membrane-bound assembly of factor VIII and factor IXa which converts coagulation factor X to factor Xa. Membrane binding of factor VI11 serves to localize ...
Vol. 266, No. 12.Issue of April 25,pp. Printed 6615-8824, in U.S.A. 1993
THEJOURNALOF BIOLOGICAL CHEMISTRY
Membrane Binding Kineticsof Factor VI11 Indicate a Complex Binding Process* (Received for publication, December 18, 1992)
Catherine BardelleS, Bruce FurieS,Barbara C. Furie, and GaryE. GilbertQTII From the $Center for Hemostasis and Thrombosis Research, Divisionof Hematology-Oncology, New England Medical Center and the Departments of Medicine and Biocbmistry, Tufts University School of Medicine, Boston, Massachusetts 02111, the §Department of Medicine, Brockton-West Roxbury Veterans Administration Medical Center, West Roxbury, Massachusetts 02132, and the VDepartments of Medicine, Brighum and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Factor VI11 is a cofactor in the tenase enzyme complex, a Factor VI11 functions as a component of the tenase enzyme complex upon phospholipid membranes. Factor membrane-bound assembly of factor VIII and factor IXa VI11 binds to phosphatidylserine-containing mem- which converts coagulation factor X to factor Xa. Membrane branes and apparently provides high affinity binding binding of factor VI11 serves to localize enzymatic activity to sites for factor IXa upon these membranes. We have the activated platelet (Nesheim et al., 1988; Gilbert et al., characterized the binding kinetics of human factor VI11 1991) or phospholipid vesicle (for reviewsee Mann et al., with phosphatidylserine-containing membranes and 1988). Membrane-bound factor VI11 provides high affinity directly compared the measured properties with those binding sites for factor IXa, theenzyme of the tenase complex, of factor V. The initial phase of association was eval- on the platelet (Ahmad et al., 1989) or phospholipid vesicle uated in a stopped-flow apparatus by fluorescence energy transferfrom aromatic residues inthe protein to surface (Duffy et al., 1992).The zymogen of factor IXa, factor dansyl-labeled phosphatidylethanolamine in the vesi- IX, does not bind tothe high affinity sites provided by cles. Association proceeded at an apparent second-or- membrane-bound factor VIII (Ben-Tal et al., 1991). Although der rate of 0.12 11”’ s” for extruded phospholipid the platelet membrane molecules that participate in the bindvesicles and 0.42 p ~ ”s-’ for sonicated vesicles under ing of factor VI11 have not been conclusively identified, availpseudo-first-order conditions in which the phospho- able data are consistent with the hypothesis that phosphatidylserine plays a critical role (Bevers et al., 1983; Gilbert et lipidconcentrationdeterminedtherate.Increased temperature resulted in more rapid association, and al., 1991, 1992). These considerations motivated studies to the effect decreased in the order extruded vesicles > elucidate the properties of factor VI11 binding to synthetic sonicated vesicles > extruded vesicles of dioleoylphos- membranes containing phosphatidylserine (Gilbert et al., pholipids, indicating that the structure of the phospho- 1990). lipid membrane contributesto the activation energy of Factor VI11 is a plasma glycoprotein of M , 280,000 which binding. The bindingof fluorescein-labeled factor VI11 circulates in a noncovalent complex with von Willebrand to membranes supported on glass microspheres(lipos- factor. Factor VI11 is homologous to factor V in amino acid pheres) was monitored by flow cytometry. Under con- sequence (Church et al., 1984; Gitschier et al., 1984; Toole et ditions in which the factor VI11 concentration determined the rate there was rapid initial association at al., 1984) and in function asa membrane-bound enzyme 6.9 11“’ s-’, accounting for half of the bound factor cofactor facilitating the activation of zymogens of the coaguVIII, and a slower component of 0.87 11”’ s-’, account- lation proteases, factor X and prothrombin,respectively (Fuing for the other half. Likewise, the dissociation of rie and Furie, 1988; Kane andDavie, 1988; Gilbert et al., 1990; factor VIII from liposphere membranes was biphasic Mann et al., 1990). The proteins share a repeating domain with a faster component of 0.010 s-’ and a slower structure of Al-A2-B-A3-Cl-C2 in which the A domains are component of 0.0012 s-’. Rates of association and dis- homologous with ceruloplasmin, the B domain is unique to sociation for factor V were similar to those for factor each protein, and the C domains are homologous with discoiVI11 and werebiphasic. Theseresults allow estimation din I, a phospholipid-binding lectin (Bartles et d . , 1982), and of the size of the phospholipid sites that interact with with a murine milk fat globule membrane protein (Stubbs et factors VIII and V and suggest that both proteins bind al., 1990). The cofactor activity of each protein is greatly to membranes via a multistep process in which rapid enhanced through limited proteolysis by thrombin, and this association is followed by a slower step yielding higherresults in the removal of the B domain, with the remaining affinity binding. heavy and light chains interacting to form a Ca2+-dependent complex. Both proteins bind with equivalent high affinity to phospholipid membranes via the “light chain” composed of the A3-C1-C2 segment. However, the membrane site recog* This work was supported in part by Grants HL42443 and the Medical Research Service of the Department of Veterans Affairs. The nized by factor VIII contains more phosphatidylserine