THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 2, Issue of January 15, pp. 815422, 1990 Printed in U. S. A.

Binding of Human Factor VIII to Phospholipid Vesicles*

(Received for publication, July 13, 1989)

Gary E. Gilbert+, Barbara C. Furie, and Bruce Furie From the Center for Hemostasis and Thrombosis Research, New England Medical Center and the Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Factor VIII, a protein cofactor involved in blood coagulation, functions in vitro on a phospholipid mem- brane surface to greatly increase the rate of factor X activation by factor IXa. Using gel filtration, rapid sedimentation, and resonance energy transfer we have studied the interaction of recombinant-derived human factor VIII with small and large unilamellar phospho- lipid vesicles composed of phosphatidylserine and phosphatidylcholine. Resonance energy transfer, from intrinsic fluorophores in factor VIII to dansyl-phos- phatidylethanolamine incorporated into vesicles, has been adapted for quantitative equilibrium measure- ments. Factor VIII binds rapidly and reversibly to small and large vesicles. At 8°C the interaction of factor VIII with small vesicles fits a simple bimolecular model with a Ko of 2 nM and a phospholipid binding site defined by 180 phospholipid monomers. At 25 ‘C the binding of factor VIII to small vesicles containing 20% phosphatidylserine can be described by an appar- ent Kn of 4 nM; the phospholipid/protein ratio at satu- ration was 170. Binding to large vesicles was demon- strated with a KD of 2 nM and a phospholipidjprotein ratio at saturation of 385. Binding was dependent upon the phosphatidylserine mole fraction and was nonlin- ear from 0 to 30% phosphatidylserine content. A direct comparison of factor VIII and factor V binding indi- cated that the affinity of factor V to phospholipid ves- icles was equivalent to that of factor VIII and that the phosphatidylserine requirement was lower. A model is proposed to explain the nonlinear phosphatidylserine dependence of binding for factor VIII.

Factor VIII is a blood coagulation protein which functions as a cofactor during coagulation. The cDNA for factor VIII indicates that factor VIII is synthesized as a single polypeptide chain containing 2,351 amino acids (Toole et al., 1984; Git- schier et al., 1984; Vehar et al., 1984). This sequence indicates a domain structure with an arrangement of Al-A2-B-A3-Cl- C2 where A represents a repeating domain that shares se- quence homology with ceruloplasmin and factor V, B repre- sents a domain unique to factor VIII, and C represents a repeating domain that shares homology with factor V and with discoidin I, a lectin (Church et al., 1984). Factor VIII is isolated from tissue culture supernatant and human plasma primarily in a two-chain form. The two chains are noncova- lently associated in a metal-dependent interaction. The

*This work was supported in part by Grants HL21543 and HL42443 from the National Institutes of Health. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- merit” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Individual National Research Service Award F32 HL07440 from the National Institutes of Health.

plasma concentration of factor VIII (M, 280,000) is about 300 PM, and it circulates in blood in a noncovalent complex with von Willebrand factor subunits at a 1:l molar ratio (Lollar and Parker, 1987). By limited proteolysis, factor VIII is cleaved by thrombin to yield an active cofactor form. Factor VIIIa functions as a cofactor for activated factor IX at a saline-phospholipid membrane interface leading to the pro- teolytic activation of factor X. The presence of phospholipid and factor VIIIa increases the reaction rate at least 4 orders of magnitude (van Dieijen et al., 1981; Brown et al., 1978). The dependence of the rate enhancement upon the presence of phospholipid is the stimulus for these studies.

Factor VIII is homologous to factor V in function as well as amino acid sequence and domain structure (Church et al., 1984) (see Kane and Davie, 1988 and Furie and Furie, 1988, for review). Both proteins are activated proteolytically by thrombin in vitro and function as cofactors with phospholipid membrane surfaces for vitamin K-dependent serine proteases. The direct interaction of factor V with phospholipids has been studied using several techniques (Pusey et al., 1982; Bloom et al., 1979; Krishnaswamy and Mann, 1988) (see Mann et al., 1988, for review). The characteristics of this interaction in- clude a requirement for acidic phospholipids, a nanomolar equilibrium constant, and rapid association at or near the collisional limit. Preliminary studies of the interaction of human factor VIII with phospholipids have been complicated by the unavailability of pure factor VIII, the lack of physical definition of the phospholipids used, and the use of nonequi- librium techniques (Bloom, 1987; Andersson and Brown, 1981). Our objective was to study the equilibrium interaction of human factor VIII with phospholipids and to compare these results with those obtained for factor V-phospholipid binding.

Measurement of resonance energy transfer has been suc- cessfully applied to study the rate of association of factor V to phospholipid vesicles (Pusey et al., 1982). The technique offers the advantage of real-time measurements without ap- parent perturbation of the interaction. In this method, the excitation of one or more intrinsic fluorophores in the protein that are in close proximity of the phospholipid membrane leads to coupled excitation of dansyl’ groups linked to phos- phoiipid in the vesicle followed by emission of energy by the excited dansyl groups. In this study we show that energy transfer also occurs between factor VIII and dansyl-labeled phospholipid vesicles. By establishing the temporal charac- teristics of the fluorescence signal we have adapted the reasonance energy transfer methodology for quantitative equilibrium studies of factor VIII-phospholipid interaction.

1 The abbreviations used are: dansvl. 5-dimethvlaminonaohthal- ene-1-sulfonyl; ELISA, enzyme-linked immunosorbent assa;; TBS, Tris-buffered saline; PS, phosphatidylserine; PC, phosphatidylcho- line; PE, phosphatidylethanolamine.

