity with Normal Protein C Antigen: Suggests a qualitative deficiency (Type II)
Important Considerations:
- Vitamin K Antagonists (e.g., Warfarin): Warfarin inhibits the production of vitamin K-dependent coagulation factors, including protein C, leading to decreased activity and antigen levels
  - Patients should be off warfarin for at least 2 weeks before testing for protein C deficiency
- Acute Thrombosis: Protein C levels may be decreased during an acute thrombotic event due to consumption
  - Testing should be performed after the acute event has resolved
- Liver Disease: Liver disease impairs the synthesis of protein C, leading to decreased activity and antigen levels
- Pregnancy and Oral Contraceptives: Can affect protein C levels

Factors Affecting Protein C Assay Results

Pre-Analytical Variables:
- Improper Collection Technique: Tissue thromboplastin contamination or hemolysis
- Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube
- Clotted Sample: Invalidates the results
- Delayed Testing: Protein C can degrade over time
- Improper Storage: Incorrect storage temperatures can affect results
Analytical Variables:
- Instrument Malfunction: Ensure proper calibration and maintenance of the coagulation analyzer
- Reagent Problems: Use fresh, properly stored reagents and follow the manufacturer’s instructions
- Interfering Substances: Heparin can interfere with some protein C assays
Patient-Related Variables:
- Warfarin Therapy: Warfarin inhibits the production of vitamin K-dependent coagulation factors, including Protein C
- Acute Thrombosis: Protein C levels may be decreased due to consumption during a thrombotic event
- Liver Disease: Impairs the synthesis of Protein C
- Pregnancy and Oral Contraceptives: Can affect Protein C levels
- L-asparaginase therapy

Troubleshooting Erroneous Results

If the protein C results are unexpected or inconsistent with the patient’s clinical presentation:
- Check the sample for clots or hemolysis
- Repeat the test on a fresh sample
- Ensure that the correct blood-to-anticoagulant ratio was used
- Verify the instrument and reagent quality control results
- Investigate potential interfering substances
- Review the patient’s medication list and medical history
- Wait for a few weeks without anticoagulation therapy (if appropriate) to repeat testing

Key Terms

Protein C: A vitamin K-dependent anticoagulant protein that inactivates Factors Va and VIIIa
Activated Protein C (APC): The active form of protein C
Protein S: A cofactor for activated protein C
Thrombomodulin: An endothelial cell receptor that binds to thrombin and activates protein C
Thrombophilia (Hypercoagulable State): An increased tendency to develop blood clots
Vitamin K-Dependent Factors: Coagulation factors that require vitamin K for their synthesis (Factors II, VII, IX, X, and Proteins C and S)

Antiphospholipids

Protein S