molecosts of publication of this article were defrayed in part by the cules than the site for factor V (Gilbert et al., 1990), and payment of page charges. This articlemust therefore be hereby factor V is not an efficient competitor with factor VI11 for marked “advertisement” in accordance with 18 U.S.C. Section 1734 these binding sites (Gilbert et al., 1992). Current evidence solely to indicate this fact. 11 Recipient of National Institutes of Health Clinical Investigator implicates different protein domains in membrane binding. Award HL02587.To whom correspondence should be addressed West Whereas the binding of factor V is mediated by both the A3 Roxbury VA Medical Center, 1400 VFW Pkwy., West Roxbury, MA domain (Kalafatis et al., 1990) and the C2 domain (Ortel et 02132. Tel.: 617-323-3427;Fax: 617-323-8786. al., 1992), the binding of factor VI11 is apparently mediated ~~
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8815
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Factor VIII Membrane Binding, a MultistepProcess
by the C2 domain (Arai et al., 1989; Foster et al., 1990). In prior studies the kinetic interactionof factor V and the proteolytically activated form, factor Va, with phospholipid vesicles have been examined (Pusey et al., 1982; Abbott and Nelsestuen, 1987; Krishnaswamy et al., 1988). The results have indicated rapid membrane binding that influenced is by the size of the vesicles used. In the present study our objective was to characterize the kinetic interaction of factor VI11 with synthetic membranes over a time scale of seconds as well as minutes to provide insight into the binding mechanism that allows rapidassociation,bindingsite specificity, and high affinity binding. EXPERIMENTALPROCEDURES
L-a-Phosphatidylcholine from eggyolk, L-a-phosphatidylserine 1,2-difrom bovine brain, 1,2-dioleoyl-sn-glycero-3-phosphocholine, oleoyl-sn-glycero-3-phosphoserine, and dansyll-labeled phosphatidylethanolamine were from Avanti Polar Lipids, Pelham, AL. Factor V was from Enzyme Research Laboratories, Southbend, IN. The specific activity of this material was 100units/mg in acoagulation assay with factor V-deficient plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it migrated as four bands of M, 200,000330,000 detectable with silver stain. Fluorescein 5-maleimide was from Molecular Probes, Eugene, OR. Factor VU1 Purification-Lyophilized factor VI11 concentrate (the generous gift of Dr. William Drohan, the American Red Cross) was reconstituted with water, the pH adjusted to 7.5, and the solution incubated with 4 mM dithioerythritol for 1 h at 22 "C. The pH was decreased to 6.0 by the addition of 0.1 M MES buffer at pH 5.0, and the factor VI11 was injected onto a Mono S column (Pharmacia LKB Biotechnology Inc.) preequilibrated with 20 mM MES, 0.15 M NaCl, 5 mM CaCI2, 0.1M betaine, 0.005% Tween 80,O.l mM dithioerythritol, pH 6.0. von Willebrand factor was eluted from the column with 0.25 M NaCl in the same buffer, and 0.75 M NaCl was used to elute factor VIII. Tubes containingfactor VI11 activity, measured by an activated partial thromboplastin time assay using factor VIII-deficient plasma, were pooled and concentrated 10-fold with a Centricon 30 microconcentrator (Amicon) at 5,000 X g, 4 "C. Aliquots of 50 pl were kept at -80 "C. As evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis our factor VI11 had a prominent silver-stained band at M, 80,000 and multiple bands in the M, 120,000-200,000 range. Factor VI11 concentration was measured by the micro BCA (Pierce) protein assay. Recombinant human factor VI11 was the generous gift of Dr. Randy Kaufman and Deborah Pittman of Genetics Institute, Cambridge, MA. Fluorescein Labeling of Factor VI11 and Factor V-Factor VI11 and factor V were labeled with fluorescein maleimide as described previously (Gilbert et al., 1991) with the following modifications. Fluorescein maleimide was diluted 1:lOO from a 50 mM stock solution in dimethyl formamide into solutions of factor V or factor VI11 at pH 7.2. After incubation for 16 h at 4 "C in the dark, betaine was added to 0.1 M,and most of the unreacted dye was removedby ultrafiltration of the solution in a Centricon 30 microconcentrator at 5,000 X g and 4 "C for 30 min. Two ml of Tris-buffered saline (TBS; 0.15 M NaCI, 0.02 M Tris-HC1, pH 7.5) containing 0.1 M betaine was then added to the retentate, and the ultrafiltration was repeated. The protein was further purified from free dye by gel filtration over a 1 X 6-cm Sephadex G-25 SF column preequilibrated in TBS containing 0.1 M betaine, 0.004% Tween 80, 2 mM CaCl,, pH 7.0. Fractions with absorbance above base line at 280 and 490 nm were pooledand tested for factor VI11 or factor V activity and protein concentration before being stored in 50-pl aliquots a t -80 "C. Vesicle and Liposphere Preparation-Small unilamellar vesicles with the composition phosphatidy1serine:dansyl-PE:phosphatidylcholine 205:75 were generated by sonication (Barenholz et al., 1977). Larger unilamellar vesicleswere prepared by extrusion (Hope et al., 1985). Phospholipid concentration was determined by phosphate assay (Chen et al., 1956). Purity of dansyl-PE was evaluated by HPLC (Chen and Kou, 1982) using a 4.6 X 250-mm silica column (Alltech Associates, Deerfield, IL) with 5-pm bead diameter. Phospholipid membranes with the composition phosphatidylserThe abbreviations used are: dansyl, 5-dimethylaminonaphthalene-I-sulfonyl; MES, 4-morpholineethanesulfonicacid; PE, phosphatidylethanolamine; HPLC, high performance liquid chromatography.
