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Glucosylceramide, a Neutral Glycosphingolipid Anticoagulant Cofactor, Enhances the Interaction of Human- and Bovine-activated Protein C with Negatively Charged Phospholipid Vesicles

2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês

10.1074/jbc.m206746200

1083-351X

Subramanian Yegneswaran, Hiroshi Deguchi, John H. Griffin,

Complement system in diseases

The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with aKd app = 16 μg/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with aKd app = 3 μg/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of ∼63 and ∼64 Å were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming κ2 = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent Kd of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6–7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal γ-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids. The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with aKd app = 16 μg/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with aKd app = 3 μg/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of ∼63 and ∼64 Å were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming κ2 = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent Kd of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6–7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal γ-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids. phosphatidylserine glucosylceramide activated protein C fluorescence resonance energy transfer l-3-phosphatidylcholine-1,2-di[1-14C]oleoyl phosphatidylcholine galactosylceramide globotetraosylceramide bovine serum albumin factor Xa factor Va (5-dimethylaminonaphthalene-1-sulfonyl-glutamylglycylarginyl chloromethylketone APC labeled in the active site with DEGR-CK Nα-fluorescein-pbenzoyl phenylalanyllysyl (Nεbromoacetyl) amide APC labeled with LWB in the active site via aNα-[(acetylmercapto)acetyl-(d-phenylalanyl)prolylarginyl chloromethylketone inhibitor APC labeled in the active site with 7-diethylamino-3-((4′-(iodoacetyl)amino)phenyl)-4-methylcoumarin via aNα-[(acetylmercapto)acetyl-(d-phenylalanyl)prolylarginyl chloromethylketone inhibitor octadecylrhodamine d-glucosyl-β1–1′-N-octanoyl-d-erythro-sphingosine N-octanoyl-d-erythro-sphingosine diphenylhexatriene 7-nitrobenz-2-oxa-1,3-diazole 6-NBD-PS, 1-palmitoyl-2-[6-[7-nitro-2–1,3-benzoxadiazol-4-yl) amino] caproyl]-sn-glycero-3-phosphoserine critical micellar concentration γ-carboxyglutamic acid donor donor and acceptor blank Blood coagulation has to be carefully regulated by balancing the procoagulant reactions with potent anticoagulant processes. Both procoagulant and anticoagulant reactions occur at a physiologically significant rate only when the respective enzymes form multicomponent complexes on lipid membrane surfaces (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1640) Google Scholar). Thus, membranes play a pivotal role in regulating blood coagulation reactions. The molecular details of mechanism(s) by which various lipids affect coagulation are not fully understood. Many groups have observed that lipid vesicles containing negatively charged phospholipids, especially phosphatidylserine (PS),1bind and markedly enhance the rate of activation of procoagulant enzymes (2Lundbland R.L. Davie E.W. Biochemistry. 1964; 3: 1720-1725Crossref PubMed Scopus (53) Google Scholar, 3Papahadjopoulus D. Hanahan D.J. Biochim. Biophys. Acta. 1964; 90: 436-439Crossref PubMed Scopus (90) Google Scholar, 4Hemker H.C. Kahn M.J.P. Nature. 1967; 215: 248-251Crossref PubMed Scopus (43) Google Scholar, 5Nelsestuen G.L. Kisiel W. DiScipio R.G. Biochemistry. 1978; 17: 2134-2138Crossref PubMed Scopus (138) Google Scholar, 6Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 7Stevenson K.J. Poller L. Thromb. Res. 1982; 26: 341-350Abstract Full Text PDF PubMed Scopus (9) Google Scholar, 8Rosing J. Speijer H. Zwaal R.F.A. Biochemistry. 