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816 Binding of Factor VIII to Phospholipid Vesicles

EXPERIMENTAL PROCEDURES Materials-Phosphatidylserine from bovine brain and dansyl-la-

beled phosphatidylethanolamine were obtained from Avanti Polar Lipids (Pelham, AL). Phosphatidylcholine from egg yolk was obtained from Sigma. Sodium deoxycholate was obtained from Calbiochem. The recombinant human factor VIII used in these studies (generously provided by Genetics Institute) was expressed in Chinese hamster ovary cells. Both the product and the secretory processing have been studied (Kaufman et al., 1988). This factor VIII preparation is com- posed primarily of a two-chain form of M, 280,000. The specific activity was 5,000 units of coagulant activity/mg, as measured using factor VIII-deficient plasma in an activated partial thromboplastin time assay (Hardesty and Macpherson, 1962). Molar concentrations were determined by absorbance at 280 nm using an I!?;&:,,, of 0.88 (Loller and Parker, 1987). Factor VIII was stored in TBS (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5) containing 0.01% Tween 80 at -80 “C and thawed only one time prior to use. Experiments were performed with a final concentration of less than 0.0002% Tween 80. Bovine factor V, obtained from Enzyme Research Laboratories (South Bend, IN), migrates as a dominant band of M, 330,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a closely associated second band representing an intermediate in the conversion of factor V to factor Va. No factor Va contaminated this preparation. The specific activity, after thrombin activation, was 100 units/mg. Molar concentrations were determined by absorbance at 280 nm using an E&$,,, of 9.6 (Nesheim et al., 1979). Both proteins had no detectable loss in specific activity after 9 months of storage at -80 “C.

Phospholipid Vesicle Preparation-Small unilamellar vesicles (hereafter referred to simply as small vesicles) were made by the method of Barenholz (1977). The final concentration of phospholipid was determined by elemental phosphorus assay (Chen et al., 1956). Large unilamellar vesicles (hereafter referred to as large vesicles) for gel filtration and rapid sedimentation experiments were prepared using a modified detergent extraction method (Enoch and Strittmat- ter, 1979). Fresh small vesicles were adjusted to 20 mM phospholipid. Sodium deoxycholate was added slowly to the vesicles to a final concentration of 8 mM and the turbidity at 600 nm recorded inter- mittently. Additional sodium deoxycholate was added until a major increase in turbidity was noted. The vesicles were placed under nitrogen at 0 “C and incubated until a visible increase in turbidity was noted. The suspension was applied to a Sephadex G-25 SF column (1 x 20 cm) preequilibrated with TBS. The visibly amber fractions were collected, filtered through a 0.45pm polyethylene filter (Milli- pore), diluted in 10 ml of TBS, and sedimented by centrifugation under nitrogen for 15 min at 100,000 X g and 25 “C. The pellet was resuspended in 1 ml of TBS using a vortex mixer and remaining detergent removed by gel filtration. The vesicle fractions were pooled, resuspended in 10 ml of TBS, and sedimented by centrifugation, as above. The pellet was resuspended in TBS using a vortex mixer. The final phospholipid concentration was determined by phosphorus analysis. Brief low temperature incubation led to further enlargement of the unilamellar vesicles, as demonstrated by increased turbidity. Centrifugation and filtration selected for large vesicles (maximum diameter 0.45 pm) which sedimented under conditions of 100,000 X g at 25 “C! for 15 min.

Large unilamellar vesicles for resonance energy transfer experi- ments were prepared by extrusion (Mayer et al., 1986). Fresh small vesicles were subjected to repeated freeze-thaw cycles by immersion alternately in liquid nitrogen and water at 37°C. The number of cycles was not less than two or more than five. After freeze-thaw enlargement the vesicles were extruded twice through polycarbonate membranes (100 pm pore size; Nucleopore) under approximately 75 p.s.i. of air pressure at 23 “C. Quantitative recovery of phospholipid was confirmed by elemental phosphorus analysis. Enlargement of the average vesicle size was confirmed by an increase in specific turbidity. This technique has been shown to produce phospholipid vesicles with a mean diameter of 100 nm (Hope et al., 1985).

Separation of Factor VIII Bound to Phospholipid Vesicles from Free Factor VIII-Gel filtration to separate factor VIII bound to large vesicles from free factor VIII was performed on a Superose 6 column using a FPLC syste,.i (Pharmacia LKB Biotechnology Inc.). The column was equilibrated in TBS, 0.1% bovine serum albumin, 0.01% Tween 20, 1 mM calcium chloride, and 0.02% sodium azide at a flow rate of 0.4 ml/min. Factor VIII activity in each fraction was measured as above.

Alternatively, factor VIII bound to large vesicles and free factor VIII were separated by rapid sedimentation, analogous to a technique

