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J. Biol. Chem., Vol. 277, Issue 11, 8861-8865, March 15, 2002

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Neutral Glycosphingolipid-dependent Inactivation of Coagulation Factor Va by Activated Protein C and Protein S^*

Hiroshi Deguchi, José A. Fernández, and John H. Griffin

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

Received for publication, October 24, 2001, and in revised form, December 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (Gb₃Cer) 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 Gb₃Cer, 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 Gb₃Cer) 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (2-5) or ascribed to the factor V polymorphism, R506Q (5-7) or to a variety of acquired conditions, e.g. oral contraceptive use (8), autoantibody against APC (9), etc. APC resistance is also associated with increased risk of ischemic stroke in subjects with normal Arg-506-factor V (10, 11). Severe deficiency of protein C or protein S causes life-threatening thrombosis (12-15). In the prospective epidemiological Atherosclerosis Risk in Communities study, protein C was an inverse risk factor for stroke and appeared to be protective (16). Recombinant APC therapy notably reduces mortality due to sepsis (17). APC exerts profibrinolytic and anti-inflammatory activities (18) as well as anticoagulant activity. Thus, the protein C pathway provides physiological antithrombotic and anti-inflammatory activities in humans, and it is important to delineate molecular mechanisms for these activities.

The coagulation system is thought to require negatively charged phospholipids. However, coagulation and anticoagulation reactions are affected differently by different membrane phospholipid components (18-23). For instance, anionic phospholipids, mainly phosphatidylserine (PS), most effectively increase prothrombinase activity; in contrast, phosphatidylethanolamine and cardiolipin enhance the APC anticoagulant pathway more than the procoagulant pathways. Among normal plasma components, high density lipoprotein notably has APC/protein S-dependent anticoagulant cofactor activities in purified clotting factor assay systems (22). Thus, the blood coagulation pathways and the opposing protein C anticoagulant system may be differentially modulated by plasma lipids and lipoproteins.

There are over 300 known glycolipids. Glycosphingolipids found in plasma include the neutral lipids, glucosylceramide (GlcCer) (glucocerebroside), lactosylceramide (LacCer/CD17), globotriaosylceramide (Gb₃Cer/CD77), and globotetraosylceramide (Gb₄Cer) (24), as well as various gangliosides (GM3, GD1a, GM2, GT1b, GD1b, GQ1b) and sulfatide (25-27). Glycolipids are important components of cell membranes, and glycolipid molecules present their highly varied saccharide residues on cell surfaces as well as on the surface of lipoprotein particles, exposing saccharides in the outer lipid leaflet to interactions with cells, antibodies, bacterial toxins, and viral envelope proteins (28, 29). Interestingly, cell surface glycosphingolipids are not distributed randomly but tend to be locally enriched in various-sized microdomains, so-called membrane rafts, that have specialized functional properties (30, 31).

Glycolipids can play critical roles as bioregulators of a variety of processes such as cell-cell adhesion, cell proliferation, cell mobility and apoptosis (28, 29). Recently, 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. To test the hypothesis that the blood coagulation pathways could be modulated by glycosphingolipids as well as phospholipids, we studied the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on APC/protein S anticoagulant activity. The incorporation of certain but not all neutral glycosphingolipids into phosphatidylcholine (PC)/PS vesicles enhanced anticoagulant activity of APC/protein S in plasma and in purified systems by augmenting factor Va inactivation due to APC cleavage at Arg-506. This surprising finding that neutral glycolipids in PC/PS vesicles enhance the anticoagulant protein C pathway provides a mechanistic rationale for the discovery (32) that deficiency of plasma GlcCer is associated with increased incidence of venous thrombosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins and Lipids-- Protein S was purified by conventional methods and by immunoaffinity chromatography (33). APC was purchased from Enzyme Research Laboratories (South Bend, IN), and bovine serum albumin (BSA) from Calbiochem-Novabiochem Corp (San Diego, CA). Factor Va was purchased from Hematologic Technologies Inc., (Essex Junction, VT). Gln-506-factor Va was isolated from the plasma of a homozygous APC-resistant patient (34) and purified by conventional methods and by immunoaffinity chromatography (35). All phospholipids and glycolipids were purchased from Sigma.