1988; 27: 8-11Crossref PubMed Scopus (60) Google Scholar, 9Pei G. Powers D.D. Lentz B.R. J. Biol. Chem. 1993; 268: 3226-3233Abstract Full Text PDF PubMed Google Scholar). It has been proposed that the high rate of activation of procoagulant enzymes in the presence of vesicles containing PS is due in large part to enhancement of substrate binding to the negatively charged surface of the phospholipid bilayer. The resulting high local concentration of substrate on the membrane surface can enhance diffusion of substrate to the activation complex in two dimensions rather than in three dimensions (10Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). Although the significance of this proposed mechanism has been controversial and alternative models have been proposed to explain many kinetic data (11Lu Y. Nelsestuen G.L. Biochemistry. 1996; 35: 8201-8209Crossref PubMed Scopus (22) Google Scholar, 12Lu Y. Nelsestuen G.L. Biochemistry. 1996; 35: 8193-8200Crossref PubMed Scopus (34) Google Scholar), current paradigms most often assume that membranes only provide a surface template for the assembly and function of the various procoagulant enzyme-containing multicomponent complexes and that the procoagulant phospholipids do not play a more active role in the blood coagulation reaction. However, it has been recently reported that submicellar concentrations of a short chain variant of phosphatidylserine, namely dicaproyl phosphatidylserine, enhances the rate of activation of both prothrombin by the prothrombinase complex and factor X by the Xase complex (13Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar, 14Gilbert G.E. Arena A.A. Biochemistry. 1997; 36: 10768-10776Crossref PubMed Scopus (15) Google Scholar). These data suggest the existence of a functionally significant binding site(s) for C6PS on one or more procoagulant plasma proteins and that the occupancy of these sites by the phospholipid molecules activates the clotting factors. If there are functionally important PS-binding sites on some procoagulant proteins, do such functionally important lipid-binding sites also exist in anticoagulant proteins? Although both procoagulant and anticoagulant reactions are markedly enhanced by the presence of negatively charged surfaces in vitro, certain lipids and lipoproteins selectively enhance anticoagulant reactions in plasma (15Smirnov M.D. Esmon C.T. J. Biol. Chem. 1994; 269: 816-819Abstract Full Text PDF PubMed Google Scholar, 16Fernández J.A. Kojima K. Hackeng T.M. Griffin J.H. Blood Cells Mol. Dis. 2000; 26: 115-123Crossref PubMed Scopus (31) Google Scholar, 17Griffin J.H. Kojima K. Banka C.L. Curtis L.K. Fernández J.A. J. Clin. Invest. 1999; 103: 219-227Crossref PubMed Scopus (206) Google Scholar). Recently, Deguchi and co-workers reported that plasma glucosylceramide (GlcCer) deficiency is a potential risk factor for venous thrombosis and that depletion or augmentation of GlcCer in normal plasma either reduces or enhances, respectively, the anticoagulant response to activated protein C (APC) (18Deguchi H. Fernández J.A. Pabinger I. Heit J.A. Griffin J.H. Blood. 2001; 97: 1907-1914Crossref PubMed Scopus (48) Google Scholar). Subsequently, they reported that certain glycolipids such as GlcCer, lactosylceramide, and globotriaosylceramide, enhance the anticoagulant response of APC (19Deguchi H. Fernández J.A. Griffin J.H. J. Biol. Chem. 2002; 277: 8861-8865Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). In this study, we have examined the mechanism by which such glycolipids may enhance the anticoagulant activity of APC. To test the hypothesis that GlcCer increases the anticoagulant activity of APC by increasing its affinity for lipid surfaces where anticoagulant reactions can occur, the binding of APC to GlcCer-containing phospholipid vesicles was examined using fluorescence resonance energy transfer (FRET). Additionally, we have also examined whether there is a unique GlcCer binding site(s) on APC, the occupancy of which augments APC activity. 