Reevaluation of the role of HDL in the anticoagulant activated protein C system in humans

Area of Science:

• Biochemistry
• Hematology
• Lipid Metabolism

Background:

• High-density lipoprotein (HDL) possesses anti-atherogenic properties.
• Plasma HDL cholesterol levels inversely correlate with coronary artery disease risk.
• HDL has been reported to act as a cofactor for activated protein C (APC) in degrading factor Va (FVa).

Purpose of the Study:

• To elucidate the precise mechanism by which HDL purportedly functions as a cofactor for APC.
• To determine if HDL itself is responsible for stimulating APC-mediated FVa degradation.

Main Methods:

• Isolation of HDL from human plasma via ultracentrifugation.
• Further purification of HDL using gel filtration chromatography.
• Assay of APC-mediated FVa degradation in different fractions.
• Assessment of prothrombinase complex assembly.
• Inhibition studies using annexin V.

Main Results:

• HDL isolated by ultracentrifugation showed APC-mediated FVa degradation-stimulating activity.
• Further purification by gel filtration separated the stimulating activity from HDL.
• The activity eluted in high-molecular-weight void volume fractions, distinct from HDL.
• The isolated active component stimulated FVa degradation by APC and prothrombinase complex assembly.
• Annexin V blocked both procoagulant and anticoagulant activities, indicating negatively charged phospholipid membranes.

Conclusions:

• HDL does not directly stimulate the APC/protein S cofactor activity.
• The previously reported HDL-associated activity in APC-mediated FVa degradation is attributed to contaminating negatively charged phospholipid membranes.
• This finding clarifies the role of HDL in coagulation and highlights the importance of rigorous purification in biochemical studies.