previously utilized for the separation of phospholipid-bound factor V from free factor V (van de Waart et al., 1983). Centrifugation condi- tions were 92,000 x g, 25 “C for 15 min in a Beckman Model L8M ultracentrifuge. Microfuge tubes (Beckman; 7 X 50 mm) with a 0.4- ml sample volume were seated in a water-filled l-ml polycarbonate ultracentrifuge tube (Beckman) and placed in a Beckman t25 ultra- centrifuge rotor. Factor VIII antigen in the supernatant and the pellet after resuspension was assayed using a sandwich-type ELISA. Im- mulon II plates (Dynatech) were coated using 50 ~1 of anti-factor VIII light chain antibody (Hybritech) at a 1:lOOO dilution in a sodium carbonate buffer, pH 9.6. After a 2-h incubation at 37°C the wells were washed three times using TBS with 0.1% Triton X-100 then treated for 2 h with 3% gelatin in TBS. Samples were placed in the wells in TBS containing 10 mM EDTA and 1% Tween 80. A calibra- tion curve was established using Mega 1 (standard factor VIII con- centrate, Office of Biologics Research and Review, Bethesda, MD) or purified recombinant factor VIII. Samples in the wells were incubated for 16 h at 23 “C. The plates were washed three times with TBS, 0.1% Triton X-100 and the biotinylated antibody Ab f8/6.4.6, an anti- factor VIII light chain antibody (generously provided by Dr. Barry Foster, Genetics Institute) in TBS, 1% gelatin, 0.1% Tween 80, was incubated for 4 h at 37°C. The wells were washed three times with TBS, 0.1% Triton X-100 prior to the addition of avidin-conjugated alkaline phosphatase (Cappel) diluted 1:lOOO in TBS, 1% gelatin, 0.1% Tween 80. After a 20-min incubation at 37°C the wells were washed three times with TBS, 0.1% Triton X-100. Alkaline phospha- tase substrate (Sigma) in diethanolamine buffer, pH 9.8, was added, and the plate was read in a Dynatech MicroELISA reader at 405 nm.

Fluorescence Measurements-Resonance energy transfer measure- ments were performed using a SLM 8000C or a Perkin-Elmer LS5 fluorescence spectrophotometer in a configuration described for the kinetic measurements of factor V association with small vesicles (Pusey and Nelsestuen, 1982). Fluorophores in the protein were excited at 280 nm. When a factor VIII molecule is in close proximity to a dansyl group covalently linked to phosphatidylethanolamine in the membrane, transfer of energy excites the dansyl group leading to emission at 545 nm. Because the efficiency of energy transfer has an inverse relation to the distance between factor VIII and the dansyl group, the resulting dansyl emission is proportional to the amount of factor VIII at the membrane surface. A Perkin-Elmer UV-35 filter was used in the excitation path to minimize second order scatter, and a high pass filter (450 nm) was used in the emission beam to further reduce scatter.

Data Analysis-A simple bimolecular equilibrium model assumes that a discrete number of phospholipid monomers in a membrane act as an independent factor VIII binding site:

KD VIII + nPL *VIII:PL,

where VIII represents factor VIII, PL represents phospholipid mon- omers, and n is the number of phospholipid monomers composing a single binding site. This can be written as

K D = wIIlwI4 [VIII:PL,] (1)

where KD is the dissociation constant describing the factor VIII- phospholipid interaction. Based upon conservation relationships, VIII = VIII, - VIII:PL, and PL/n = PL,/n - VIII:PL, where VIII is the amount of free factor VIII, VIII, is the total factor VIII, VIII:PL, is the factor VIII-phospholipid complex, PL/n is the concentration of unoccupied binding sites, and PLJn is the total quantity of phospho- lipid binding sites. Substituting for the free factor VIII and free phospholipid and solving for bound factor VIII yields the quadratic relationship

[VIII:PL.] = (2)

K~ + [VIII,] + [PLJ~I f Jw, + ~VIII,I + IPL+)* - 4w111~wwi

2

in which only the subtraction operation gives physically meaningful values.

To interpret these fluorescent data, we assumed that the fluores- cence-transfer signal associated with the binding of each factor VIII molecule to the phospholipid vesicle surface was the same, regardless of the amount of factor VIII already bound to the surface. Thus, the fluorescence signal is related to the fraction of factor VIII molecules

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Binding of Factor VIII to Phospholipid Vesicles

occupying phospholipid binding sites by the relationship

I _ [VIII:PL,] F Fm - W-&l (3)

where F, is the corrected fluorescence signal and F, is the corrected fluorescence signal associated with saturation of all factor VIII bind- ing sites (PL,/n). The fluorescence signal, Foss, was corrected for the fluorescence contribution of factor VIII alone (F = Foss - F& and for the loss of dansyl fluorescence over time due to photobleaching. Therefore,

F,=;-l

where Fb is the dansyl fluorescence in the absence of factor VIII corrected for measured photobleaching. Thus F, represents a ratio of fluorescence after the addition of factor VIII to the fluorescence due to direct excitation of phospholipid-linked dansyl. The initial value is adjusted to zero by the subtraction of 1. This relationship can be adapted for comparison to fluorescence measurements by substituting the right-hand side of Equation 2 for VIII:PL, in Equation 3. Solving for F,,

F K,+ [VIII,] + [PL,/n]- ~(KD+ [VIII,] + (PL,/n])*-4[VIII,][PLJn] m

WWnl

This relationship is analogous to the model used to evaluate the binding of factor Va to small vesicles (Krishnaswamy and Mann, 1988).

The parameters F,, KD, and n were obtained by fitting Equation 4 to fluorescence data using the Marquardt algorithm. The software program, FitAll (MTR Software, Toronto), which utilizes the Mar- quardt algorithm for least squares regression analysis of nonlinear equations, was modified to evaluate Equation 4. Quality of fit was evaluated by visual examination of fit, convergence of best fit param- eter values when higher or lower initial parameter estimates were used, and by visual examination of residuals to the fitted line. In experiments where varying the initial parameter estimates did not yield convergent results (due to a low signal to noise ratio) parameters were evaluated by visual examination of fit as parameters were varied (as specified in figures and table).

Data from the rapid sedimentation assay were corrected for the fraction of factor VIII which sedimented in the absence of phospho- lipid vesicles and analyzed by fitting to Equation 2.