Preparation of Glycolipid/Phospholipid Vesicles-- Purified lipids in chloroform were mixed, and following evaporation of the organic solvent the lipid mixture was hydrated at a concentration of 6.25 mM in Tris buffered saline (TBS) and freeze-thawed 10 times using liquid nitrogen. Then each final solution of vesicles was prepared by extrusion 15 times through a 0.2-µm filter (Osmonics, Livermore, CA) (32). The concentration of PS was 10% (w/w)) unless otherwise noted. The concentrations of glycolipid and PC recovered in vesicles were determined by orcinol-based sugar test (Sigma) (36) and an enzymatic colorimetric method for choline (Phospholipids B kit, Wako Chemical USA, Inc., Richmond, VA), respectively.

Vesicle Characterization by Gel Filtration-- PC/PS and PC/PS/GlcCer vesicles in TBS were applied to columns containing Sephacryl S 1000 (Amersham Biosciences) (fractionation range: M_r 5 × 10⁵ to 5 × 10⁸) and Superose 6 (Amersham Biosciences) (fraction range: M_r 5 × 10³ to 5 × 10⁶) equilibrated with TBS containing 5 mM CaCl₂. PC/PS and PC/PS/GlcCer vesicles were incubated with 5 mM CaCl₂ for 15 min in TBS and then applied to the column containing Sephacryl S 1000. The column was eluted with equilibrating buffer, and the lipid was measured as light scattering at 236 nm. Blue dextran (_w 2,000,000) and BSA were also applied to the column as molecular weight markers and were monitored as absorbance at 280 nm.

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. Aliquots were withdrawn at various indicated times, and the inactivation reaction was quenched by adding EDTA prior to determination of residual factor Va activity. The residual factor Va activity was quantitated using prothrombin time clotting assays and standard log-log calibration curves of clotting time versus factor Va concentration generated using purified factor Va and factor V-deficient plasma (22, 37).

Curve Fitting of Time Courses of Factor Va Inactivation-- Time courses of factor Va inactivation by APC and protein S were determined by following the loss of procoagulant cofactor activity of factor Va. Curve fitting of the time cour e of factor Va inactivation was done as described previously (38, 39) using the following equation (Equation 1),

(Eq. 1)

in which Va_t is the cofactor activity determined at time t, Va₀ is the initial cofactor activity determined before APC is added, B is the cofactor activity of factor Va_int (expressed as fraction of the cofactor activity of native factor Va) ("int" denotes intermediate), k₅₀₆ is the rate constant for the cleavage at Arg-506, k₃₀₆ is the rate constant for the cleavage at Arg-306 in factor Va_int, and k'₃₀₆ is the rate constant for the cleavage at Arg-306 in native factor Va. The rate constants and the cofactor activity of factor Va were obtained by fitting the data using non-linear curve fit by Prizm3.0 software (Graph Pad Software, Inc, San Diego, CA). Time courses of normal factor Va inactivation were fitted with a fixed value for k'₃₀₆ determined from the inactivation time course for Gln-506-factor Va fit to a single exponential.

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 µl) were mixed with normal plasma (25 µl), APC (34.5 nM final), and/or exogenous protein S (36 nM final) or buffer (TBS containing 0.5% BSA) (30 µl) and incubated for 3 min at 37 °C. Then, factor Xa (50 µl) (0.3 nM final) in buffer containing 30 mM CaCl₂ was added to initiate clotting, and clotting times were recorded using an Amelung KC4 micro-coagulometer (Sigma).

Gel Analysis of Factor Va Inactivation-- To study the effect of glycolipids on the factor Va cleavage pattern by APC/protein S, SDS-PAGE analysis was performed. APC (45 nM) and/or protein S (117 nM) or buffer was incubated for 2 or 30 min with purified factor Va (400 nM), 270 µg/ml lipid vesicles containing either PC/PS (90%/10%) or PC/PS/ GlcCer (80%/10%/10%) and 5 mM CaCl₂. Reactions were quenched by adding Tris/HCl, 10 mM EDTA, 2.5% SDS, 25% glycerol, 1.7 mM dithiothreitol, pH 6.8. Then SDS-PAGE was performed using 4-12% Bis-Tris polyacrylamide gels with MOPS buffer (Novex, San Diego, CA). The factor Va proteolytic fragments were visualized on the gels by Colloid blue stain (Novex).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Vesicle Size-- The distribution of vesicle size was determined by gel filtration using Sephacryl S 1000 with an exclusion limit at 400 nm diameter or particle size of M_r >50,000,000. Blue dextran (Sigma) (mean M_r 2,000,000, mean particle size 300 nm diameter) and BSA (M_r 66,000) eluted at 215 ml and 320 ml, respectively (Fig. 1). In TBS containing 5 mM CaCl₂, PC/PS and PC/PS/GlcCer vesicles eluted at 290 ml with identical peak profiles (Fig. 1). Both PC/PS and PC/PS/GlcCer vesicles in TBS without CaCl₂ eluted at 290 ml, and their peak profiles were identical (data not shown). These results indicate that these two populations of vesicles had the same size distribution in the presence or absence of CaCl₂. When using Superose 6 for gel filtration, elution profiles for PC/PS and PC/PS/GlcCer vesicles were identical (data not shown). Thus, the size distribution analyses of vesicles showed there was no detectable difference for the size distribution between PC/PS (90%/10%) and PC/PS/GlcCer (80%/10%/10%) vesicles.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Size distribution of PC/PS and PC/PS/GlcCer vesicles by gel filtration chromatography. PC/PS () and PC/PS/GlcCer () vesicles were chromatographed on a Sephacryl S 1000 column. Blue dextran (w 2,000,000) and BSA (w 66,000) were used as molecular weight markers.

