Abstract
To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.
Highlights
Poor anticoagulant response to activated protein C (APC),1 termed APC resistance, is detected in 20 –50% of venous thrombosis patients [1], and it can be idiopathic [2,3,4,5] or ascribed to the factor V polymorphism, R506Q [5,6,7] or to a variety of acquired conditions, e.g. oral contraceptive use [8], autoantibody against APC [9], etc
We found that deficiency of the plasma glycolipid, GlcCer, is a potential risk factor for venous thrombosis and that GlcCer can enhance inactivation of factor Va by APC and protein S [32], indicating a new potential function for neutral glycosphingolipids, namely the ability to modulate blood coagulation systems on cell membranes or on lipoprotein particles
Blood coagulation pathways and the anticoagulant protein C pathway can be modulated by plasma lipids and lipoproteins, TABLE I GlcCer-dependent enhancement of APC cleavage at Arg-506 and
Summary
Proteins and Lipids—Protein S was purified by conventional methods and by immunoaffinity chromatography [33]. Factor Va Inactivation Assay—To study the time course of factor Va inactivation by APC alone or APC/protein S, mixtures containing various lipid vesicles (e.g. PC/PS (90%/10%, w/w), PC/PS/GlcCer (80%/10%/ 10%), or vesicles containing glycolipids or PC at varying concentrations) were incubated with APC, protein S, and factor Va in TBS, 5 mM CaCl2 containing 0.1% BSA at 37 °C. Factor Xa-1-stage Clotting Assay—The procoagulant and anticoagulant properties of vesicles containing GlcCer were determined using factor Xa-initiated clotting assays and normal plasma with exogenously added APC and/or protein S. For these assays, GlcCer-containing vesicles at varying doses (50 l) were mixed with normal plasma (25 l), APC (34.5 nM final), and/or exogenous protein S (36 nM final) or buffer (TBS containing 0.5% BSA) (30 l) and incubated for 3 min at 37 °C. The factor Va proteolytic fragments were visualized on the gels by Colloid blue stain (Novex)
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