RESULTS

Gel filtration of a mixture of factor VIII and large vesicles indicated that factor VIII activity eluted in the void volume with large vesicles composed of PS/PC. In the absence of phospholipid, factor VIII activity was observed solely in the included volume. Furthermore, factor VIII did not elute with large vesicles composed of only PC. These results provide qualitative evidence that factor VIII binds to PS-containing phospholipid vesicles. The nonequilibrium rapid sedimenta- tion technique was used to measure factor VIII bound to large phospholipid vesicles. In these experiments, the amount of factor VIII antigen sedimented at varying concentrations of large phospholipid vesicles was determined. The data con- firmed that factor VIII binds to PS-containing phospholipid vesicles and had little, if any, affinity for vesicles composed only of PC. The binding data were fitted to the binding predicted by a bimolecular solution phase equilibrium model, Equation 2. The mean values for the dissociation constant, KD, and the binding site size, n, averaged from two experi- ments were 8 nM and 500 phospholipid monomers per factor VIII, respectively. The analysis of nonequilibrium data by comparison to an equilibrium model provides an upper limit for values of Ko. For this reason, an equilibrium technique was developed to study factor VIII-phospholipid vesicle inter- action.

Pusey et al. (1982) have utilized resonance energy transfer to measure factor V binding to dansyl-containing phospho- lipid vesicles. We have demonstrated that this transfer phe- nomenon also occurs between factor VIII and phospholipid vesicles (Fig. 1). The emission peak of factor VIII is decreased upon addition of dansyl-labeled small vesicles while the dansyl emission peak increases in intensity. These changes indicate energy transfer to the dansyl group. This technique has been previously applied only for real-time association studies and not for equilibrium measurements (Pusey et al., 1982). There- fore, we have evaluated factors that might alter fluorescence during a period in excess of the time necessary for equilibrium measurements. Such factors include photoinactivation of the acceptor fluorophore, dansyl-PE; effects of protein binding on phospholipid structure and, thus protein-phospholipid in- teraction; the effect of dansyl-PE on protein binding; the kinetics of protein-phospholipid interaction; and the reversi- bility of protein-phospholipid interaction.

Photoinactivation of the dansyl group under the experi- mental conditions used was 0.2%/30-s time measurement. For this reason cumulative fluorescence measurements were lim- ited to 6 min of irradiation at 280 nm, or a total of 12 measurements for a maximum net loss of 2.5%. Photoinacti- vation was measured for each set of conditions and the ob- served fluorescence corrected.

In order to establish kinetic constraints the rate of approach to equilibrium was estimated by monitoring fluorescence in a continuously stirred small vesicle sample as factor VIII was injected. A plateau was reached by 12 s. In addition, serial fluorescence measurements initiated at least 10 s after factor VIII addition yielded a constant value.

Since factors VIII and V binding to phospholipid vesicles might induce phospholipid rearrangement, with consequent

300 400 500

x em, (nm)

FIG. 1. Energy transfer between factor VIII and dansyl- labeled phospholipid vesicles. Factor VIII, 20 nM, was excited at 280 nm and the emission spectra recorded before (---) and after (-) addition of 3 &M dansyl-labeled small vesicles. That the de- crease in the intensity of factor VIII fluorescence represents transfer to dansyl moieties on the vesicles is demonstrated in the inset where the emission spectra of 3 pM dansyl-labeled small vesicles at a higher signal amplification is shown before (-.-.-) and after (-) the addition of 20 nM factor VIII; X,, 280 nm. The uertical arrow indicates the intensity difference measured in these studies. The fluorescence spectrophotometer was configured with a Perkin-Elmer UV-35 filter in the excitation path; a long pass filter (cutoff 450 nm) was added in the emission path in the experiment depicted in the inset.

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818 Binding of Factor VIII to Phospholipid Vesicles

dansyl-dansyl quenching, we evaluated protein-induced fluo- rescence loss. Fluorescence loss after the addition of factors V and VIII were similar; therefore factor V was employed to delineate the process. Time-dependent quenching is demon- strated in Fig. 2A. The process is slow, with a half-time of 5- 20 min at 25 “C. In these experiments, the addition of protein increases the rate of fluorescence loss, as monitored by exci- tation at 280 nm and emission at 545 nm. Fig. 2B presents the same experiment, but with excitation at 340 nm, directly

0 10 20

Tine (minutes)

1.1 - B

0.8 -

0 10 20

Time (minutes)

I 0 3 6

Time (minutes)

FIG. 2. Protein-induced changes in the phospholipid vesi- cles that result in fluorescence loss. A, loss of resonance transfer signal. Increase in fluorescence signal with factor V addition and time-dependent change in fluorescence from large vesicles in the presence (*) and absence (0) of factor V (3 nM)i A.,, 280 nm; L,,, 545 nm. Large vesicles were composed of dansvl-PE:PS:PC (5:20:75) at a phosphokpid monomer concentration of 1 ;M. B, time-dependent change in fluorescence is due to dansyl-dansyl quenching rather than loss of energy transfer signal. Samples and conditions were identical to A except that large vesicles were irradiated at 340 nm, exciting the dansyl groups directly and bypassing the protein-dansyl excitation. The experiment was performed in the presence (*) and absence (0) of factor V as above; A,,, 340 nm; A,,, 545 nm. C, fluorescence loss reduction to the range of experimental error. By decreasing the dansyl-PE mole fraction to 0.025 and decreasing the time of the experiment, the quenching loss becomes less than the loss due to photobleaching of dansyl groups and was within experimental error of the control sample. Excitation and emission wavelengths as in B so that the observed signal is from direct dansyl excitation rather than energy transfer. Total phospholipid concentration, 1 j&M. Factor V was added to the cuvette incrementally after each fluorescence reading (total range O-40 nM final concentration) simulating an equilibrium binding experiment (+) and without protein addition (0).