5-Dimethylaminonaphthalene-1-sulfonyl-glutamylglycyl arginyl chloromethylketone (DEGR-CK) was purchased from Calbiochem (La Jolla, CA). Na-fluorescein-p benzoyl phenylalanyl-lysyl (Ne bromoacetyl) amide (LWB) was prepared as described before (20Yegneswaran S. Fernández J.A. Griffin J.H. Dawson P.E. Chem. Biol. 2002; 9: 485-494Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). 7-Diethylamino-3-((4′-(iodoacetyl)amino)phenyl)-4-methylcoumarin and octadecylrhodamine (OR) were obtained from Molecular Probes (Eugene, OR). Bovine brain phosphatidylcholine (PC), bovine brain PS, and human spleen GlcCer were obtained from Sigma.d-Glucosyl-β1–1′-N-octanoyl-d-erythro-sphingosine (C8β-d-glucosyl ceramide),N-octanoyl-d-erythro-sphingosine (C8-ceramide), 1-palmitoyl-2-[6-[7-nitro-2–1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C16,6 NBD-PC), and 1-palmitoyl-2-[6-[7-nitro-2–1,3-benzoxadiazol-4-yl) amino]caproyl]-sn-glycero-3-phosphoserine (C16,6-NBD-PS) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).l-3-Phosphatidylcholine-1,2-di[1-14C]oleoyl ([14C]PC) was purchased from Amersham Biosciences. The chromogenic substrate Spectrozyme PCa (American Diagnostica, Greenwich, CT), normal human plasma, factor V-deficient plasma (George King Bio-Medical Inc., Overland Park, KS), and Innovin® (Dade, Milano, Italy) were obtained. Human APC was obtained from Enzyme Research Laboratories (South Bend, IN). Bovine APC was obtained as a gift from Dr. Mary J. Heeb, The Scripps Research Institute (La Jolla, CA). Factor Va (fVa) and factor Xa (fXa) were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Bovine and human APCs were active site-specific-labeled with either fluorescein (20Yegneswaran S. Fernández J.A. Griffin J.H. Dawson P.E. Chem. Biol. 2002; 9: 485-494Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), dansyl (21Krishnaswamy S. Williams E.B. Mann K.G. J. Biol. Chem. 1986; 261: 9684-9693Abstract Full Text PDF PubMed Google Scholar), or coumarin dyes and purified from the excess reagents according to procedures published earlier (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). For the fluorescein and coumarin labeling, Nα-fluorescein-p benzoyl phenylalanyl-lysyl (Nε bromoacetyl) amide and 7-diethylamino-3-((4′-(iodoacetyl)amino)phenyl)-4-methylcoumarin were used, respectively, instead of the 5-iodoacetamido fluorescein. Multicomponent vesicles of PC/PS (9:1 w/w ratio), PC/PS/GlcCer (8:1:1), PC/GlcCer (9:1), PC/PS/galactosylceramide (GalCer) (8:1:1), PC/PS/globotetraosylceramide (PC/PS/Gb4Cer; 8:1:1), and 100% PC were prepared according to procedures described earlier (18Deguchi H. Fernández J.A. Pabinger I. Heit J.A. Griffin J.H. Blood. 2001; 97: 1907-1914Crossref PubMed Scopus (48) Google Scholar). Briefly, pure lipids in chloroform were mixed in the appropriate ratios, and then the chloroform was evaporated under nitrogen gas to give 1 mg of solid lipid mixture in a vial. This lipid mixture was then resuspended in 1 ml of buffer containing 50 mm Tris (pH 7.4) and 150 mm NaCl. The suspension was quick-frozen in liquid nitrogen and thawed immediately in a 37 °C water bath. This freeze-thaw cycle was repeated 10 times. Finally, the suspension was transferred to an extrusion chamber (Osmonics, Livermore, CA), and vesicles were prepared by extruding the solution through a 0.22-μm filter (15 passes through the filter). A [14C]PC tracer was added to each preparation at the beginning of the procedure to facilitate determination of postextrusion phospholipid recovery. The concentration of glycolipids recovered was determined by measuring sugar head groups of the glycolipids using an orcinol-based colorimetric assay (18Deguchi H. Fernández J.A. Pabinger I. Heit J.A. Griffin J.H. Blood. 