The Binding of Factor Va to Phospholipid Vesicles*

THE JOURNAL OF BOLOGCAL CHEMSTRY 1988 by The American Society for Biochemistry and Molecular Biology, nc. Vol. 263, No. 12, ssue of April 25, pp. 5714-5723,1988 Printed in U.S.A. The Binding of Factor Va to Phospholipid Vesicles* Sriram Krishnaswamy and Kenneth G. Mann$ (Received for publication, June 15,1987) From the Department of Biochemistry, Health Science Complex, University of Vermont, Burlington, Vermont 545 The analysis of free sulfhydryl groups in factor Va Factor Va functions in the blood clotting cascade as a using dithiobis-(nitrobenoic acid) (DTNB) indicated cofactor protein in the activation of the ymogen prothrombin the presence of one accessible thiol in each of the two catalyed by factor Xa (1-4). Factor Va is produced by the subunits of the cofactor. ntact factor Va contained one action of thrombin on the pro-cofactor, factor V, which cirreadily accessible sulfhydryl group under native con- culates in plasma as a large, asymmetric, and monomeric ditions and approximately two such groups after de- protein and possesses minimal cofactor activity (3-7). Upon naturation. A comparison of the rate of modification proteolysis by thrombin, factor V is converted to the active of the accessible thiol in factor Va under native conditions to those observed with the isolated subunits incofactor as a consequence of the cleavage of three peptide dicated that the thiol present in component D of the bonds (6,8). The resultant factor Va molecule (Mr = 168,) cofactor was readily accessible to reaction with DTNB. is a heterodimer, composed of an NH2-terminal-derived heavy Factor Va was reacted with the sulfhydryl-directed chain (Ar = 94,; component D) and a COOH-terminalfluorophore N-( 1-pyrene)maleimide, resulting in the derived light chain (M, = 74,; component E) which remain concomitant loss of the accessible thiol with no detect- associated via divalent cation-mediated interactions (6, 8). A able alteration in the activity of the cofactor. This large central domain comprising approximately 5% by weight fluorescent derivative of factor Va (Pyr-Va) was used of the pro-cofactor is released in the form of two heavily to examine the binding of factor Va to phospholipid glycosylated activation peptides (9). The newly formed cofacvesicles by fluorescence polariation. Fluorescence po- tor is then available to interact with the serine protease factor lariation of the pyrene moiety increased saturably Xa in the presence of calcium ions on a phospholipid or when Pyr-Va was titrated with increasing concentra- cellular surface to form the prothrombinase complex of the tions of vesicles composed of phosphatidylcholine and blood clotting cascade. phosphatidylserine (PS). Systematic analysis of the An understanding of the constraints that govern the assembinding of Pyr-Va to PCPs (75% phosphatidylcholine, bly of the prothrombinase complex and the expression of its 25% PS) indicated that the binding interaction was activity requires a detailed and quantitative description of the characteried by a dissociation constant of 2.7 X lo- discrete protein-protein and protein-lipid interactions that M with 42 mol of PCPs bound per mol of Va at saturation. The data obtained by varying the PS content of the vesicles are consistent with the interpretation that the Va-combining site on the vesicle surface is composed of a discrete number of PS molecules. The bind- ing of Pyr-Va to PCPs was independent of added calcium ion and could be reversed by the addition of unlabeled Va or isolated component E but not by component D. Analysis of the displacement curves indicated that native factor Va or isolated component E and Pyr-Va mutually excluded each other on the vesicle surface with identical affinities. Competition experiments conducted using component E digested by factor Xa or the isolated derivative peptides indicated that the cleavage of component E by factor Xa had no effect on the PCPs binding properties of this subunit. Further, the data obtained with the isolated peptides suggest that the lipid-binding domain of component E is present in the amino-terminal region of this subunit. * This work was supported by National nstitutes of Health Grants HL-34575 and HL-3558 (to K. G. M.) and by FRST Award HL- 38337 (to S. K.). Presented in preliminary form at the Xth nternational Congress of Thrombosis and Haemostasis, Brussels, Belgium, July 6-1, 1987, and published in abstract form (55). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. 