exciting dansyl and bypassing factor V excitation. This ex- perimental design excludes energy transfer as a source of the observed fluorescence changes. This confirms that the fluo- rescence increase with factor V addition seen in Fig. 2A is due to energy transfer rather than a perturbation of dansyl envi- ronment and that the slow fluorescence loss, by contrast, is due to a change in dansyl environment in the vesicle mem- brane. This quenching occurred at 25 “C and at 13 “C but not at 8°C (where decreased lipid fluidity would be seen). A 2- fold reduction in the mole fraction of PE-dansyl in the phos- pholipid vesicle composition, 0.05-0.025, decreased the rate of quenching 4-fold. The demonstration that quenching is dependent upon phospholipid bilayer temperature and that the rate of quenching behaves as a function of the dansyl mole fraction squared are consistent with (but do not prove) quenching due to a slow two-dimensional phospholipid re- arrangement increasing local density of dansyl chromophores. These results suggest that fluorescence loss due to dansyl rearrangement would be minimized by performing experi- ments at 8 “C. Furthermore, at a lower mole fraction of dansyl- labeled PE, 0.025, and a short time between incremental protein addition (0.5 min), there was no measurable quench- ing (Fig. 2C). Absence of measurable quenching was demon- strated with small vesicles of the same composition for both factor V and factor VIII. Again, excitation at 340 nm selectiv- ity excites dansyl rather than factor V fluorophores, and thus no increase in fluorescence due to energy transfer is observed. Under these conditions the equilibrium measurements were obtained without the introduction of a correction factor.

Dansyl-labeled PE had no effect on factor VIII binding to phospholipid vesicles. This was established by a series of binding experiments that compared factor VIII binding to dansyl-PE-labeled small vesicles in the presence and absence of identical small vesicles without dansyl-PE. When the two sets of data were normalized for the absolute quantity of dansyl in each sample, the binding curves were superimposa- ble, indicating that dansyl-labeled PE had no effect upon factor VIII binding.

The reversibility of factor VIII binding to phospholipid was established by measuring the decrease in fluorescence signal resulting from addition of unlabeled small vesicles to a sample containing dansyl-PE labeled small vesicles and factor VIII (Fig. 3). Data are normalized to F, obtained from a saturation experiment and compared to the dissociation predicted by the solution phase model, Equation 4. These results indicate that factor VIII binding to phospholipid is reversible.

Data from a resonance energy transfer binding experiment at 25 “C, using small vesicles, are shown in Fig. 4A. The curve indicates the best fit of Equation 4 to the data. The bimolec- ular solution phase model adequately predicts the binding. The dissociation constant, KD, obtained was 3.1 f 0.5 nM, and the phospholipid/protein ratio at saturation was 166 f 4. The factor VIII-phospholipid interaction is a surface-solution interaction rather than a solution phase interaction. In spite of this, Equation 4, which assumes a simple solution interac- tion, provides an excellent fit to resonance energy binding data. Similar results were obtained when binding studies were performed at 8 “C (Fig. 4B). Under these conditions the best fit to the model yielded a K. of 2 nM and a phospholipid/ protein ratio at saturation of 180.

We evaluated factor V binding to small vesicles at 25 “C to determine whether this interaction fit Equation 4 and to compare the binding parameters, Kn and n, with those char- acteristic of factor VIII (Fig. 5). The curve indicates the best fit of the data to Equation 4. The value obtained for the KD was 2.9 nM, and the value for n was 114. These data indicate

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Binding of Factor VIII to Phospholipid Vesicles

01 1 2 3 4

[PLI qdvl)

FIG. 3. Reversibility of factor VIII binding to small unila- mellar vesicles. Factor VIII was added to a cuvette containing small vesicles of dansyl-PE:PS:PC (molar ratio 2.5:20:77.5) at a final con- centration of 1 PM and the fluorescence recorded at 545 nm. Small vesicles without dansyl-PE were subsequently added and a fluores- cence measurement was made 3 min later. The fluorescence values presented represent a ratio of fluorescence after addition to the fluorescence before addition. The curve represents the predicted fluorescence decrease from the bimolecular equilibrium model (Equa- tion 4) utilizing values for &, n, and F, obtained from best fit to a saturation binding experiment with the same small vesicle prepara- tion. Decrease in fluorescence indicates a reversible interaction with the phospholipid (PL) bilayer. The ability of nonlabeled vesicles to compete for factor VIII binding indicates that the dansyl-phosphati- dylethanolamine label is not responsible for factor VIII binding.

application of the resonance transfer method to equilibrium binding of factor V to phospholipid vesicles. The results obtained are similar to the equilibrium measurements of Krishnaswamy and Mann (1988). In addition, these results indicate that factors V and VIII bind to phospholipid vesicles with similar affinity.

Large and small unilamellar phospholipid vesicles have measurable differences in their factor V binding characteris- tics (Abbott and Nelsestuen, 1987). Therefore we compared the binding of factor VIII to large vesicles with approximate diameter of 100 nm to the binding of factor VIII to small vesicles (approximate diameter of 20 nm). The binding of factor VIII to large vesicles is shown in Fig. 6. The fluores- cence signal is smaller with large vesicles, decreasing the signal to noise ratio in binding experiments. When the dis- sociation constant and binding site size were determined by fitting the data to Equation 4, a KD of 2 nM and a binding site of 385 phospholipid monomers were obtained.