The Influence of GlcCer in PC/PS Vesicle on Factor Va Inactivation by APC-- To test whether the incorporation of GlcCer in PC/PS vesicles has a direct effect on factor Va inactivation by APC, studies were performed as described under "Experimental Procedures." Inactivation of purified factor Va by APC was dose dependently enhanced by the presence of GlcCer (0-20%, w/w) in PC/PS vesicles containing 10% PS (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   GlcCer in phospholipid vesicles enhances factor Va inactivation by APC. Purified factor Va (1 nM final) was incubated with APC alone (0.94 nM final) and various multicomponent vesicles to allow factor Va inactivation for 5 min at 37 °C, and then residual factor Va activity was determined using clotting assays as described under "Experimental Procedures." Concentration of GlcCer in PC/PS vesicles was 0% (), 10% (), 15% (), or 20% (). PS was 10% (w/w), and PC (90-70%) was varied as needed.

Glycosphingolipid Enhancement of Plasma Anticoagulant Response to APC/Protein S-- To examine the effects of plasma-neutral glycosphingolipids on APC and/or protein S anticoagulant activity in normal plasma, factor Xa-1-stage assays were performed after addition of Glc-containing neutral glycosphingolipids, including GlcCer, LacCer, Gb₃Cer, Gb₄Cer, and GalCer in PC/PS vesicles. When GlcCer in PC/PS was added to plasma in the presence of APC, it caused dose-dependent prolongation of the clotting assay greater than the prolongation observed for APC plus PC/PS alone (Fig. 3A). GlcCer and other tested glycosphingolipids did not affect the baseline clotting time. When protein S alone was added in the absence of APC, there were no differences in clotting times for PC/PS compared with PC/PS/GlcCer vesicles (data not shown). LacCer and Gb₃Cer in PC/PS vesicles also prolonged clotting times greater than PC/PS vesicles in the presence of APC (Fig. 3A). However, Gb₄Cer and GalCer in PC/PS vesicles at up to 200 µg/ml total lipid did not enhance APC action (Fig. 3A). Above 250 µg/ml total lipid, both GalCer and Gb₄Cer in PC/PS vesicles did enhance APC activity (data not shown). When clotting time prolongation by APC was determined at varying glycolipid levels in PC/PS vesicles, as little as 1% of Gb₃Cer gave enhancement of APC activity (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Anticoagulant response of plasma to APC is enhanced by neutral glycosphingolipids in phospholipid vesicles. A, neutral glycosphingolipids in PC/PS vesicles were added to normal plasma aliquots that were then assayed using factor Xa-1-stage clotting assays in the presence and absence of added APC. Clotting times are shown for the presence of APC (upper curves above 90 s) and for baseline controls without APC addition (lower families of curves around 60 s). Vesicles contained 10% PS, 0 or 10% glycosphingolipid, and 80 or 90% PC. PC/PS (), PC/PS/GlcCer (), PC/PS/LacCer (), PC/PS/Gb3Cer (), PC/PS/Gb4Cer (), and PC/PS/GalCer (). B, multicomponent vesicles (172 µg/ml) containing the indicated weight percent of neutral glycosphingolipid were tested in factor Xa-1-stage assays with or without APC added. Vesicles contained 10% PS and PC as needed. PC/10%PS/GlcCer () and PC/PS/Gb3Cer () in the presence of APC, and PC/PS/GlcCer () and PC/10%PS/Gb3Cer () in the absence of APC are shown.