5714 occur within the complex. The use of specific active sitedirected fluorescent reporter groups has been extremely useful in the delineation of the interactions between factor Xa or activated protein C and the other constituents of prothrombinase (11, 14, 15). Due to the lack of fluorescent reporter groups that could be incorporated with some degree of specificity into factor Va, studies of the interaction between factor Va and phospholipid membranes have been conducted using light scattering (16-18), electron microscopy (19), and none- quilibrium techniques including kinetics (17, 2) and rapid sedimentation of lipid-bound factor Va by centrifugation (21). Recent studies have utilied factor Va that has been modified with sulfhydryl-directed fluorophores to estimate the orientation of factor Va on the phospholipid surface using fluores- cence energy transfer techniques (22). Data obtained with two fluorophores indicated that the modified sites were not significantly perturbed by the interaction of the cofactor with phospholipids or with other components of the prothrombinase complex (22). The results of several studies have indicated that the factor V- or the factor Va-phospholipid interaction requires the presence of negatively charged phospholipids (16, 211, is not dependent on the presence of exogenously added calcium ions (16), and in the case of factor Va is primarily mediated by component E of the cofactor (18,2,21). Fluorescence energy transfer studies indicate that component D of factor Va is approximately 9 A away from the phospholipid surface, leading to the speculation that factor Va projects radially away from the lipid surface (22). Two aspects of the interaction between factor Va and phospholipds remain controver-

sial. Conflicting reports are available in the literature regarding the contributions of hydrophobic and electrostatic interactions towards the overall stabiliation of the factor Vaphospholipid complex (18, 2, 21). n addition, estimates of the dissociation constant for this interaction obtained by several groups have varied greatly, ranging from lo-'' M on the basis of kinetic studies (17) to M on the basis of light scattering (16, 18). Some of these results have led to the hypothesis that factor Va binding to platelets (Kd = 1"' M) is intrinsically different from the binding of factor Va observed to phospholipid vesicles and may involve a factor Va receptor (23). On the basis of indications that factor Va could be labeled with some degree of specificity using sulfhydryl-directed fluorophores (22), efforts were directed towards the specific modification of accessible sulfhydryl residues in factor Va using fluorophores with long excited state lifetimes. The present study describes the specific modification of factor Va with N-(1-pyrene)maleimide and the use of this adduct (Pyr- Va)' to examine the quantitative aspects of the interaction between factor Va and phospholipid vesicles using fluorescence polariation. EXPERMENTAL PROCEDURES Materials Hepes, 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB), L-a-phosphatidylcholine (hen egg), and L-a-phosphatidylserine (bovine brain) were from Sigma. N-(1-Pyrene)maleimide adsorbed to Celite (1% w/w) was from Molecular Probes. D-Phenylalanylprolylarginyl chloromethyl ester (PPACK) was from Behring Diagnostics. Phospholipid vesicles (PCPs) usually composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine or of the indicated composition were prepared as described previously (18, 24), except that vesicles were prepared in 2 mm Hepes,.15 M NaCl, ph 7.4. Vesicles were usually used in experiments within 3 days of preparation. The fluorescent a-thrombin inhibitor dansylarginine-n-(3-ethyl-1,5-pen- tanediy1)amide (DAPA) was prepared by described methods (4). Bovine factor V was isolated as described previously (4,5). Bovine a- thrombin was prepared according to the procedure of Lundblad et al. (25). Bovine factor Xa was prepared from factor X treated with the purified factor X activator from Russell's viper venom (12). Factor Va was prepared from partially purified preparations of factor V by immunoaffinity chromatography as described previously (26-28). Briefly, factor V was partially purified from 8 liters of bovine blood using the procedures described by Nesheim et al. (4) with the omission of the Cihacron Blue chromatography step. This material was resuspended in 2 mm Hepes,.15 M NaC, ph 7.4, and activated in 2 aliquots by the addition of thrombin to 9 units/ml and incubation at 37 "C for 1 min. The activation was terminated by the addition of PPACK (3 FM), followed by the addition ofcac1, (2mM). The activated mixture was applied to the immunoaffinity column (1-ml packed bed, 3 mg of gg) and factor Va purified using the procedures described previously (26). The best yields observed by this procedure were 12-175 Aao units. solated components D and E of factor Va were prepared by fast protein