The binding of factor V and the vitamin K-dependent blood coagulation proteins to phospholipid vesicles has been re- ported to require acidic phospholipids. The highest binding affinity has been reported using a high mole fraction of PS. We tested the relationship between the mole fraction of PS and factor VIII binding. Fig. 7, top, indicates the dependence of factor VIII binding to small phospholipid vesicles on the mole fraction of PS. Fig. 7, bottom, indicates the dependence of bovine factor V binding to small vesicles on the mole fraction of PS. As indicated, factors VIII and V binding to small vesicles depends upon the mole fraction of PS in the vesicles. However, factor VIII requires a higher concentration of PS than does factor V before optimal binding is observed. These results suggest that the factor VIII binding site includes more PS monomers than does the factor V binding site. Table I lists the number of molecules bound at saturation estimated by fitting Equation 4 to the set of data for each mole fraction of PS and, in addition, by comparing the maximal fluores-

-0.1’ 1

0 ~vIUI~’ (nM)

fVIIfl, (nM)

FIG. 4. Binding of factor VIII to small vesicles. Vesicle com- position was PS:PC:PE-dansyl, 20:78.5:2.5. The results of a binding experiment at 25 “C is displayed in A. The line indicates the best fit of the bimolecular model, Equation 4. Fitted values from this exper- iment were KD, 3.7 + 0.4 nM; n, 166 -C 4 phospholipid monomers per factor VIII molecule. Total phospholipid concentration was 3 PM. Binding at lower temperature, 8”C, is shown in B. Results of two experiments and best-fit curves are displayed. Fitted values were KD, 1.6 + 0.2 nM (0) and 2.7 rt 0.5 nM (0); n, 180 + 12 (a) and 179 + 5 (0) phospholipid monomers per factor VIII molecule. Total phospho- lipid concentration: l , 1 FM; 0, 3 pM.

the parameters K. and n are not influenced by the value of F,, these two methods give independent results.

DISCUSSION

Factor VIII is a plasma protein that functions as a cofactor in the activation of factor X by factor IXa. This cofactor function, increasing the rate of factor X activaion by at least 4 orders of magnitude (van Dieijen et al., 1980; Brown et al., 1978), is demonstrable in the presence of phospholipid mem- branes. The dependence of the rate enhancement upon the presence of phospholipid membranes was the stimulus for

cence at each mole fraction of PS. Because best fit values for these studies. We have demonstrated binding of factor VIII

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820 Binding of Factor VIII to Phospholipid Vesicles

w = -0.1 I

1.0

, 0.5 ILL-*

0.c 20 40 60

[VI , (nM)

FIG. 5. Binding of factor V to small vesicles at 25 ‘C. Vesicle composition was PS:PC:PE-dansyl, 20:78.5:2.5. The line indicates the best fit of the bimolecular model, Equation 4. Fitted values were Ko, 2.9 -+ 0.4 nM; n, 114 + 3 phospholipid monomers per factor V molecule. Values obtained are very similar to those obtained for factor VIII at this vesicle composition (Fig. 4 and Table I) with a slightly smaller value for n.

FIG. 6. Factor VIII binding to large unilamellar vesicles at 25 OC. Vesicle composition was PS:PC:PE-dansyl, 20:78.5:2.5. Two binding experiments are displayed. Phospholipid concentrations: 0, 1 pM; 0, 3 pM. Curves represent fits to Equation 4. The lower curue was fitted mathematically, and the upper curue was fitted visually. KD for the fitted curves are 1.8 nM and 2.5 + 1 nM for the upper and lower curves, respectively. n was 385 and 385 f 50 phospholipid monomers per factor VIII molecule, respectively.

to phospholipid membranes of small vesicles and large vesi- cles. The interaction of factor VIII with phospholipid vesicles is reversible and characterized by an apparent Ko of 4 nM. The presence of phosphatidylserine in the vesicles is required for binding.

0 0 10

[Factor :] (nM) 30 40

FIG. 7. Effect of PS-fraction in vesicles on factors VIII and V binding to phospholipid at 25°C. Protein binding to small vesicles as a function of the mole fraction PS. All small vesicles contained 0.025 dansyl-PE, the indicated fraction of PS, and PC. The mole fractions of PS are: 0.20, w 0.10, 0, 0.05, +; 0.025,O; 0.00, 0. Top, factor VIII; bottom, factor V.

A previous study of the binding of factor VIII antigen to small vesicles demonstrated qualitative binding which was preferential for small vesicles over von Willebrand factor (Lajmanovich et al., 1981; Andersson and Brown, 1981). Bloom (1987) demonstrated that recombinant human factor VIII bound to PS deposited on microELISA plates by organic solvent evaporation. Quantitative estimates were made; the dissociation constant was 11 pM and the number of PS monomers per factor VIII molecule bound was 1754. The dissociation constant measured is 2 orders of magnitude lower and the phospholipid/protein ratio at saturation is an order of magnitude higher than our data indicate. We suspect that the difference is due to the physical state of phospholipid prepared by evaporation on a polystyrene surface. The recom- binant-derived factor VIII utilized in this study is represent- ative of plasma factor VIII in terms of specific activity, amino- terminal sequence, sensitivity to thrombin and protein C, von Willebrands factor binding, and carbohydrate content. With- out apparent consequence, the molecular weight of the B domain is slightly higher than plasma factor VIII (Kaufman et al., 1988), presumably due to complexity of carbohydrate branching. The B domain is removed proteolytically upon factor VIII activation and can be deleted or substituted for without apparent functional consequence (Pittman et al., 1989; Toole et al., 1986). Hence it is unlikely that this differ- ence affected experimental results. Although prior study of factor VIII binding to lipid surfaces has been limited, consid- erable work has been performed on the interaction of factor V and phospholipid vesicles. Given the structural and func- tional homology of factors VIII and V these experiments provide a quantitative basis for the comparison of the phos- pholipid binding properties.