Effect of Neutral Glycosphingolipids on Factor Va Inactivation by APC-- GlcCer, LacCer, Gb₃Cer, Gb₄Cer, and GalCer at various concentrations (0, 1, 5, 10%) in PC/PS vesicles containing 10% PS were compared for their ability to enhance factor Va inactivation by APC/protein S. GlcCer at 5 and 10% in PC/PS vesicles markedly enhanced inactivation of factor Va (Fig. 4A). The apparent potency of PC/PS vesicles containing 10% GlcCer was approximately an order of magnitude greater than PC/PS vesicles. Gb₃Cer was notably more potent than GlcCer. Surprisingly, as little as 1% Gb₃Cer in PC/PS vesicles significantly enhanced factor Va inactivation even at 1 µg/ml total lipid, and 5% Gb₃Cer was very effective (Fig. 4D). The stimulating effects of LacCer were generally similar to those of GlcCer (Fig. 4C). When GalCer in PC/PS vesicles was studied, the effects on factor Va inactivation by APC/protein S were generally modest (Fig. 4B). Gb₄Cer in PC/PS vesicles showed essentially no significant effect on factor Va inactivation (Fig. 4E) compared with Gb₃Cer, LacCer, or GlcCer. Thus, various neutral glycosphingolipids in phospholipid vesicles directly enhance inactivation of purified factor Va by APC/protein S, and the enhancement of the APC anticoagulant action by neutral glycolipids was differentially dependent on the saccharide moieties.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Factor Va inactivation by APC/protein S is variably enhanced by different neutral glycosphingolipids in phospholipid vesicles. Purified factor Va (1.5 nM) was incubated with APC (0.16 nM final)/protein S (18 nM final) and varying lipid vesicles to allow factor Va inactivation for 5 min, and then the residual factor Va activity was determined. Neutral glycosphingolipid in vesicles (weight %) containing 10% PS and varying PC was 0% (), 1% (), 5% (), or 10% (). PC/PS vesicles contained: GlcCer (A), GalCer (B), LacCer (C), Gb3Cer (D), or Gb4Cer (E).

Effect of GlcCer on Limited Proteolysis of Factor Va by APC-- To study qualitatively the well known cleavages of factor Va by APC/protein S, SDS-PAGE analyses of factor Va inactivation with both PC/PS and PC/PS/GlcCer vesicles were performed. In the presence of both PC/PS and PC/PS/GlcCer vesicles, APC caused a decrease of the band seen for the heavy chain (105 K_d) and an increase of products with apparent M_r of 60/62, 45, 30, and 26/28 K_d. When cleavage patterns for GlcCer/PC/PS vesicles were compared with those for PC/PS vesicles, the various factor Va fragments apparently derived from the heavy chain comigrated (Fig. 5). The factor Va light chain mobility was not affected by APC/protein S in the presence of either PC/PS or PC/PS/GlcCer vesicles. Thus, GlcCer incorporation into PC/PS vesicles promoted a pattern of limited proteolysis of factor Va by APC/protein S that was indistinguishable from that caused by PC/PS vesicles.

View larger version (77K): [in this window] [in a new window]   Fig. 5.   Effect of GlcCer on limited proteolysis of factor Va by APC. Factor Va (400 nM) was incubated with APC (2.2 nM)/protein S (36 nM) in the presence of PC/PS or PC/PS/GlcCer vesicles (270 nM) at 37 °C. The reaction was stopped at 2 and 30 min, and SDS-PAGE was used to visualized factor Va polypeptides by staining with Colloid Blue. Lane 1, control factor Va (0 time); lanes 2 and 3, samples contains PC/PS/GlcCer vesicles after incubations for 2 or 30 min; lanes 4 and 5, samples contains PC/PS vesicles after incubations for 2 or 30 min.