liquid chromatography using the procedures previously detailed (15), except that purified factor Va was used as the starting material. solated component E was cleaved with factor Xa to produce component E% by the following procedure. Purified component E (.73 pm) was incubated with.2 pm factor Xa in 2 mm Hepes,.15 M NaCl, 2 mm CaCl, (total volume = 11 ml) for 2 b at 37 "C. Preliminary experiments had indicated that these conditions were adequate for the quantitative cleavage of component E. Proteolysis was terminated by the addition The abbreviations used are: Pyr-Va, factor Va derivatied with months. N-(1-pyrene)maleimide; DAPA, dansylarginine-n-(3-etbyl-1,5-pentanediy1)amide; Hepes, 4-(2-hydroxyethyl)-l-piperaineethanesulfonic acid; DTNB, 5,5'-dithiobis-(2-nitrobenoic acid); PCPs, vesicles composed of L-a-phosphatidylcholine and L-a-phosphatidylserine; PS, L-a-phosphatidylserine; PPACK, D-phenylalanylprolylarginyl chloromethyl ester; TNB, 5-thio(2-nitrobenoic acid); EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Binding of Factor Va to Phospholipid Vesicles 5715 of PPACK (46 p ~ followed ) by incubation on ice for an additional 1 min. The activation mixture was applied to the immunoaffinity matrix described above (1-ml packed bed, 3 mg of gg) which also retains isolated component E and component EX. (27). Bound protein was eluted from the matrix using the same procedures used to isolate factor Va. The yield of component Exs was 3.7 mg (65%). The two peptides that comprise component Ex. (Mr = 3, and 48,) were isolated by reverse-phase high pressure liquid chromatography by the procedures previously described (27). Fractions containing the isolated M. = 3, peptide or the M, = 48, peptide were pooled, dialyed extensively into 2 mm Hepes,.15 M NaC1, 2 mmcac12, ph 7.4 (4, volumes), and used in binding studies. The purity of all protein preparations was evaluated using polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide bond reduction with 2-mercaptoethanol by the procedures described by Laemmli (29). Due to the significant variability in the reported extinction coeffi- cients for factor Va and its isolated subunits (8,18,3), thextinction coefficients of these proteins were determined by differential refractometry in the analytical ultracentrifuge by the procedures described by Babul and Stellwagen (31). The determined extinction coefficients (E;&%) were factor Va, 1.74; component D, 1.24; and component E, 2.23. The concentration of component EX. was determined using the extinction coefficient for component E. The molecular weights used were factor Va, 15,; component D, 94,; and component E, 74, (18). For the other bovine proteins, the extinction coefficients (E:&%) and molecular weights used were prothrombin, 1.44 and 74, (32); factor Xa, 1.24 and 45,3 (33, 34); and factor V,.96 and 33, (5). Titration of Sulfhydryls The concentration of accessible sulfhydryls was determined under native or denaturing conditions with DTNB (35) using a CARY 219 double-beam spectrophotometer. For continuous titrations, sample cuvettes contained between 3.6 and 1.3 pm protein in 2 mm Hepes,.15 M NaC1, 2 mm CaCi2, ph 7.4. Sulfhydryl modification was initiated by the addition of DTNB (2. mm) to both the sample and reference cuvettes, and the formation of 5-thio-(2-nitrobenoic acid) (TNB) was followed continuously by the increase in absorbance at 412 nm. Absorbance readings were converted to molar concentrations of sulfhydryls modified using a molar extinction coefficient of TNB of 13,6 at this wavelength (35). Static titrations were performed by preparing reaction mixtures in triplicate (.8-ml final volume) containing between 1.2 and 5.2 pm protein in 2 mm Hepes,.15 M NaCl, 2 mm CaCZ, ph 7.4, and 2. mm DTNB (native) or in 2 mm Hepes,.15 M NaC1,4. M guanidine-hc, 6.7 mm EDTA, and 2. mm DTNB (denaturing). The reaction mixtures were incubated in the dark at room temperature for 2 h before the absorbance of the samples was determined at 412 nm. Readings obtained 3 h after initiation indicated that a stable, limiting absorbance was reached within 2 h. n all cases, the reference solution was identical to the sample solution, except that it lacked protein. Modification of Factor Va with N-(1 -Pyrene)maleimide Factor Va in 2 mm Hepes,.15 M NaC1, 5 mm CaCZ, ph 7.4 (1.5 mg/ml, 1. ml), was treated with 138 mg of N-(1-pyrene)maleimide on Celite (1% w/w) and incubated in the dark at 4 "C for 24 h. The mixture was then briefly centrifuged to pellet the Celite, and the precipitate was washed three times with the incubation buffer to recover all factor Va present. The recovered factor Va (final volume approximately 4 ml) was separated from excess dye by gel filtration in the dark using a 1.5 