Reevaluation of the role of HDL in the anticoagulant activated protein C system in humans
Cecilia Oslakovic, … , Eva Norstrøm, Björn Dahlbäck
Cecilia Oslakovic, … , Eva Norstrøm, Björn Dahlbäck
Published April 12, 2010
Citation Information: J Clin Invest. 2010;120(5):1396-1399. https://doi.org/10.1172/JCI42260.
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Brief Report Hematology

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

Abstract

HDL has anti-atherogenic properties, and plasma levels of HDL cholesterol correlate inversely with risk of coronary artery disease. HDL reportedly functions as a cofactor to the anticoagulant activated protein C (APC) in the degradation of factor Va (FVa). The aim of the present study was to elucidate the mechanism by which HDL functions as cofactor to APC. Consistent with a previous report, HDL isolated from human plasma by ultracentrifugation was found to stimulate APC-mediated degradation of FVa. However, further purification of HDL by gel filtration revealed that the stimulating activity was not a property of HDL. Instead, the stimulating activity eluted completely separately from HDL in the high-molecular-weight void volume fractions. The active portion of these fractions stimulated FVa degradation by APC and supported the assembly of factor Xa and FVa into a functional prothrombinase complex. Both the procoagulant and anticoagulant activities were blocked by addition of annexin V, suggesting that the active portion was negatively charged phospholipid membranes. These results demonstrate that HDL does not stimulate the APC/protein S effect and that the activity previously reported to be a property of HDL is instead caused by contaminating negatively charged phospholipid membranes.

Authors

Cecilia Oslakovic, Eva Norstrøm, Björn Dahlbäck

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Figure 3

Annexin V inhibits both anti- and procoagulant activities.

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Annexin V inhibits both anti- and procoagulant activities. Fractions 16–...
Fractions 16–17 from the void of the size exclusion chromatography of HDL were incubated in the absence or presence of 100 nM annexin V with 2.5 mM CaCl2 at 25°C for 15 minutes. Remaining anti- and procoagulant activities were tested using a FVa inactivation assay (A) or prothrombinase assay (B). (A) Final concentrations during inactivation of FVa were 20 pM FVa, 0.5 nM APC, 14.5 nM protein S, using a 30-minute inactivation time. Fractions were diluted 5-fold during inactivation of FVa, and values are expressed as percent of controls without APC. (B) In the prothrombin activation, final concentrations were 210 pM FVa, 2.5 nM FXa, 0.5 μM prothrombin, using a 2-minute activation time. The samples were then diluted and the generated thrombin tested as described in Methods. The tested fractions were diluted 10-fold during the activation of prothrombin. Values are expressed as mean ± SD from repeated experiments (n = 2).