The Effect of Protein S and GlcCer on Inactivation Factor Va-- Inactivation of membrane bound factor Va by APC occurs via a biphasic reaction that consists of an initial rapid phase that is caused by Arg-506 cleavage (k₅₀₆) and a slow phase that is caused by Arg-306 cleavage (k₃₀₆) (38). A value for the fraction of intermediate factor Va activity following cleavage at Arg-506 was first determined to be 0.56 under the experimental conditions employed (data not shown). A value for the apparent second-order rate constant for cleavage at Arg-306 in the absence of Arg-506 cleavage was determined from the time course of inactivation of Gln-506-factor Va (data not shown), which was fit to a single exponential equation. This value of k'₃₀₆ was fixed in the equation used to fit the inactivation time courses of factor Va (38, 39). The rate constants obtained by fitting the time course curve for PC/PS (90/10) vesicles and APC alone in Fig. 6 to a biphasic exponential were k₅₀₆ = 2.2 × 10⁷ M¹ s¹ and k₃₀₆ = 2.0 × 10⁶ M¹ s¹. When the same experiment using PC/PS vesicles was performed in the presence of 18 nM protein S, k₅₀₆ was hardly affected (3.0 versus 2.2 × 10⁷ M¹ s¹), whereas k₃₀₆ was 5-fold greater (9.6 versus 2.0 × 10⁶ M¹ s¹) as described before (38, 39) (Fig. 6A). For normal Arg-506-factor Va, GlcCer in PC/PS vesicles increased the rate constants for factor Va inactivation for k₅₀₆ by 4-fold (9.1 versus 2.2 × 10⁷ M¹ s¹) and k₃₀₆ by 2.1-fold (4.2 versus 2.0 × 10⁶ M¹ s¹) (Table I). In the presence of protein S, GlcCer in PC/PS vesicles increased the rate constants of APC-catalyzed cleavage 13-fold for Arg-506 (39 versus 3.0 × 10⁷ M¹ s¹) and 2.4-fold for Arg-306 (24 versus 9.6 × 10⁶ M¹ s¹). Thus, GlcCer increased the rate of APC-catalyzed cleavage at both Arg-506 and Arg-306 in the presence and absence of protein S, and GlcCer remarkably had the biggest effect on cleavage at Arg-506 in the presence of protein S. 

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of protein S and GlcCer on factor Va inactivation by APC. Purified factor Va (1.5 nM, final) was incubated with APC (0.3 nM final)/protein S (18 nM final) and multicomponent lipid vesicles (23 µg/ml) to allow factor Va inactivation for times indicated at 37 °C. Then, residual factor Va activity was quantitated as described under "Experimental Procedures." The factor Va activity observed at 0 time was defined as 100%. Reaction mixtures contained: PC/PS (90/10) + APC (); PC/PS (90/10) + APC/protein S (); PC/PS/GlcCer (80/10/10) + APC (); and PC/PS/GlcCer (80/10/10) + APC/protein S ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Blood coagulation pathways and the anticoagulant protein C pathway can be modulated by plasma lipids and lipoproteins, and membrane surfaces accelerate the proteolytic inactivation of factor Va by activated protein C. Anionic PS is believed to be a particularly procoagulant phospholipid (19-22, 40), whereas phosphatidylethanolamine, high density lipoprotein, and cardiolipin are apparently, under certain conditions, anticoagulant lipid cofactors that act by enhancing the anticoagulant action of APC (21-23). The present study has two major findings. First, the neutral glycolipids GlcCer, LacCer, and Gb₃Cer in PC/PS vesicles enhance APC/protein S anticoagulant activity without significantly enhancing procoagulant activity in coagulation assays using human plasma. Second, in purified assay systems, GlcCer, LacCer, and Gb₃Cer incorporated into PC/PS vesicle showed dose-dependent enhancement in factor Va inactivation by APC/protein S. These surprising findings that certain neutral glycosphingolipids in PC/PS vesicles enhance the anticoagulant protein C pathway provide a mechanistic rationale for the discovery that deficiency of plasma GlcCer is associated with increased incidence of venous thrombosis (32) because it is shown that uncharged, neutral lipids such as GlcCer can enhance APC activity.

The mechanism of factor Va inactivation in a purified system based on the rate constant analysis showed unique properties of GlcCer in PC/PS vesicles, which were different from pure phospholipid vesicles. SDS-PAGE analysis showed no differences in the cleavage patterns of factor Va by APC/protein S for PC/PS vesicles compared with PC/PS/GlcCer vesicles, suggesting that GlcCer did not alter the cleavage sites in factor Va by APC/protein S. GlcCer in PC/PS vesicles enhanced the rates of factor Va cleavage by APC at both Arg-506 and Arg-306. Interestingly, in the presence of protein S, GlcCer in PC/PS vesicles increased 13-fold the cleavage rate at Arg-506 compared with a 2.4-fold increase for Arg-306 (Table I), in contrast to the small effect of protein S on Arg506 for phospholipid vesicles (38). Since there is dose dependence of protein S cofactor activity and plasma free protein S (145 nM) is much higher than that used here (18 nM), these protein S-dependent effects of GlcCer could be much larger in vivo, especially on some membrane microdomains enriched in certain neutral glycosphingolipid (see below). Thus, GlcCer has the possibility to modulate the specific Arg cleavages responsible for factor Va inactivation on membrane or lipoprotein surfaces.