X 5-cm column of Sephadex G-25-15 at room temperature equilibrated in 2 mm Hepes,.15 M NaC1, 5 mm CaClZ, ph 7.4. Fractions containing protein were pooled and precipitated by the addition of solid ammonium sulfate (8% saturation), collected by centrifugation (5, X g, 2 min), resuspended in 1 mm Tris, 1 mm borate, 1 mm CaC12, 5% (v/v) glycerol, ph 8.3, and stored at -2 "C. Pyr-Va prepared by this procedure is stable with respect to its activity and fluorescence properties for at least 6 Activity Measurements The cofactor activity of factor Va or of modified factor Va (Pyr- Va) was evaluated using clotting assays (4) or by the direct measurement of prothrombin activation catalyed by prothrombinase using the fluorescent a-thrombin inhibitor DAPA (4, 36). Titration curves were constructed exactly as described (12) to obtain the apparent

5716 Binding of Factor Va to Phospholipid Vesicles equilibrium constants for the interaction of factor VA or Pyr-Va with factor Xa in the presence of PCPs. Fluorescence Measurements of Pyr- Vu Fluorescence Spectra-Fluorescence measurements were made using an SLM 8 photon-counting fluorescence spectrophotometer equipped with hardware and software modifications from On-Line Systems, Jefferson, GA. The uncorrected fluorescence excitation spectrum of Pyr-Va was obtained by scanning the excitation monochromator between 26 and 36 nm (band pass = 2 nm) and monitoring emission intensity at 396 nm (band pass = 16 nm) with a longpass filter (Schott KV-37) in the emission beam. Four hundred points were collected over the excitation range with 1 readings averaged per datum, using a time constant of 1 ms. Spectra were obtained using 5 X M Pyr-Va in 2 mm Hepes,.15 M NaCl, 2. mm CaCl', ph 7.4, in the presence or absence of 6 p~ PCPs. Fluorescence polariation spectra were obtained using the instrument described above operated in T-format (37) with Glan-Thompson polariing filters in the excitation and the two emission beams. The spectra were collected using the ratio of the signal from the two emission detectors, and 32 readings were averaged per datum to increase the sensitivity of detection. Polariation values as a function of excitation wavelength were calculated after two passes of the excitation monochromator to correct for channel bias using the equations available in the literature (39). Fluorescence Polariation Titrations-The excitation wavelength was 33 nm (band pass = 8 nm), and the emission wavelength was 396 nm (band pass = 16 nm). Long-pass filters (Schott KV 389; %T3,, =.4 and %T, = 4.1) were placed in the two emission beams to minimie scattered light artifacts. Fluorescence titrations were performed using 1 X 1-cm quart cuvettes with the sample chamber maintained at 25 "C. Cuvette temperature wasclosely monitored using a thermocouple probe in an adjacent cuvette. Buffers (with the exception of protein solutions) were filtered using.45-pm filters to reduce solution scatter. Protein solutions were centrifuged in an Eppendorf centrifuge for 5 min at 4 "C to remove particulate material. All titrations were performed using a 2.-ml initial sample volume with the indicated reactants, and microliter additions were made of the varied component. Polariation values were measured approximately 2 s after each addition by the integration of the signal at each position of the excitation polarier for a total of 1 s, and the final values recorded represent the means of between 8 and 1 repeated readings. Total fluorescence intensity measured as n + 24 (38) was also recorded after each addition in some experiments. n competition experiments, polariation values were recorded 4 min after each addition of the titrant to ensure that the new equilibrium had been established. Frequently, the establishment of a stable reading was confirmed by a second series of readings. Corrections were not made for fluorophore dilution or inner-filter effects as the final reaction volume never exceeded 2.15 ml and the absorbance of the sample at the exciting wavelength never exceeded.4. Data Analysis Kinetic Analysis of the nteraction Between Factor Xu and Factor Vu-The interaction between factor Xa and phospholipid-bound factor Va or Pyr-Va was evaluated using the assumptions and equations previously described (12). The initial, steady-state rates of prothrombin activation obtained at two fixed concentrations of factor Xa and variable concentrations of factor Va or Pyr-Va were analyed by nonlinear least squares regression analysis according to Equations 1-3 described in Ref. 12, to obtain estimates for the apparent dissociation constant and stoichiometry describing the interaction between factor Xa and the cofactor. Analysis of the Modification of Factor Vu by DTNB-The