2001; 97: 1907-1914Crossref PubMed Scopus (48) Google Scholar). For the plane-to-plane FRET experiments, vesicles containing the acceptor dye octadecylrhodamine OR were also prepared as above except that the desired volume from a solution of OR in ethyl acetate was added to the lipid mixture prior to drying with nitrogen. The concentration of OR was determined as before as was ς, the OR acceptor density at the vesicle surface (23Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar). The procoagulant and anticoagulant properties of vesicles containing GlcCer were determined using fXa-initiated clotting assays with exogenously added human or bovine APC. For these assays, GlcCer-containing vesicles at varying doses (50 μl) were mixed with normal plasma (25 μl), human or bovine APC (34.5 nm final), or buffer (TBS containing 0.1% BSA, 30 μl) and incubated for 3 min at 37 °C. Then, fXa (50 μl, 0.3 nm final) in buffer containing 30 mmCaCl2 was added to initiate clotting, and clotting times were recorded using an Amelung KC4 microcoagulometer (Sigma). The response to APC was expressed as a ratio of clotting times that was calculated by dividing the clotting time in the presence of APC by the baseline clotting time in the absence of APC. Modified dilute prothrombin time-based assays were also performed as previously described (17Griffin J.H. Kojima K. Banka C.L. Curtis L.K. Fernández J.A. J. Clin. Invest. 1999; 103: 219-227Crossref PubMed Scopus (206) Google Scholar, 18Deguchi H. Fernández J.A. Pabinger I. Heit J.A. Griffin J.H. Blood. 2001; 97: 1907-1914Crossref PubMed Scopus (48) Google Scholar). Briefly, 7.5 μl of plasma was mixed with varying concentrations of C8-GlcCer or C8-Cer and incubated for 3 min at 37 °C with fibrinogen (0.6 mg/ml final) and APC (8.7 nm final) plus protein S (28 nm final) or buffer (100 μl total). Clotting times were measured after addition of 50 μl of recombinant tissue factor (Innovin® from DADE, Miami, FL) diluted 1:64 in Tris-buffered saline containing 0.1% BSA and 30 mm CaCl2. To study the effect of GlcCer on APC-dependent inactivation of factor Va, APC (0.94 nm) in 50 mm Tris (pH 7.4), 150 mmNaCl, and 5 mm CaCl2 was incubated for 5 min at 37 °C with factor Va (1 nm) in the presence or absence of various concentrations of PC/PS, PC/PS/GlcCer, PC/GlcCer, or PC vesicles, respectively. Then the reaction was quenched by the addition of a 2-fold molar excess of EDTA over CaCl2, an aliquot of the reaction mix was withdrawn, and the residual factor Va activity was determined using a prothrombin time-clotting assay using a fVa-deficient plasma. All spectral measurements were made using a SLM AB2 or a SLM 8100 spectrofluorometer (SLM-Aminco, Rochester, NY) as described earlier (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar). Fluorescein, dansyl, and coumarin dye emissions were detected at excitation and emission maxima of 490, 340, and 390 nm and 525, 540, and 462 nm, respectively. All experiments were performed using 5 × 5-mm quartz cuvettes. Sample were mixed, and adsorption of proteins to cuvette walls was minimized as described (24Dell V.A. Miller D.L. Johnson A.E. Biochemistry. 1990; 29: 1757-1763Crossref PubMed Scopus (63) Google Scholar, 25Ye J. Esmon N.L. Esmon C.T. Johnson A.E. J. Biol. Chem. 1991; 266: 23016-23021Abstract Full Text PDF PubMed Google Scholar). The interaction of APC with various preparations of multicomponent lipid vesicles was monitored using plane-to-plane FRET between donor dyes in the active site of APC and acceptors on the membrane surface. Either fluorescein-labeled APC (Fl-APC), or dansyl-labeled APC (DEGR-APC) served as donors and rhodamine imbedded on the membrane surface served as acceptors. FRET experiments were performed as before (26Hackeng T.M. Yegneswaran S. Johnson A.E. Griffin J.H. Biochem. J. 2000; 349: 757-764Crossref PubMed Scopus (18) Google Scholar) except that the D (donor-containing)- and DA (donor and acceptor)-containing cuvettes initially received 100 nm of the donor DEGR-APC (or Fl-APC), whereas cuvettes A (containing acceptor) and B (blank) received 100 nm unlabeled EGR-APC (or FPR-APC) (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Spectral parameters including quantum yields (Φ), spectral overlap integrals (JDA), and distance of 50% energy transfer (Ro) for these experiments are described as before (23Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar, 22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 26Hackeng T.M. Yegneswaran S. Johnson A.E. Griffin J.H. Biochem. J. 2000; 349: 757-764Crossref PubMed Scopus (18) Google Scholar, 27Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. FASEB J. 1996; 10: A663Google Scholar, 28Isaacs B.S. Husten E.J. Esmon C.T. Johnson A.E. Biochemistry. 1986; 25: 4958-4969Crossref PubMed Scopus (29) Google Scholar, 29Koppaka V. Lentz B.R. Biophys. J. 1996; 70: 2930-2937Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 30Shen L. Shah A.M. Dahlback B. Nelsestuen G.L. Biochemistry. 1997; 36: 16025-16031Crossref PubMed Scopus (29) Google Scholar, 31Yegneswaran, S., The Molecular Architecture of the Anticoagulant Membrane-bound Activated Protein C Enzyme and Its Dependence on the Cofactor Protein S and Phospholipid Composition.Ph.D. dissertation, 1997, Texas A&M University, College Station, TX.Google Scholar). The distance of closest approach (R) was calculated using the relation Equation 1QD/QDA=1+(πσRo2/2)(Ro/R)4Eq. 1 where QD/QDA is the ratio of donor quantum yields in the presence and absence of acceptor and ς is the density of acceptor chromophores (OR) at the membrane surface. Four samples were prepared in parallel for each energy transfer experiment: cuvette D (containing donor) and cuvette DA (containing donor and acceptor) each received 100 nm of coumarin-labeled APC (donor), whereas cuvettes A (containing acceptor) and B received 100 nm unlabeled FPR-APC. The net initial emission intensity (Fo) was obtained by the subtraction of the signal of B from D, DA, and A. Samples D and B were titrated with short chain lipids lacking the acceptor dye NBD, whereas samples DA and A were titrated with short chain lipids conjugated to the NBD acceptor. The net intensity of D, DA, or A (FD, FDA, andFA, respectively) was obtained by subtracting the signal from the B cuvette and correcting for dilution. To correct for any signal in DA sample due to direct excitation of the acceptor, the net dilution-corrected emission intensity from A was subtracted from the DA sample signal. Making the reasonable assumption that the absorption of coumarin in the active site is unaffected by the presence of NBD-labeled lipids, the ratio of donor quantum yields in D and DA samples is given by Equation 2, QD/QDA=[FD/FD]/[(FDA−FA)/(FDA−FA)o]Eq. 2 where F is the net dilution-corrected emission intensity of a sample at some point in the titration and the subscript “o” is used to donate the initial intensity of the sample. For titrations in the presence of C8-GlcCer, 100 nmcoumarin-APC was first incubated with 15 μm C8-GlcCer before point-to-point FRET experiments were performed with NBD-labeled phospholipids as described above. The distance R between coumarin donor in the active site groove of APC and NBD acceptor conjugated to the phospholipid was determined using the relation in Equation 3,E=Ro6/(Ro6+R6)Eq. 3 where Ro is the distance at which FRET is 50% efficient and E is the efficiency of FRET given by Equation 4.E=1−(QDA/QD)Eq. 4 The critical micellar concentration (CMC) of C8-GlcCer in 50 mmHepes (pH 7.4), 150 mm NaCl, and 5 mmCaCl2 was determined using diphenyl hexatriene (DPH) fluorescence and 90 ° light scattering. DPH is an aggregation-sensitive dye and has been used to determine the CMC of lipids (13Koppaka V. Wang J. Banerjee M. Lentz B.R. Biochemistry. 