We have modified the resonance energy transfer technique

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Fraction PS”

Binding of Factor VIII to Phospholipid Vesicles 821

TABLE I

Factor VIII and factor V: binding as a function of phospholipid vesicle composition

Factor VIII Factor V

KD nb Fm..IFmc KD n

llM

0.30 2.2 + 0.4d 0.20 3.7 + 1.6' 0.15 4.2 + 0.5d 0.10 4,2/ 0.05 4,2 0.025 4,2 0 NA

ILM

89 3I 1 0.69 5.0 + 2.6d 68f 3 170f 20 0.53 3.9 + 1.2' 129*20 242f12 0.41 3.7 + 0.4d 171f 5 760+ 300" 0.16 6 26Ok 24

1400 + 600" 0.04 6 159 f 28 >5000 0.04 6 380 f 300

NA 0.01

’ The small unilamellar vesicles were composed of PS as indicated, 0.025 dansyl-labeled PE and PC. * n is the molar ratio of phospholipid to protein at saturation. ‘F,,,,, represents the maximum measured fluorescence. F, was determined by assuming that at a high mole

fraction of PS, where the number of protein molecules bound is limited by the physical space requirement the protein phospholipid ratio is 47. Thus the maximum fluorescence value with the mole fraction of PS equal to 0.30 is 0.69.

d Error values represent standard error of fit. e Mean f S.E. from four experiments. f Values of n for the curves with PS fraction less than 0.15 were determined from fitting the data to Equation 4

with an assigned value of & corresponding to the mean value obtained with vesicles of a higher mole fraction of PS the same day.

g Error values are + range from two experiments.

described by Pusey et al. (1982) for kinetic measurements in order to provide a new technique for equilibrium measure- ments. This technique is sensitive, allowing analysis with protein concentration in the nanomolar range. Results ob- tained for the dissociation constant of factor V with phospho- lipid vesicles are in close agreement with those obtained by Krishnaswamy and Mann (1988). Whereas their technique requires modification of factor Va but not the phospholipid vesicles, our method requires modification of the phospholipid vesicles but not the protein. The similarity of the results of both methods offers further evidence of the validity of the determined values.

A significant difference between the phospholipid binding of factors VIII and V was observed when the fraction of phosphatidylserine was less than 20%. For factor VIII the quantity of protein bound has a nonlinear dependence upon the mole fraction of PS. For factor V the required mole fraction of PS is smaller and the dependence upon PS is approximately linear. Factor V bound to vesicles at a PS fraction too low to support factor VIII binding. In an attempt to understand this divergent nonlinear dependence we ex- amined the adherence of the data to a probability-based lipid binding model.

Assume that a protein collides with a discrete region of a phospholipid vesicle membrane, the “contact region.” Physi- cal constraints on the membrane will assure that the contact region is composed of an approximately constant number of phospholipid head groups, N. Assume, also, that if a minimum number of head groups, m, or more are PS, then collision with the contact region will result in binding; otherwise the colli- sion will be unsuccessful and the protein will return to solu- tion. The fraction of contact regions which are capable of binding is given by a probability function,

P(m; N, PI = i j=m *! (;I x)! p’ 0 - pJN - x (5)

where p represents the mole fraction of PS and x is the number of PS molecules within the contact region.

Fig. 8 demonstrates the behavior of the model in compari- son to data obtained (Table I). While a binding site has been defined as the ratio of phospholipid to protein at saturation, the size of the contact region has been established by visual

1

0.5 a

0 0 0.2 0.4

Mole Fraction PS

FIG. 8. Factor VIII binding capacity compared to a proba- bility model. Factor VIII binding capacity from Table I compared to predicted capacity as the probability, P, of binding to any contact region. The closed boxes indicate capacity determined by maximum measured fluorescence, and the closed circles indicate capacity (in- versely proportional to rz) determined from fit of Equation 4 to fluorescence data. For fluorescence and fitted values a maximum binding capacity, when the number of molecules bound is limited by physical crowding, was determined by the assumption of a minimum value for n, 47 phospholipid monomers per factor VIII molecules. Values displayed are a ratio of measured values to this theoretical maximum. The curve represents a visual fit of the probability function P(m; N, p) (Equation 5) for the case where m = 3, N = 13, and p corresponds to the mole fraction PS displayed on the abscissa.

fitting of the experimental data to the probabilistic model. The contact region size, iV, for the figure is 13 phospholipid monomers. Fitting the model to the data, the minimum num- ber of PS monomers, m, for a factor VIII binding site is 3. These results indicate that, despite the similarities between factors VIII and V, the chemical surfaces that define their phospholipid binding properties are different.

An important question remains as to whether the binding of factor VIII to cell surfaces is mediated by protein receptors or whether factor VIII binding to membranes is mediated by phospholipids. The exposed surface of platelet membranes has a lipid composition of less than 2% PS. After stimulation the composition changes to between 4 and 13% depending upon the stimulus (Bevers et al., 1983). Recently, factor VIII has been shown to bind preferentially to activated platelets over resting platelets (Nesheim et al., 1988; Muntean et al.,

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a22 Binding of Factor VIII to Phospholipid Vesicles

1987). The molecular basis for this transition remains un- known. However, the range over which the platelet membrane PS content changes is sufficient to alter the ability of factor VIII to bind to phospholipid vesicles.