modification of factor Va and its isolated subunits by DTNB was carried out under conditions where the modifier was in vast excess over the protein. Absorbance readings obtained as a function of time were therefore analyed according to a single exponential to obtain fitted values for the pseudo-first order rate constant and maximal the extent (amplitude) of modification. Analysis of the Binding of Modified Factor Vu to Phospholipid Vesicles by Fluorescence Polariation-Fluorescence polariation values obtained using fixed concentrations of Pyr-Va (fluorophore), and variable concentrations of PCPs vesicles (titrant) were analyed according to Equation 7 described in Ref. 15. The assumption used in the derivation of this expression is that the titrant (PCPs) contains multiple, identical, independent, and noninteracting sites that the fluorophore can occupy. Values of fluorescence polariation obtained at increasing concentrations of titrant were converted to anisotropy values using the relationship described by Weber (39). As the change in fluorescence anisotropy is directly proportional to the concentration of the PCPs-bound Pyr-Va, the data were analyed according to Equation 1 below, where [P,] is the total concentration of titrant (PCPs), [F,] is the total concentration of fluorophore (Pyr-Va), i is the number of moles of fluorophore combining per mole of titrant at saturation, Kd is the dissociation constant describing the interaction between fluorophore and titrant, ro is the anisotropy observed in the absence of titrant, r,, is the maximum anisotropy observed when all fluorophore is bound to the titrant, and robs is the observed anisotropy in the presence of a given concentration of titrant. Fluorescence data obtained at varying concentrations of PCPs at two or more fixed concentrations of Pyr-Va were evaluated according to Equation 1 by unweighted nonlinear least squares regression analysis (4) to obtain fitted values for i, Kd, and r-. Analysis of the Displacement of PCPS-bound Pyr-Vu by Competitors-Consider the competitive equilibria: and where i moles of fluorophore (F) combine per mole of PCPs (P) to give F,P or where j moles of lipid-binder (C) combine per mole of PCPs to give c,p governed by the dissociation constants Kdl and Kd. The formation of FiP is associated with a fluorescence signal while the formation of CjP is not accompanied by a directly detectable change in fluorescence. n the presence of fixed concentrations of fluorophore ([F,]) and PCPs ([P,]) and increasing concentrations of Competitor ([C,]), the concentration of C,P will increase, with a concomitant decrease in the total concentration of PCPs available for fluorophore to interact with. Hence, increasing concentrations of competitor ([C,]) will result in a decrease in the concentration of PCPs-bound fluorophore bound and in a decrease in the observed anisotropy. At infinite concentrations of competitor, anisotropy will approach ro. When the competitor and fluorophore interact with identical sites on PCPs (i.e. i = j), the concentration of total lipidbinding protein ([L,]) present in the reaction mixture expressed in terms of the binding efficiency of the fluorophore will be given by Equations 4 and 5. [Ltl = [Ftl + A.[Ctl (4) Equations 4 and 5 are based on the assumption that the competitor and fluorophore combine with identical sites on the vesicle surface. Therefore, for a given concentration of phospholipids, the concentrations of competitor and fluorophore bound to the vesiclewillbe determined by the total concentrations of the two ligands and the dissociation constants for the two interactions. The proportionality constant A, allows the expression of [C,] in terms of lipid-binding capacity that is equivalent to the lipid-binding activity of [F,]. When Kdl = K,, A = 1. The concentration of lipid-binding protein bound to PCPs ([Lb]) is given. [L*l = (i.[pt] [Lt] + Kdl) - J(i.[pt] [Lt] Kd1)' - 4.i.[pt]'[Lt1 2 The observed anisotropy will be determined by Equation 7 below: LLtJ (6)

1.2 1. > 3.8 2.6 n E.4 - =.2 w 1. n.8 U LA!.6 6 \. 