1996; 35: 7482-7491Crossref PubMed Scopus (62) Google Scholar). Two samples were prepared in parallel for each experiment. The first sample (S) contained DPH (15 μm final) in 50 mm Hepes (pH 7.4), 150 mm NaCl, and 5 mm CaCl2, whereas the second sample contained a dye-free B. DPH fluorescence was monitored at 365 nm excitation and 460 nm emission, respectively. The net initial emission intensity termedFo was obtained by subtracting the initial intensity of B from the initial intensity S. The samples S and B were then titrated with increasing concentrations of lipids. Relative fluorescence intensity (F/Fo), calculated using Equation 5, was plotted against lipid concentration. F/Fo=(FS−FB)/(FS−FB)oEq. 5 F is the net dilution-corrected emission intensity of a sample at some point in the titration and the subscripto is used to denote the initial intensity of the sample. A plot of F/Foversus lipid concentration gave two linear slopes, and the intercept of the two linear slopes was taken as the CMC of C8-GlcCer. The CMC of C8-GlcCer in the presence of 100 nm FPR-APC was similarly determined. The CMC of C8-GlcCer was also determined by 90 ° light scattering using a SLM AB2 spectrofluorometer (SLM-Aminco, Rochester, NY) with emission and excitation wavelengths of 320 nm. Experiments were performed with a band pass of 2 nm on both excitation and emission light paths. All buffers for light scattering were prepared dust-free by filtration using a 0.22-μm Acrodisc syringe filter units (Pall Gelman Laboratory, Ann Arbor, MI) and by centrifuging dust particles using a Microfuge before light-scattering experiments. The scatter intensity (I) of monomeric C8-GlcCer is much lower than multimeric aggregates or micelles of C8-GlcCer, and the concentration at which the scatter intensity increased sharply was taken as the CMC of GlcCer. The CMC of C16,6-NBD phosphatidylserine and C16,6-NBD phosphatidylcholine were determined by monitoring the steady-state fluorescence anisotropy of the NBD reporter for increasing concentrations of lipid. NBD anisotropy decreases sharply upon lipid aggregation due to homo-FRET (27Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. FASEB J. 1996; 10: A663Google Scholar). NBD anisotropy was measured using Glan-Thompson prism polarizers placed on both the excitation and emission beam paths at 466 and 536 nm excitation and emission wavelengths, respectively. The emission intensity measured when the sample was excited by vertically plane-polarized light and the emission detected through a horizontal polarized light was termedIVH. IVV,IHH, and IHV were defined analogously. Anisotropy (r) was determined using the relation in Equation 6,r=(IVV−GIVH)/(IVV+2GIVH)Eq. 6 where the grating factor G equalsIHV/IVH. The effect of GlcCer on the anticoagulant response of plasma to human and bovine APC was tested using fXa-induced clotting assays (Fig.1). Both PC/PS and PC/PS/GlcCer vesicles enhanced the anticoagulant response of both species of APC. The steeper slope for the GlcCer-containing vesicles compared with the vesicles lacking GlcCer (Fig. 1) indicates that incorporation of GlcCer into PC/PS increased the anticoagulant response of both species of APC. A GlcCer-dependent enhancement of APC activity was also observed in a tissue factor-induced dilute prothrombin-clotting assays (data not shown) showing that GlcCer serves as a lipid cofactor for both human and bovine APC. The anticoagulant activity of APC observed in a fXa-induced clotting assay, which is due to the inactivation of fVa by APC, was studied using four different vesicle surfaces, 100% PC, PC/PS (9:1 w/w), PC/GlcCer (9:1), and PC/PS/GlcCer (8:1:1) (Fig.2). Under the experimental