ine:dansyl-PE:phosphatidylcholine20:5:75 supported by glass microspheres (lipospheres) were prepared as described (Gilbert et al., 1992). Briefly, glass microspheres of 1.6-pm nominal diameter (Duke Scientific, Palo Alto, CA) were cleaned by sonication in 0.02% filtered Sparkleen (Fisher) and small particles removed by sedimentation at 80 X g for 3 min in aswinging bucket rotor. Microspheres were passed through a laser-etched polycarbonate membrane with 3-pm diameter pores (Nucleopore) to remove larger particles. The cleaned, sizerestricted microspheres were rinsed for 1 h in a stream of deionized, distilled water while submerged in a sonication bath and then suspended in 3 ml of TBS with small unilamellar phospholipid vesicles (0.3 mM) with a composition as above. The mixture was sonicated in the bath sonicator for 5 min and allowed to incubate for 30 min with mixing by inversion. Microspheres were pelleted at 80 X g, resuspended by vortex mixing in TBS, 0.1% bovine serum albumin, and washed five times by repetition of the same procedure. Lipospheres were counted using a Coulter ZM particle counter, stored at 4 "C, and used within 48 h of synthesis. Stopped-flowFluorescence Rate Measurements-Rates were obtained by following the increase of fluorescence energy transfer from tryptophan residues in the protein to dansyl-PE in the vesicle when factor VI11 or factor V in TBS containing 0.5 mM CaC12, 0.005% Tween 80 and vesiclesweremixed ina stopped-flow apparatus (Applied Photophysics). This apparatushas a dead time of less than 1 ms and enhanced optics yielding an effective aperture of f 3.4 for the observation photomultiplier. The excitation monochromator was set at 280 nm, 7 nm band pass, with light supplied from a 150-watt xenon arc lamp. Emission was monitored by using a 505-nm cutoff filter. Temperatures of the driving syringes andthe observation chamber were maintained with a circulating water bath. Each determination represents 7-15 averaged experiments. After confirmation that experimental conditions were pseudo-first-order (see "Results") data were fitted by nonlinear least squares regression analysis to a single component exponential curve using Applied Photophysics software and an Archimedes 410/1 computer. Flow Cytometry Measurements-Factor VIII-vesicle association and dissociation were measured using a Coulter EPICS-profile I1 apparatus. Data acquisition was triggered by forward light scatter with all photomultipliers in the log mode. Noise was reduced during analysis by eliminating events with forward and side scatter values different from those characteristic of the lipospheres. Only experiments in which the fluorescence histogram indicated a log normal distribution, as judged by inspection, were analyzed quantitatively with mean log fluorescence converted to linear fluorescence. For dissociation measurements 2 nM fluorescein maleimide-labeled factor VI11 was incubated for 25 min with 1 X lo6 lipospheres/ml in TBS containing 0.5 mM CaC12and 0.1% bovine serum albumin. Dissociation rates were determined by measuring the mean factor VIII fluorescence per liposphere immediately before and atvarious times after the addition of a 10-fold excess of unlabeled factor VIII. Association rates were determined from following the increase of factor VIII fluorescence bound to lipospheres at various times after the addition of 2 nM fluorescein maleimide-labeled factor VI11 to 1 X lo6 lipospheres/ml. When more than 5 min elapsed between measurements, uniform suspension of lipospheres was assured by gently swirling the tube. Samples were protected from direct light between measurements. Data were analyzed as described under "Results" using the nonlinear least squares capacity of Sigma Plot. Quality of fit was evaluated by visual examination of fit and residuals. RESULTS
Binding of factor VI11 to a phospholipid membrane may be monitored by measuring the transfer of fluorescence energy from aromatic residues within the protein to dansyl-labeled phosphatidylethanolamine (dansyl-PE) incorporated into the membrane (Gilbert et al., 1990). This technique was used to measure the rateof association of factor VIII to phospholipid vesicles in a stopped-flow apparatus. The homogeneity of dansyl-PE was evaluated by HPLC as described under "Experimental Procedures."Asinglepeak was detected with retention time intermediatebetween phosphatidylcholine and phosphatidylethanolamine. In contrast to native phospholipids, this lipid absorbed light at 286 nm, as well as 203 nm,indicatingthepresence of the dansyl moiety. Preliminary kinetic experiments were performed to
ractor VIII Membrane Multistep Binding, a determine whether the fluorescence emission from phospholipid vesicles containing dansyl-PE was influenced by rapid dilution. We observed a fluorescence decrease of 3.5% over the first 3 s following 1:1 dilution in the stopped-flow appaSample mixing within thestopped-flow chamratus (Fig. U). ber, complete within 1 ms, could not correlate to a fluorescence losson this time scale. When vesicles containing dansylPE were mixed with vesicles containing no fluorescent probe the fluorescence loss was decreased. When the ratio of unlabeled to labeled vesicles was greater than 1:l we observed a fluorescence increase rather thana decrease. n
I [Unlabeled Phosphollpldl PM
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FIG. 1. Effect of dilution upon fluorescence of dansyl-PE containing vesicles. Panel A, a decrease in dansyl fluorescence followed a 1:l dilution of vesicles with TBS in the stopped-flow apparatus (see "Results"). This decrease in fluorescence was affected by the presence of unlabeled vesicles in the TBS. When the concentration of dansyl-PE-containing vesicles matched the concentration of vesicles without dansyl-PE the fluorescence loss was essentially negated. When dansyl-PE-containing vesicles were mixedwith higher concentrations of vesicles lacking dansyl-PE the net fluorescence increased. Data and fitted curves are shown in the absence of unlabeled phospholipid, but only fitted curves are shown for experiments in which unlabeled vesicleswere included. Panel B, when dansyl groups were excited at 340 nm the presence of factor VI11 did not affect the dilution-associated loss of fluorescence. Stopped-flow observations were initiated immediately after 1:l mixture of two solutions, one containing dansyl-PE-labeledphospholipid vesicles, 16 PM phospholipid, the other containing TBS supplemented by phospholipid vesicles lacking dansyl-PE or factor VI11 as indicated. Phospholipid and protein concentrations refer to the final concentration. The buffer in the second drive syringe was TBS, 0.5 mM CaC12, and 0.005% Tween 80. The buffer in the first syringe, with dansyl-PEcontaining phospholipid vesicles, wasidentical except for the absence of Tween 80. Tween 80 does not affect the fluorescence emission of dansyl-labeled phospholipid vesicles (data not shown) and was included because factor VI11 adheres to thewalls of a silica observation chamber in its absence (data not shown).