4 n ;.2 A Binding of Factor Va to Phospholipid Vesicles 5717 dd O w 1.2 c w 1. n.8 U w.6 - \.4 v) n.2 w - x 5 1 15 2 25 3 TME (mid 2 4 6 8 12 1 TME (mid FG. 1. Modification of accessible sulfhydryl groups in factor Va and its isolated subunits. Reaction mixtures contained A, 1.25 pm factor Va; E, 6.54 pm component D; or C, 3.6 pm component E in 2 mm Hepes,.15 M NaC1, 2 mm CaCl,, ph 7.4. Modification of accessible sulfhydryls was followed at 412 nm following the addition of DTNB (2. mm) as described under Experimental Procedures. The lines are drawn according to single exponentials (pseudo-first order kinetics) with fitted rate constants and amplitudes of: A,.224 f.1 rnin,.988 f.5 mol of sulfhydryls/mol of factor Va. E, TABLE Quuntitatwn of accessible sulfhydryls Thiols identified f S.E. Protein Native Denatured Factor Va.95 f.1 2.35 f.8 Component D 1.3 -C.2 1.49 f.15 Component E.85 f.15 1.18 f.8 Pyr-Va.8 &.9 1.1 f.9 Accessible thiols were quantitated using DTNB as described under Experimental Procedures. The values represent the mean k S.E. of between three and eight separate measurements. where rob., ro, and r,, indicate the same quantities described for Equation 1. Polariation/anisotropy data obtained at a fixed concentration of fluorophore and two or more concentrations of PCPs at varying concentrations of competing ligand were analyed by un- 1 weighted nonlinear least squares regression analysis according to 15 1 5 2 25 3 35 4 Equations 6 and 7 using values of i, ro, and r,, determined from TME experiments evaluated according to Equation 1, in order to obtain (mid fitted values of Kdl and Kd2. This technique avoids the use of complicated Newton-Raphson iteration procedures that are required for the solution of similar equations derived for competing equilibria (41). c 1.2,m, All fitted data were evaluated by the visual inspection of residuals to the fitted line, by the standard errors of the determined constants, by the overall root mean squared deviation, the correlation between fitted parameters, the lack of dependence of fitted parameters on initial estimates and by the Durbin-Watson statistic (42). RESULTS Quantitation and Reactivity of the Free Sulfhydryls in Factor Vu and the solated Subunits of the Cofactor-The reaction between DTNB and accessible cysteine residues in factor Va or the isolated subunits of the cofactor (components D and E) are illustrated in Fig. 1. Under native conditions, DTNB readily reacts with factor Va (Fig. la), and the modification process is reasonably defined by a single exponential. The fitted constants for this process correspond to the modifica- tion of approximately 1 mol of sulfhydryls/mol of factor Va with a tlh of 3.1 min. To further localie this reactive side chain to one of the two subunits of the cofactor, the reactivity of isolated components D and E with respect to DTNB was examined. The data illustrated in Fig. 1, B and C indicate that both isolated subunits contain accessible, free sulfhydryls under native conditions. The modification of isolated component D by DTNB (Fig. 1B) is well defined by a single exponential indicating 1.4molof accessible thiol/mol of protein modified with a tlh of 3.6 min. The close agreement between the rates and extent of modifications of the intact cofactor and isolated component D suggests that the readily accessible cysteine present in the cofactor resides in component D. solated component E is modified at a much slower rate by DTNB (Fig. C) with a tlh of 21 min. The reaction between DTNB and component E illustrated in Fig. 1C deviates systematically from pseudo-first order kinetics, raising the possibility of the presence of differentially reactive residues in this subunit. Kinetic complexity could arise from the presence of several differentially accessible thiol groups or from protein heterogeneity. The latter possibility seems likely as preparations of component E contain variable amounts (usually minor) of cleavage products (27). The quantitation of free sulfhydryls present in factor Va and its isolated subunits under native and denaturing conditions is summaried in Table 1. Collectively, the data indicate that factor Va contains two free sulfhydryls with each of its subunits contributing one.192 &.1 rnin, 1.4 f.2 mol of sulfhydryls/mol of component D; and C,.33 &.1 rnin,.967 *.12 mol of sulfhydryls/ mol of component E.

5718 Binding of Factor Va to Phospholipid Vesicles cysteine. n the absence of denaturants, the single cysteine present in component D is readily accessible to modification by sulfhydryl-directed reagents, while the cysteine in component E remains refractory to modification prior to chain separation. Modification of Factor Vu with N-(1 -Pyrene)muleimide- Factor Va was reacted with the sulfhydryl-directed fluorophore N-(1-pyrene)maleimide to yield Pyr-Va and separated from unreacted dye as described under Experimental Procedures. The reaction conditions were chosen to maximie the stability of the cofactor and to minimie the reaction between free amino groups on the protein and the imido carbonyl group (43). n three separate experiments using different preparations of factor Va and the published molar extinction coefficient of the chromophore of 17,378 at 339 nm (43), the number of moles of pyrene found incorporated per mole of factor Va were.83,.88, and.91. Parallel experiments performed using