conditions used, APC inactivated very little fVa on 100% PC vesicles and PC/GlcCer vesicles, whereas PC/PS and PC/PS/GlcCer vesicles supported APC-dependent inactivation of fVa. The APC-dependent inactivation of fVa was most efficient using 400 μg/ml of PC/PS/GlcCer vesicles where ∼70% of fVa activity was lost in 5 min (Fig. 2). For PC/PS vesicles, 40% of fVa activity was lost under similar experimental conditions. Therefore, these data show that GlcCer enhanced APC-dependent inactivation of fVa in the presence of 10% PS. To test the hypothesis that GlcCer alters interaction of human APC with membranes, the binding of human APC to lipid vesicles containing GlcCer was compared with binding to vesicles lacking GlcCer. For these experiments, FRET was used to monitor APC binding to lipid vesicles (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar). APC labeled in the active site with either a fluorescein (Fl-APC) or a dansyl (DEGR-APC) reporter served as a donor, whereas rhodamine dyes in OR at the aqueous-lipid interface served as the acceptor. When human Fl-APC was titrated with PC/PS vesicles, only a very small (3%) decrease in fluorescein emission was observed (data not shown). Likewise, a very small change in fluorescein emission was also detected when Fl-APC was titrated with PC/PS/GlcCer, PC, or PC/GlcCer vesicles (data not shown). However, when Fl-APC was titrated with PC/PS (or PC/PS/GlcCer, PC, or PC/GlcCer) vesicles containing OR acceptor, the fluorescein intensity decreased until sufficient phospholipids were added to bind all of the Fl-APC (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This OR-dependent decrease in fluorescein intensity results largely from FRET from the fluorescein dye in the active site of APC to the OR at the membrane surface (22Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To facilitate data analysis, the data were normalized and expressed as a ratio of donor quantum yields in the presence (QDA) or absence (QD) of the acceptor using Equation 1. When no acceptor-containing vesicles were present, the ratio ofQDA/QD was 1, whereas in the presence of these lipids, the value ofQDA/QD was less than 1 due to FRET. Fig. 3A presents data for titrations of human Fl-APC using four different vesicles containing similar OR acceptor density. The ratio ofQDA/QD showed a similar dependence on lipid concentration for the PC/PS, PC, and PC/GlcCer vesicles titrations, decreasing and reaching a plateau at ∼30 μg/ml vesicle concentration. However, the same plateau value was reached at a much lower lipid concentration in the PC/PS/GlcCer titration. These data suggest that PC/PS, PC, and PC/GlcCer vesicles have approximately the same affinity for human APC, whereas PC/PS/GlcCer vesicles have a greater affinity for Fl-APC. Apparent dissociation constant (Kd app) calculations based on curve fitting showed that the affinity for Fl-APC was 5-fold greater for the PC/PS/GlcCer vesicles (3 μg/ml) compared with PC/PS vesicles (16 μg/ml). Thus, the incorporation of GlcCer into PC/PS vesicles significantly increased the affinity of vesicles for APC. Although vesicles composed of only PC are inactive in our functional assays (see Fig. 2), these vesicles bound to Fl-APC with almost the same affinity as the functionally active PC/PS vesicles as reported in an earlier study (27Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. FASEB J. 1996; 10: A663Google Scholar). This discrepancy between the functional and the binding data is presumably due to the poor binding affinity of fVa, the substrate of APC, for PC vesicles leading to the functional inactivity of PC vesicles (28Isaacs B.S. Husten E.J. Esmon C.T. Johnson A.E. Biochemistry. 1986; 25: 4958-4969Crossref PubMed Scopus (29) Google Scholar, 29Koppaka V. Lentz B.R. Biophys. J. 1996; 70: 2930-2937Abstract Full Text PDF PubMed Scopus (34) Go