Process
8817
We hypothesized that a fraction of thedansyl-PE was partitioning between phospholipid vesicles and bulk solution and that thefluorescence loss resulted from a changing environment encountered by dansyl-fluorophores as theymoved from vesicles t o bulk solution. The presence of free dansylP E in phospholipid vesicle preparations was confirmed by two experiments. First, dansyl-PE-labeled vesicles were gel filtered on a 1 X 30-cm column of Sephadex G-25 SF, and the eluate was monitored at 280 nm. The vesicle peak eluted in the void volume followed by a second peak approximately 1% as large as the first. The presence of dansyl in both peaks was confirmed by characteristic fluorescence excitation and emission spectra. In a second experiment extruded vesicles containing 83 ,.LM dansyl-PE were mixed with smaller sonicated vesicles, 5 mM, lacking dansyl. After 5 min the vesicle populations were separated by gel filtration over a Superose S6 column. Thesonicated vesicle fractionscontained 15% of total dansyl fluorescence. Thus, dansyl-PE, as a component of phospholipid vesicles, may partition to bulk solution and may rapidly reincorporate into othervesicles. We performed additional control experiments to determine whether the binding of factor VIII influenced the dilutionrelated loss of dansyl-PE fluorescence (Fig. 1B).Fluorescence excitation was a t 340 nm rather than 280 so that intrinsic fluorophores of factor VI11 would remain unexcited andfluorescence energy transfer would not contribute to the emission signal. We compared the dilution-related loss of dansyl-PE fluorescence with and without 6 nM factor VIII. Visual inspection indicated an equivalent rate and magnitude of fluorescence decrease. When both curves were fitted to a firstorder model the fitted constantsfor rate and amplitude were within 15% of one another, leading us to conclude that the presence of factor VIII did notsignificantly affectthe dilutionrelated fluorescence loss. For subsequent factor VI11 binding experimentsthedilution-related fluorescenceloss was recorded and subsequently subtracted from the netincrease in fluorescence resulting from factorVI11 binding. In measurements of factor VI11 binding sufficient phospholipid was used to ensure that membrane binding sites were in excessrelative tofactor VI11 (Gilbert et al., 1990). Upon mixing factor VI11 with phospholipid vesicles an increase in fluorescence was observed (Fig.2 A ) . Fluorescence change was maximal over the first 0.2 s and approached the horizontal asymptote by 0.5 s. After subtraction of the dilution-related fluorescence loss these data fita first-order association model s -' with a second-order association constant,k,, of 0.42 '"p (Fig. 2B). Increasing the phospholipid concentration 2-fold increased the rate of fluorescence change 2-fold but did not change the magnitudeof fluorescence change. Increasing the factor VI11 concentration 2-fold yielded a 2-fold increase in fluorescence signal but did not alter the rate of fluorescence change.Theseobservationsvalidatethepseudo-first-order binding model. The rate of association of factor VIII with phospholipid vesicles was independent of the specific activity of the factor VI11 preparation. Plasma-derived factorVIII, one preparation with a specific activity of 500 units/mg and another with a specific activity of 1,200 units/mg, and recombinant factor VIII, with a specific activity of 2,000 units/mg, yielded fluorescence signals with an amplitude proportional to the factor VI11 specific activity. However, the factor VIII-phospholipid association rateswere identical. These results emphasize that under the experimental conditionsemployed we were observing pseudo-first-order association in which the phospholipid concentration is rate-determining. We determined whether, like the light chain of activated
Factor VIII Membrane Binding, a Multistep Process with the larger vesicles (kl 0.12 pL"' s-'). The association rates of factor V to small vesicles and to larger vesicles were also compared (Fig. 4). The association of factor V to phospholipid vesicles was fitted by a first-order model after correction for the fluorescence decrease associated with phospholipid membrane dilution. The fluorescence increase attributed to factor V binding to membranes reached a plateau in aboutone-thirdthetime for small vesicles as for larger vesicles. The fitted curves correlate to association constants that are %fold faster to sonicated vesicles than to extruded vesicles (Table I). The effect of temperature upon the rate of factor VIIImembrane association was determined using both small sonicated vesicles and larger vesicles. As shown in Fig. 5A, the
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Seconds FIG. 2. Detection of the time-dependent association of factor VI11 with phospholipid vesicles. Panel A , an increase in dansyl fluorescence emission was observed upon mixing factor VI11 with sonicated, dansyl-labeled phospholipid vesicles. Panel B , when the fluorescence decrease resulting from vesicle dilution was subtracted from the increase that occurred upon mixing factor VI11 with vesicles, the resulting curve is well fitted by a single exponential. Stoppedflow observations were initiated immediately after a 1:l mixture of two solutions, each containing factor VI11 or phospholipid vesicles, for final concentrations of 6 nM and 8.5 pM, respectively.
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FIG. 4. Comparison of the association of factor V to sonicated vesicles and to larger extruded vesicles. Factor V was or larger extruded vesicles mixed with sonicated vesicles (upper curve) (lower curve), and the resultant fluorescence increase was observed. The fitted curves (smooth lines) represent the best fit for a model with a single exponential and indicate a faster association rate for small vesicles even though the concentration of larger vesicles is higher. Final concentrations were: factor V, 8.8 nM; phospholipid, 4.2 p~ in upper curue and 8.5 p~ in lower curve. Other experimental conditions are described in the legend to Fig. 1.
TABLE I Association constantsfor factors VIII and V binding to phospholipid vesicles Experiments were performed under pseudo-first-order conditions a t 25 "C. The second-order rate constantswere calculated by dividing the fitted first-order constant by the phospholipid concentration. I 0
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k, (25 'C) pM-, s-l
FIG. 3. Comparison of the association of factor VI11 to sonicated vesicles and to larger extruded vesicles. Factor VI11 was mixed with sonicated vesicles (uppercurue) or larger extruded vesicles (lower curue), and the resultant fluorescence increase was observed. The fitted curves (smooth lines) represent the best fit for a model with a single exponential. Final concentrations were: factor VIII, 6.4 nM; phospholipid, 8.5 p ~ Other . experimental conditions are described in the legend to Fig. l.