fluorescein-5-maleimide, tetramethylrhodamine-5-maleimide, rhodamine X maleimide, and 6-acryloyl-2-dimethylaminonaphthalene also indicated that be- tween.7 and.9 mol of fluorophore were incorporated per mol of factor Va over the incubation period. Visualiation under UV illumination of factor Va modified by the latter fluorophores following sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the fluorophore was primarily incorporated into component D. Very faint fluorescence was found associated with species of lower molecular weight, corresponding to degradation products of component D. The quantitation of free sulfhydryl groups in Pyr-Va listed in Table indicates that the modification of factor Va by N-(1- pyrene)maleimide results in the concomitant reduction of reactive cysteine residues in the cofactor. The data are consistent with the interpretation that reaction of factor Va with N-( 1-pyrene)maleimide results in the covalent modification of a readily accessible cysteine residue present in component D. Assessment of the Activity of Pyr-Vu-Measurements of residual clotting activity following reaction of factor Va by the sulfhydryl reagents described above indicated that the modification reaction had little or no effect on the activity of the cofactor. These results are identical to those reported FG. 2. Fluorescencexcitation and polariation spectra of Pyr-Va or the Pyr-Va-PCPs complex. The dependence of total fluorescence intensity (A) or fluorescence polariation (B and C) on excitation intensity was determined using reaction mixtures containing 5 X M Pyr-Va in 2 mm Hepes,.15 M NaC1, 2. mm CaC12, ph 7.4, with no PCPs (A and B) or with 6 p~ PCPs (C). The emission monochromator was centered at 396 nm (band pass = 16 nm), and the excitation wavelength was varied between 26 and 36 nm (band pass = 2 nm). The fluorescence excitation spectrum for the Pyr-Va. PCPs complex was essentially identical to that obtained with Pyr-Va alone (spectrum A) and is not illustrated for the purpose of clarity. >- - w v) W - w W u W u v) W CY 3 J LL. -. 1. u 1.5 1.2.9.6.3 previously following the reaction of factor Va with iodoacetamido derivatives (22). To rule out the possibility that the observed clotting activity of Pyr-Va resulted from the presence of a small population of differentially modified molecules of high specific activity, the ability of the modified protein to function as a cofactor in the prothrombinase complex was directly assessed. Titration curves, generated by the dependence of the initial rate of prothrombin activation (rate/[xa]) on the concentration of native factor Va or Pyr-Va at two fixed concentrations of factor Xa (data not shown) were essentially superimposable and yielded an apparent dissociation constant of8.55 x f:.98 x 1 and a stoichiometry of.91 _+.5 mol of cofactor bound per mol of factor Xa at saturation. These values are identical to those obtained for human and bovine prothrombinase (3, 12) and indicate that Pyr-Va is identical to the native cofactor with respect to its biological activity. Fluorescent Properties of Pyr- Vu-The uncorrected fluorescence excitation spectrum of Pyr-Va obtained in the presence or absence of PCPs is illustrated in Fig. 2. The spectrum is characteried by absorbance maxima centered at 33 nm and at 347 nm, characteristic of pyrene (43), with a large absorbance maximum at 28 nm reflecting extensive energy transfer between aromatic amino acid side chains and the pyrene moiety. The emission spectrum (not shown) was characteried by maxima at 375,395, and 415 nm with no evidence for excited-state complex formation at 47 nm. As pyrene is a highly environmentally sensitive fluorophore (43), the lack of influence of PCPs on the fluorescence spectra indicates that the site of modification is not detectably perturbed by the association of Pyr-Va with PCPs. The dependence of fluorescence polariation of Pyr-Va in the presence or absence of PCPs on excitation wavelength is also illustrated in Fig. 2. True polariation spectra under vitrified conditions could not be obtained due to the possible effect of ethylene glycol or other additives on vesicle integrity or the possible interference with interaction between Pyr-Va and PCPs. The spectra illustrated in Fig. 2 indicate that the addition of saturating concentrations of PCPs resulted in a significant increase in fluorescence polariation in a wavelength-dependent fashion with a maximal increase (approximately.48) centered at 1 1 26 28 3 3236 34 WAVELENGTH <nm>. 4. 3.2. 1 -. 1 -. 2 r T c n m V n m n m D r w n U N w U

