Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Protein S acts as a cofactor for activated protein C (APC) and possesses independent anticoagulant properties.
• Variations in reported APC-independent anticoagulant activity of Protein S suggest underlying complexities.
• Protein S directly inhibits prothrombin activation by interacting with factors Xa, Va, and phospholipids.

Purpose of the Study:

• To investigate the heterogeneity of purified Protein S preparations.
• To identify the molecular forms of Protein S responsible for its APC-independent anticoagulant activity.
• To elucidate the relationship between Protein S forms and their phospholipid-binding affinities.

Main Methods:

• Separation of Protein S forms based on differential phospholipid-binding affinities.
• Native polyacrylamide gel electrophoresis (Native PAGE) to analyze Protein S forms.
• Size-exclusion chromatography to characterize multimeric Protein S structures.

Main Results:

• Two distinct forms of Protein S were identified: a high-affinity form (<5%) and a low-affinity form (>95%).
• High-affinity Protein S exists as multimeric forms, exhibiting reduced mobility on Native PAGE.
• Multimeric Protein S demonstrated a 100-fold greater APC-independent anticoagulant activity compared to the abundant monomeric form.
• APC-independent anticoagulant activity correlated with the content of multimeric Protein S in purified preparations.
• Multimeric Protein S was not detected in normal human plasma.

Conclusions:

• Purified Protein S contains distinct monomeric and multimeric forms with differing phospholipid affinities and anticoagulant activities.
• Multimeric Protein S is the primary driver of APC-independent anticoagulant activity in purified preparations.
• The absence of multimeric Protein S in plasma suggests its limited role in plasma-mediated anticoagulation.