factor V (Abbott and Nelsestuen, 1987), factor VI11 binds more rapidly to small vesicles than to large vesicles. Small vesicles (approximately 0.02-pm diameter) were prepared by sonication, and larger vesicles(0.1-pm nominal diameter) were prepared by extrusion through polycarbonate membranes. As shown in Fig. 3, factor VI11 associates about %fold more rapidly with the smaller vesicles (kl0.42 p ~ "s-') than
K-' Sonicated Extruded
E. kcal mol"
Factor V
k, (25 "C) p M - l s-l
E. kcal mol-'
K-' 0.42 f 0.02' 11.4 f 0.9' 0.54 2 0.02 0.12 f 0.01 13.6 f 0.7
7.9 f 0.9
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* 1.3
DOPS/DOPCd 0.15 +. 0.04' 9.4 f 0.7 0.14 & 0.02 4.5 f 0.8 "All vesicles had a composition of phosphatidylserine: dansy1phosphatidylethanolamine:phosphatidylcholine of 20:5:75. * Mean f range for four determinations. e Activation energy (EA) was determined by best fit using nonlinear least sauares reeression analvsis. The error value is the standard deviation of fit. Dioleovluhosuhatidvlcholineand dioleovluhosDhatidvlserinewere substituted for egg yolk phosphatidylcholine-andbovine brain phosphatidylserine in extruded vesicles. e Single determination from eight averaged experiments f standard deviation of fit.
Factor VIII Membrane Multistep Binding, Process a Temperature ("C) 30
40
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8819 dylcholine and bovine brain phosphatidylserine for both factor VI11 and factor V (Table I). However, variation of temperature had a less pronounced effect upon the association rates of both proteins to these vesicles than tolarger vesicles synthesized using egg yolk phosphatidylcholine (Fig. 6). The fit curves for activation energies corresponding to the best factor VI11 and factor V were lower than those obtainedwith extruded vesicles or sonicated vesicles. These results support the hypothesis thatlipid packing and order affect the activation energy of membrane binding for both factor VI11 and factor V. We have recentlydeveloped a technique for the preparation of a phospholipid bilayer upon the surface of glass microspheres; we refer t o these coated glass beads as lipospheres (Gilbert et al., 1992). Equilibrium binding studies indicate that factor VIII binding to liposphere membranes isequivalent to factorVI11 binding to phospholipid vesicles. To evaluate factor VI11 binding to phospholipid membranes over a period of 60 min, we studied factor VIII-liposphere interaction by flow cytometry. This method offered direct detection of protein-membrane interaction in which the signal used to detect binding is stableover at least 60 min. Factor VI11 was labeled with fluorescein maleimide and the kineticsof interaction of this conjugate with membranes determined using lipospheres. In these experiments the concentrationof factor Temperature
("C)
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FIG. 5. Effect of temperature upon the association of factors VI11 and V with phospholipid vesicles. Arrhenius plots of the stopped-flow association rates indicate a larger effect of temperature upon the association of factor VI11 (panel A ) with extruded vesicles (0)than with sonicated vesicles (W). Likewise, the effect of temperature upon the association of factor V (panel B ) withsonicated vesicles (W) was somewhat less than the effect upon association with extruded vesicles (0).Lines are the least squares fit. Final concentrations were: factor VIII, 11 nM; factor V, 9 nM; sonicated phospholipid vesicles in panels A and B, 4.4 and 3.8 WM, respectively; extruded phospholipid vesicles in panels A and B, 10.8 and 13.0 PM, respectively. association rates increased by more than 10-fold over a temperature range of 5-40 "C. The activation energies from the best fit curves were 11.4 kcal mol" K" for small vesicles and 13.6 kcal mol" K" for larger vesicles (Table I).Likewise, the effect of temperature upon the rates of factor V association with small and larger phospholipidvesicles was studied (Fig. 5 B ) . Although the increase in association rate was less for factor V than for factor VIII,a similar pattern was observed in that the activation energy was lower by 1.5 kcal mol" K" for small vesicles than forlarger vesicles (TableI).The differences in association rates and activation energies for factor VI11 binding to small vesicles versus largervesicles suggested that lipid packing and order within the membrane may effect binding characteristics. To distinguish the effects of lipid packing and order from effects attributable to thediffering radii of vesicles, extruded vesicles (0.1-pm nominaldiameter) composed of dioleoylphosphatidylcholine anddioleoylphosphatidylserine were employed. The decrease in lipid density and order imposed by the bulky dioleoyl lipid chains alters these membrane properties in the direction of the lower lipid density and order found in small vesicles. At 25 "C the association rate was comparable to larger vesicles made from egg yolk phosphati-
0.1
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FIG. 6. Effect of temperature upon the association of factors VI11 and V to vesicles of dioleoylphospholipids. Arrhenius plots of the stopped-flow association rates of factor VI11 (panel A ) and factor V (panel B ) to extruded vesicles of dioleoylphosphatidylserine:dansyl-PE:dioleoylphosphatidylcholine 205:75. The effect of temperature upon the association rate to these vesicles was less than the effect upon extruded or sonicated vesicles of egg yolk phosphatidylcholine and bovine brain phosphatidylserine (Fig. 4). Final concentrations were: factor VIII, 11 nM; factor V, 11 nM; phospholipid, 10.0
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Factor VIII Membrane Binding,
a Multistep Process
