Fast functional protein C assay using Protac, a novel protein C activator

Area of Science:

• Biochemistry
• Hematology
• Clinical Chemistry

Background:

• Protein C (PC) is a crucial vitamin K-dependent anticoagulant protein.
• Accurate measurement of PC levels is essential for diagnosing and managing thrombotic disorders.
• Existing methods for PC determination can be complex or lack specificity.

Purpose of the Study:

• To develop and validate a simple, rapid clotting assay for quantitative determination of functional protein C (PC) in plasma.
• To assess the assay's performance in healthy individuals, patients with liver disease, and those on warfarin therapy.
• To evaluate the assay's specificity for functional PC, distinguishing it from inactive forms.

Main Methods:

• Conversion of PC to activated PC (APC) using Protac, an activator from Agkistrodon contortrix contortrix venom.
• Degradation of factors V and VIII by generated APC in protein C-depleted plasma.
• Measurement of activated partial thromboplastin time (APTT) prolongation, proportional to PC concentration.

Main Results:

• A mean PC level of 97.1% was observed in 33 healthy individuals.
• Excellent correlation (r = 0.986) was found between the clotting assay and an enzyme-immunoassay in 33 patients with liver disease.
• The assay showed lower PC levels in warfarin-treated patients (19.8%) compared to immunological assays (55.3%), indicating specificity for functional PC and insensitivity to heparin < 1 U/ml.

Conclusions:

• The Protac-based clotting assay provides a simple and rapid method for quantitative determination of functional protein C.
• The assay demonstrates good correlation with established methods and specificity for active PC, differentiating it from inactive forms.
• Its simplicity and suitability for routine use make it valuable for monitoring patients with abnormal PC levels, including those under anticoagulation.