In summary, we have demonstrated by three independent techniques that human factor VIII binds to the surface of PS- containing phospholipid vesicles. We have shown that reso- nance energy transfer, utilizing intrinsic fluorescence of factor VIII to excite dansyl labels in a phospholipid vesicle mem- brane, can be adapted for equilibrium studies of the binding of factor VIII to the phospholipid surface. Factor VIII binding is rapid and reversible and fits a simple bimolecular equilib- rium model. The apparent dissociation constant and the pro- tein/phospholipid ratio at saturation are similar to values reported for factor V with a lower protein/lipid ratio for large vesicles than for small vesicles. Factor VIII exhibits a nonlin- ear binding relationship to the mole fraction of PS and has different PS requirements than does factor V binding. Further studies comparing the phospholipid binding properties of factors VIIIa and VIIIi with factor VIII and the assembly of factor IXa on membrane surfaces containing factor VIII should provide insight into the structure and function of the enzyme complex. Elicitation of the molecular mechanism whereby factor VIII binds to phospholipid membranes should allow a more direct evaluation of the importance of this interaction to function on a cell or platelet surface.

Acknowledgmerzts-We thank Dr. Barry Foster for helpful discus- sions in the early phase of this work, Dr. Philip Rosoff for extensive use of his Perkin Elmer LS5 fluorometer, and Dr. Paul Robb for use of his IBM computer on which nonlinear curve fitting was performed.

REFERENCES

Abbott, A. J., and Nelsestuen, G. L. (1987) Biochemistry 26, 7994- 8003

Andersson, L.-O., and Brown, J. E. (1981) Biochem. J. 200, 161-167 Barenholz, Y., Gibbes, D., Litman, B. J., Gall, J., Thompson, T. E.,

and Carlson, F. D. (1977) Biochemistry 16,2806-2810 Bevers, E. M., Comfurius, P., and Zwaal, R. F. A. (1983) Biochim.

Biophys. Acta 736, 57-66 Bloom, J. W. (1987) Thromb. Res. 48,439-448 Bloom, J. W., Nesheim, M. E., and Mann, K. G. (1979) Biochemistry

l&4419-4425 Brown, J. E., Baugh, R. F., and Hougie, C. (1978) Thromb. Res. 13,

893-900

Chen, P. S., Toribara, T. Y., and Warner, H. (1956) Anal. Chem. 28, 1756-1758

Church, W. R., Jernigan. R. L., Toole, J., Hewick, R. M., Knopf, J., Knutson, G. J. Neaheim, M. E., Mann, K. G., and Fass, D. N. (1984) Proc. N&l. Acad. Sci. U. S. A. 81, 6934-6937

Enoch, H. G., and Strittmatter, P. (1979) Proc. N&l. Acad. Sci. U. S. A. 76, 145-149

Furie, B., and Furie, B. C. (1988) Cell 53,505-518 Gitschier, J., Wood, W. I., Goralka, T. M., Wion, K. L. Chen, E. Y.,

Eaton, D. H., Vehar, G. A., Capon, D. J., and Lawn, R. M. (1984) Nature 312,326

Hardesty, R. M., and Macpherson, J. C. (1962) Thromb. Diath. Haemorrh. 7, 215

Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Biochim. Biophys. Actu 812,55-65

Kane, W. H., and Davie, E. W. (1988) Blood 71, 539-555 Kaufman, R. J., Wasley, L. C., and Dorner, A. J. (1988) J. Biol. Chem.

263,6352-6362 Krishnaswamy, S., and Mann, K. G. (1988) J. Biol. Chem. 263,5714-

5723 Lajmanovich, A., Hudry-Clergeon, G., Freyssinet, J.-M., and Mar-

guerie, G. (1981) Biochim. Biophys. Actu 678, 132-136 Lollar, P., and Parker, C. G. (1987) J. Biol. Chem. 262,17572-17576 Mann, K. G., Jenny, R. J., and Krishnaswamv, S. (1988) Annu. Reu.

Bio&m. 57,915 _

Mayer, L. D., Hope, M. J., and Cullis, P. R. (1986) Biochim. Biophys. Acta 858,161-168

Muntean, W., Leschnik, B., and Haas, J. (1987) Thromb. Res. 45, 345-354

Nesheim. M. E., Mvrmel. K. H.. Hibbard. C.. and Mann, K. G. (1979) J. Biol.’ Chem; 254,568-517’

Nesheim, M. E., Pittman, D. D., Wang, J. H., Slonosky, D., Giles, A. R., and Kaufman, R. J. (1988) J. Biol. Chem. 263, 16467-16470

Pittman. D. D.. Marauette. K. A.. and Kaufman. R. J. (1989) Thromb. Haem&tasis’62,3~ ’ ’

Pusey, M. L., Mayer, L. D., Wei, G. J., Bloomfield, V. A., and Nelsestuen, G. L. (1982) Biochemistry 21, 5262-5269

Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., Buecker, J. L., Pittman, D. D., Kaufman, R. J., Brown, E., Shoemaker, C., Orr, E. C., Amphlett, G. W., Foster, W. B., Coe, M. L., Knutson, G. J., Fass, D. N., and Hewick, R. M. (1984) Nature 312,342-347

Toole, J. J., Pittman, D. D., On, E. C., Murtha, P., Wasley, L. C., and Kaufman, R. J. (1986) Proc. N&l. Acad. Sci. U. S. A. 83, 5939- 5946

van de Waart, P., Bruls, H., Hemker, H. C., and Lindhout, T. (1983) Biochemistry 22,2237-2242

van Dieijen, G., Tans, G., Rosing, J., and Hemker, H. C. (1981) J. Biol. Chem. 256, 3433-3442

Vehar, G. A., Keyt, B., Eaton, D., Rodriguez, H., O’Brien, P. P., Rotblat, F., Oppermann, H., Kech, R., Wood, W. I., Harkins, R. N., Tuddenham, E. G. D., Lawn, R. M., and Capon, D. J. (1984) Nature 312,337-342

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G E Gilbert, B C Furie and B FurieBinding of human factor VIII to phospholipid vesicles.

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