VI11 was in excess over the phospholipid concentration, and first 5 min while the remainder decreased slowly over 70 min. the factorVI11 concentration was below the equilibrium bind- These dissociation data, inconsistent with a single exponening constant to assure pseudo-first-order binding conditions. tial decay model, were well fitted to a two-component expoAs shown in Fig. 7, the initial rate of interaction of factor nential decay process. The dissociation constant, k l , was 10 s-', and k2 was 1.2 X s-'. Similar experiments VI11 with lipospheres was rapid, as predicted by stopped-flow X fluorescence results. However, the fluorescence signal contin- were performed to study the dissociation of factor V from ued to rise longer than predicted if only a rapid first-order lipospheres (Fig. 8B). After fluorescein-labeled factor V was association was operative. Indeed, the datawere not consist- incubated for25 min with lipospheresexcess unlabeled factor ent with a first-order model of association, suggesting instead V was added. These dissociation data were also well fitted to that there was a second, slower phase of membrane binding adouble exponential decay model. The initial dissociation not detectableby the energy transfer. The rate of association constant, kl, was 6-fold faster than k2 (Table 11). As an additional control we wished to determine whether was well fitted toa double exponential model. The calculated association constants corresponding to the best fit curve in the rate of dissociation of fluorescein-labeled factor VI11 was influenced by the presence of excess unlabeled factor VI11 Fig. 7 A indicate a kl of 6.9 phi-' s-l and a k2 of 0.87 pM-' s-'. The rateof association of fluorescein maleimide-labeledfactor upon the membrane. Accordingly, dissociation experiments were performed under conditions in which the total factor V to lipospheres was also examined (Fig. 7 B ) . Like factor VIII, there was a rapid initial component of association fol- VI11 concentration was very low. Following a 25-min incubalowed by a slower second component. This process was well tion of fluorescein maleimide-labeled factor VI11 with lipowas diluted 20-fold, and serial fitted by a double exponential curve in which the kl was 20- spheres the incubation mixture fluorescence measurements were obtained (Fig. 9). Dissociafold faster than thek2 (Table 11). The liposphere binding assaywas also employed to measure tion was fitted by adouble exponential modelwith rate dissociation in the presence of excess the rate of dissociation of factor VI11 from membranes. After constants comparable to a 25-min incubation of fluorescein maleimide-labeled factor unlabeled factor VIII. Control lipospheres, incubated with the final concentrationof fluorescein-labeled factor VIII, 0.15 nM, VI11 with lipospheres, a 15-foldexcess of unlabeled factor VI11 was added, and the factor VI11 fluorescence associated for 70 min exhibited final fluorescence equivalent to lipowith lipospheres was measured over time. As shown in Fig. spheres with which the factor VI11 concentration had been 8A, approximately 40% of fluorescence disappeared over the decreased 20-fold (not shown). Thus, equilibrium had been
30 25 20 15 10
FIG. 7. Association of factors VI11 and V with liposphere membranes. The rate of association of fluorescein maleimide-labeled factor VI11 with lipospheres was measured by flow cytometry (panel A ) . Fluorescence increased rapidly for the first 3 min and more slowly for the following 30 min. Like factor VIII, fluorescein maleimide-labeled factor V bound to liposphere membranes with a rapid initial component and a slower second component ( p a n e l B ) .The data were fitted by a model with two exponential components of equal amplitude, depicted as a smooth line. Final concentrations were: factor VIII, 2 nM; factor V, 3 nM; lipospheres, 1 X 106/ml.
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50
882 1
Factor VIII Membrane Binding, a Multistep Process reached in the dissociation experiment. During the dissociation experiment the sample was divided after 24 min, and an excess of unlabeled factor VI11 was added to one portion. Liposphere fluorescence decreased more rapidly in this sample and reached a final value within 10% of background, indicating that at least 90% of the factor VI11 was reversibly bound over this time period. DISCUSSION
We have examined the membrane binding kineticsof factor VI11 using two differenttechniques. Stopped-flow fluorescence experiments, utilizing fluorescence energy transfer, allowed binding rate determination over 10 s when the phospholipid concentration was rate-determining. We find that TABLEI1 Kinetic constants for factors VIIZ and V interacting with lipospheres Experiments were performed under pseudo-first-order conditions in which the protein concentration determined the rate. The secondorder association constants were calculated by dividing the apparent association constants by the protein concentration. Dissociation
Association
k:!
kl
pM-'S-'
Factor VI11 Factor V
6.9 f 0.7" 0.87 ? 0.28 6.1 f 0.8 0.27 f 0.02
k2
k1
X
10 f 2 3.3 f 0.5
103 s-l
1.2 f 0.4 0.57 f 0.05
Mean f range for two determinations.
70
the initial phase of membrane binding, detected by fluorescence energy transfer, is rapid with a calculated second-order constant of 0.42 pL"' s-l for sonicated vesicles. This rate is similar to the rate of factor V binding. Our results for membrane binding of factor V are in agreement with prior measurements (Pusey et al., 1982). Although experimental conditions were different, the resulting association constants are within 2-fold of one another. (Prior results were analyzed as the rateper membrane binding site, with a binding site defined as 235 phospholipid molecules, the ratio of phospholipid to protein when the membrane surface was saturated. We have analyzed data as the rate per phospholipid molecule so that rates in this report mustbe multiplied by 235 for comparison.) Likewise, rates reported in this work are close to those reported for the activated form of factor V binding to phospholipid vesicles (Abbott and Nelsestuen, 1987; Krishnaswamy et al., 1988). The decrease in the fluorescence of dansyl-PE-labeled phospholipid vesicles which followed rapid dilution was not anticipated by our prior experience nor by reports of stopped-flow experiments in which factor V associated with similar vesicles (Pusey et al., 1982; Abbott and Nelsestuen, 1987). The energetically favorable structure of phospholipid membranes is supported by interactions between dipolarionic phospholipid head groups as well as by hydrophobic interaction between fatty tails. Because the dipolarionic head group of phosphatidylethanolamine is changed by dansyl derivatization to a
I
60 50 40
30 20
FIG. 8. Dissociation of factors VI11 and V from liposphere membranes. After a 25-min incubation of 2 nM fluorescein-labeledfactor VI11 (panel A ) or factor V (panel B ) with lipospheres excess unlabeled protein was added, and fluorescence measurements were obtained at various times. Data were fitted by a model with two exponential components of equal amplitude, depicted as a smooth line.Final concentrations were: 1 X 10' lipospheres/ml, 34 nM native factor VI11 in (panel A), and 156 nM native factor V in (panel B ) .