The biological activities of glycosphingolipids are partially determined by their sugar head groups and partially by their lipid moieties (41). The ability of neutral glycosphingolipids to enhance APC/protein S anticoagulant activity in plasma and in purified factor Va inactivation assays as presented here also demonstrated specificity for the conformation and composition of the saccharide moieties. Anticoagulant activity in normal plasma appeared to be, at least in part, specific for D-Glc linked covalently to Cer because GlcCer was anticoagulant in PC/PS vesicles whereas GalCer was not. Gb₄Cer containing Gal-Gal-Gal-Glc in PC/PS vesicles did not have anticoagulant activities, whereas Gb₃Cer containing Gal-Gal-Glc had strong anticoagulant cofactor effects. Different saccharide orientations of the tetrasaccharide in Gb₄Cer compared with the trisaccharide in Gb₃Cer has been demonstrated (42), and such structural differences may relate to glycosphingolipid anticoagulant properties.

Glycosphingolipids may play important in vivo roles for the blood coagulation system. Based on a pilot clinical study showing that the mean plasma GlcCer level is lower in venous thrombosis patients than in controls (32), we suggested that GlcCer could be important for maintaining an antithrombotic state in vivo. Moreover, an Escherichia coli-derived verotoxin, verotoxin B, is known to be a Gb₃Cer-specific-binding protein that is a pathological factor for the hemolytic uremic syndrome. In patients with hemolytic uremic syndrome, activation of the coagulation system and glomerular thrombotic microangiopathy are observed, and these effects are mediated by the binding of verotoxin B to Gb₃Cer (43). Administration of verotoxin B causes fibrin deposition in an animal model (44). Therefore, we speculated that the blockade of Gb₃Cer in the kidney by verotoxin B may locally stimulate the coagulation system, at least in part, by inhibiting Gb₃Cer-dependent enhancement of APC/protein S activities.

Most glycosphingolipids are distributed in membranous structures in the cell and are usually located in the external leaflet rather than the internal leaflet of the bilayer membrane of cells. Glycosphingolipids tend to form glycolipid-enriched microdomains, sometimes called rafts, which are enriched with cholesterol and sphingolipid and which contain relatively less phospholipids than other areas of the plasma membrane (28-31). We recently demonstrated that GlcCer increases the affinity of APC for phospholipid vesicles and that GlcCer binds directly to APC.² Thus, we speculate that GlcCer or the other neutral glycosphingolipids shown here to enhance APC activity may be localized in some, but not all, microdomains on certain cells or in a subset of lipoproteins where they could enhance the binding of APC to a subset of microdomains. Consequently, this hypothesized subset of microdomains might be considered "antithrombotic microdomains" because they could bind and localize APC to interact efficiently with protein S and to enhance factor Va inactivation. Moreover, we speculate that a subset of glycosphingolipid-enriched microdomains could also mediate anti-inflammatory activities of APC by increasing the affinity of receptors for APC on certain cells. The existence of antithrombotic microdomains with neutral glycosphingolipids that enhance affinity for APC is purely speculative at this point and merits future experimental assessment.

	ACKNOWLEDGEMENTS

We are grateful to Young Mee Lee for skillful technical assistance and for helpful discussion with Drs. M. J. Heeb, A. J. Gale, and S. Yegneswaran.

	FOOTNOTES

^* This work was supported in part by Grants R37HL52246 and R01HL21544 from the National Institutes of Health and an American Heart Association Postdoctoral Fellowship (Western States Affiliate) (to H. D.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Inst., MEM180, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8220; Fax: 858-784-2243; E-mail: jgriffin@scripps.edu.

Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M110252200

² S. Yegneswaran, H. Deguchi, and J. H. Griffin, unpublished results.

	ABBREVIATIONS

The abbreviations used are: APC, activated protein C; PS, phosphatidylserine; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb₃Cer, globotriaosylceramide; Gb₄Cer, globotetraosylceramide; PC, phosphatidylcholine; BSA, bovine serum albumin; TBS, Tris-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; GalCer, galactosylceramide; _w, weight average M_r.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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