10
.,
,-
0
20
10
50
40
30
60
70
TIME (min)
60
50 40
30
20
o ! 0
20
1
I
40
60
I
80
120 100
TIME (min)
I
1
I
140
160
8822
Factor VIII Membrane Binding,a Multistep Process
experiments in which the factor V concentration affected the association rate used a concentration of factor V exceeding the equilibrium binding constant (Krishnaswamy et al., 1988), or only the first portion of the association curve, reflecting the rapid component, was analyzed (Abbott and Nelsestuen, 1987). Comparison of the rapid, first component of membrane binding determinedby the two methods allows an estimateof the size of the phospholipid site that interacts with factor VIII, the “contact region” as defined previously (Gilbert et al., 1990). This differs from the membrane binding site defined 0 20 40 60 EO by Pusey et al. (1982) and represents the numberof phosphoTIME (rnin) lipid head groups with whicha proteinmustinteract for FIG. 9. Dissociation of factor VI11 from liposphere mem- binding tooccur. Assuming that thediffusion of phospholipid branes. After a 25-min incubationof 3 nM fluorescein-labeled factor vesicles and lipospheres is slow, compared with factor VIII, VI11 withlipospheres the mixture was diluted20-foldwith TBS. the numberof lipid molecules in an average membrane interBound factor VI11 fluorescence was measured at various times. After action site is estimatedby determining the ratioof the factor 24 min the sample was divided into two aliquots, and an excess of unlabeled factor VI11 was added to one (arrow). Further dissociation VIII-dependent association rate to the phospholipid-depend58 for association with the of fluorescein-labeled factor VI11 was monitored in the absence (m) ent association rate. This ratio is larger vesicles used,implying that a membranesitethat and presence (0)of excess unlabeled factor VIII. Final unlabeled factor VI11 concentration was 20 nM. interacts with factorVI11 contains 58 total phospholipidmolecules. Because phospholipid forms a bilayer, only the outer half will actually interact with factor VIII. Thus, a membrane bulkier, anionic moiety we hypothesized that the partition region containing 29 phospholipid molecules is postulated as coefficient of dansyl-PE for phospholipid membranes may be different than phosphatidylethanolamine so that significant the average unit site on with which factor VI11 interacts in dansyl-PE may be free in solution. This hypothesis implies binding to a membrane. The ratio is 16 for association with that the decrease in fluorescence following rapid dilution is a the 0.02-pm diameter sonicated vesicles. Because two-thirds leaflet of sonicated vesicles consequence of an increase in free dansyl-PE bulk in solution. of the phospholipid is in the outer Our hypothesis was supported by the capacity of unlabeled (Huang and Mason,1978) a n average membrane site containvesicles to preventfluorescence loss, bythe demonstrationof ing 11 phospholipid molecules is implied. This estimate is free dansyl-PE invesicle preparations, andby the demonstra- close to a prior estimate of the membrane contactregion size tion of substantial transfer of dansyl-PE between different of 13 phospholipid molecules (Gilbert et al., 1990). Both populations of vesicles. Because dansyl-PE is amphipathic, estimates suggest that the membrane site that functions in of free dansyl-PE was probably found in micelles in which the the adsorption of factor VI11 is smaller than the ratio phospholipid to protein when the vesicle membrane is satuclose association of dansyl groups would lead to self-quenching. We have demonstrated that thefluorescence loss associ- rated with factor VI11 molecules (Gilbert et al., 1990). For molecules per membrane ated with vesicle dilution is not influenced by the binding of factor V, the estimated phospholipid factor VIII. Prior studies indicated that the presenceof dan- adsorptionsite is 18 forlarger vesicles and 8 for smaller syl-PE in a phospholipid membrane does not affect binding vesicles, implying that a region of smaller size interacts with process factor V. These estimates are also smaller than prior estimates of factor VI11 (Gilbert et al., 1990) so that the binding is not likely to be influenced by reequilibration of dansyl-PE. that defined a binding site as the ratio of phospholipid to Therefore, the kinetic factorVI11 binding data in this report protein when the membrane surfaceis saturated with protein 1987; KrishnaswamyandMann, have been corrected by subtracting the fluorescence loss as- (AbbottandNelsestuen, 1988). sociated with vesicle dilution in control experiments. Both the association and the dissociation of factor VI11 The use of membranes supported by glassmicrospheres (lipospheres) (Gilbert et al., 1992) allowed determination of from liposphereswere biphasic. These dataled us to postulate the association rate under conditions in which the factorVI11 that at equilibrium bound factor VI11 is present in two or two concentration determined the rateof membrane association. more states.Although the data are compatible with either types of binding sites or a multistep membrane binding procThis experiment cannot be done by the stopped-flow method because of the large amount of protein required. Data from ess the implicitsimplicity of a two-component liquid crystalline membrane leads us to prefer the model in which there these experiments confirmed a rapid initial association rate classes of but also indicated a slower second component of membrane are several bindingstepsratherthandifferent binding (Table 11). Dissociation of factors VI11 and V from binding sites (Fig. 10). In the first step factor VI11 adsorbs lipospheres was also biphasic, with a rapid initial component rapidly to a membrane. The adsorbed factor VI11 may either and a slower second component. Although the slow second dissociate or undergo a second binding step that results in component of phospholipid vesicle binding has not been re- higher affinity binding. The net effect is rapid adsorption, ported previously for factor VIII, a slow component of disso- with the quantity of loosely bound factor VIII quickly apciation of factor Va from vesicles has been reported (Abbott proaching equilibrium through a rapid dissociation compoandNelsestuen, 1987). The composite rate of membrane nent, followed by a slow increase in bound protein asloosely association to anddissociation from platelet microparticles is bound molecules undergo a second step of binding and are no slower than the rapid component of association t o phospho- longersubject torapid dissociation. Therequirementfor a slow association membrane phosphatidylserine suggests that the first step is lipid vesicles, suggesting thatthereis process, as well as a rapid process, on the platelet membrane mediated by electrostatic interaction between phosphatidylto which factors VI11 and V bind (Gilbert et al., 1991). Tech- serine and factor VIII. Two mechanisms that may subsenical features of prior experiments probably limited sensitiv- quently contribute to the binding process are insertion of a hydrophobic protein domain into the membrane and clusteritytodetection of the slower second component.Kinetic
Factor VIII Membrane Binding, a Multistep Process
8823
tails of dioleoylphosphatidylcholine, with a double bond at position C9,pack less densely in a membrane than egg yolk phosphatidylcholine (Tamm andMcConnell, 1985). Although the association rate to these vesicles was comparable to extruded vesicles of egg yolk phosphatidylcholine and bovine brain phosphatidylserine at 25 "C the activation energy was lower than with extruded and sonicated vesicles. These results indicate that alterationof membrane structure contributesa substantial portion of the activation energy of binding. The increased rateof factor VI11 adsorption withelevated temperature may reflect increased opportunity for interaction between factor VI11 and glycerol, carbonyl, or acyl moities that become moreaccessible fromthemembrane surfacewith temperature-induced disorder. Support for thishypothesis arises froma recent study inwhich the capacityof membranes with low phosphatidylserine content to support membrane FIG. 10. Two-step membrane binding model for factor VIII. binding of factor V and assembly of theprothrombinase FactorVIII (VZZZ) is depictedcollidingwithandadhering to a complex correlated with thedegree of fatty acid chain unsatphospholipid membrane ( a l ) .The loosely adherent factor VI11 has a uration (Govers-Riemslaget al., 1992). Abbott and Nelsestuen higher likelihood of dissociating from the membrane ( d l ) than progressing througha second step of adherence (aZ),which may or may (1987) have hypothesized that themore rapid association rate notinvolve lateral motion of the factor VI11molecule.After the observed with sonicated vesicles is related to hydrodynamic second adherence step the likelihood of dissociation from the mem- principles that apply when one particlecollides with a small brane is lower than after the first adherence step ( d 2 ) . The white binding site ona large secondparticle. Further understanding arrows indicate the possibility that the second adherencestep involves of the role of these hydrodynamic properties awaits a better a domain of factor understanding of the contribution of membrane structural clustering of specific phospholipid molecules about VIII. differences to binding ratesso that therelative contributions of these factors may be distinguished. ing of specific lipids aboutmembrane-boundfactor VIII. Although factor VI11 and factorV are homologous in strucPhotoactivatable, lipophilic probes labeled the light chain of ture and in function as membrane-bound cofactors and alfactor V when it was bound to phospholipidvesicles, suggest- thoughthe equilibrium membranebindingaffinitiesare ing that a portion of thelightchainpenetratesintothe equivalent, there are differences between the membrane bindmembrane (Krieget al., 1987; Lecompte et al., 1987). Homol- ing properties. Factor VI11 requires more phosphatidylserine ogy between factors VI11 and V makes itlikely that membrane per binding site (Gilbert et al., 1990), and most of the membinding of factor VI11 is also supported, in part, by hydropho- brane binding sites for factor VI11 are specific for factor VIII bic interaction between the light chainof factor VI11 and the in the presence of excess factor V (Gilbert et al., 1992). We membrane. Clustering of phospholipid molecules about an find that the initial phase of membrane adsorption for factor adsorbed factorVI11 molecule may also contribute to binding V was less influenced by temperature than the adsorption of affinity, analogous tothatreported for proteinkinase C factor VIII, suggesting a less stringent requirement for tem(Newton and Koshland,1989) and the protein kinaseC com- perature-induced membrane irregularities. The second phase plex (Bazzi and Nelsestuen,1991). In supportof a mechanism of binding was more rapid for factor VI11 than for factor V. involving lipid rearrangement are prior data indicating that Corresponding to this, bothdissociation constants were three membrane bindingof factors VI11 and V initiate self-quench- times faster for factor VI11 than for factor V. Clear interpreing of fluorescence from dansyl-PE (Gilbertet al., 1990). tation of this difference awaits further evidence of the mechThe reason for more rapid association of factors VI11 and anism involved in the second phase of membrane binding. V to sonicatedvesicles compared withlarger extruded vesicles In summary, we have observed that membrane binding of is uncertain. Prior data indicate that sonicated vesicles will bothfactor VI11 andfactor V is characterized by a slow bind twice as much factor VIII, per unit of phospholipid, as component as well as a rapid component. The overall time extruded vesicles of the same composition (Gilbert et al., course of associationand dissociation with lipospheres is 1990). If it is assumed that membrane association sites are similar to thatobserved with platelet microparticles (Gilbert also twice as numerous then the association rate for extruded et al., 1991), suggesting that a slow component of membrane vesicles is 70% as fast as the rate for sonicated vesicles, and binding may be characteristic of a biologic membrane that the difference seems less remarkable. However, in addition to provides receptors for factor VI11 and factor V. The second the more rapid association ratesat 25 "C the sonicated vesicles component of association contributes tohigher affinity binddifferedfrom extruded vesicles in having association rates ing but may also contribute to specificity observed in memless influenced by temperature. In combination these differ- brane bindingof factor VI11 (Gilbert et al., 1992). Studies with ences suggest that the membrane structureof sonicated vesi- lipids of varying degrees of lateral mobility and with fluorescles influences factor VI11 binding. Sonicated phospholipid cent lipids may further elucidate the mechanism contributing vesicles have a larger portion of the phospholipid in the outer to this second step of membrane binding. membrane leaflet and morespacebetween the ionic head Acknowledgments-We are grateful to Dr. Mark Zeidel for use of groups in the outer membrane leaflet (Huang and Mason, 1978). Correlating to the greaterspace between head groups, the Applied Photophysics stopped-flow fluorescence apparatus. We thank Dr. George Buschand Sue Bennett for use of the Coulter Epics the outer leaflet of sonicated vesicles is less ordered than Profile I1 flow cytometer and Diane Drinkwater for excellent techphospholipids in larger vesicles (Sheetz and Chan,1972). 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