Binding of Factor Va to Phospholipid Vesicles 5719 33 nm. On the basis of these observations, all polariation titrations were performed using an excitation wavelength of 33 nm. As polariation values between 32 and 36 nm (Fig. 2) are significantly higher than those expected for a long-lifetime probe such as pyrene, the fluorescence properties of Pyr-Va were further characteried to provide greater assurance regarding the properties of the fluorescent species. Perrin plots of Pyr-Va and the Pyr-Va. PCPs complex are illustrated in Fig. 3. The extrapolated values of Po for Pyr-Va alone and for the Pyr-Va. PCPs complex were.268 and.31 respectively, well within the range of values observed with other proteins modified with N-(1-pyrene)maleimide (43-45). The difference in the extrapolated Po values of Pyr-Va in the presence and absence of PCPs, indicates that the probe exhibits decreased segmental mobility when modified Va is bound to the phospholipid surface. Determination of the fluorescence excited-state lifetime of Pyr-Va by multifrequency phase modulation spectroscopy (data not shown) indicated the presence of three lifetime components with the following lifetimes and fractional amplitudes: 129 ns,.138; 9 ns,.67; 1 ns,.127. The observed lifetimes are consistent with those measured for other proteins modified with N-( 1-pyrene)maleimide (43-45). Furthermore, the preponderance of the 9-ns lifetime component is consistent with the observed polariation values of Pyr-Va. The Binding of Pyr- Vu to Phospholipid Vesicles-Titration curves obtained using fixed concentrations of Pyr-Va and varying concentrations of PCPs are illustrated in Fig. 4. Fluorescence polariation increased saturably with increasing concentrations of PCPs, from a value of.218 in the absence of PCPs to a limiting value of.267 at saturating concentrations of phospholipids. The distinct saturation of the titration curves illustrated in this figure rules out the possibility of artifactual increases in polariation due to increased light scattering at high lipid concentrations. The lines in Fig. 4 are drawn according to the fitted constants that are presented in Table 11. The observed dissociation constant is significantly different from previously reported values for this interaction (16-21) but specifically pertains to the temperature and ph of the present experimental conditions. The stoichiometry of the Pyr-Va PCPs interaction is within the range previously observed (16,18), and could be dictated by packing constraints imposed by vesicle surface area and/or a discrete site requirement for the binding of factor Va to the vesicle surface. 4.7 L 1 3.9 v- a > 3.7 1 " 3.5 3. 3 3. 1 17 2 23 26 29 32 35 38 41 44 T/n (K/cp) FG. 3. Perrin plots for Pyr-Va and the Pyr-VaaPCPS com- plex. Reaction mixtures contained 2 X lo-' M Pyr-Va in 2 mm Hepes,.15 M NaC1, 2. mm CaC2, ph 7.4, with no additions () or with 2 p~ PCPs (). Temperaturewasvaried between 3.7 and 33.1 "C and the lines are drawn according to the Perrin equation with fitted values of Po =.268 (Pyr-Va) and Po =.31 (Pyr-Va. PCPs.) J.27 L J.21 1 2 4 6 8 1 12 14 16 CPCPS (pm) FG. 4. Titration of Pyr-Va with PCPs. Reactionmixtures M () Pyr-Va in 2 mm contained 8.5 X lo-* M () or 1.7 X Hepes,.15 M NaC1, 2. mm CaCl,, ph 7.4. Fluorescence polariation measurements were made as described under "Experimental Procedures"afterincremental additions of PCPs. The lines aredrawn according to Equation 1, with fitted constants of & = 2.72 X lo-' f.47 X lo-' M and a stoichiometry of 41.9 f.5 mol of PCPs bound permol of Pyr-Va at saturation. The concentration of PCPs is expressed as the concentration of monomeric phospholipids. TABLE 1 Dependence of the Pyr- Va-PCPs interaction on vesicle composition Equilibrium constants were calculated by nonlinear least squares fitting to Equation 1 using titration curves obtained at two fixed concentrations of Pyr-Va. h9.e.' 1. NDf 15 45 f 6.954 4.77 f 1.44 72.4 f 2.2 1.9 25 41 f 2 1.1 2.72 f.47 41.9 f.5 1.5 3 48 f 2.975 3.44 f.38 35.7 f.3 1.7 4 396 f 2 1. 6.59 f 2.1 18.4 f.7 7.4 PCPs vesicles with the indicated PS composition. Ar = rma. - ro FJF, = ratio of fluorescence intensity at saturating PCPs to fluorescence intensity in the absence of PCPs. Moles of total PS per mole of Pyr-Va at saturation. e Moles of total phospholipid per mole of Pyr-Va at saturation. ND, not determined. Assuming packing constraints contribute little to the saturation of the vesicle surface and that approximately 6% of the phospholipids are exposed on the vesicle surface (18), the lipid-combining site for factor Va would comprise approximately 26 monomeric phospholipd (PC + PS) molecules. Reversibility of the Pyr- Vu. PCPs nteraction-the reversibility of the lipid-binding measured by polariation was examined by preforming the Pyr-Va- PCPs binary complex and systematically varying the concentration of unmodified factor Va. Titration curves obtained using 1.5 X M Pyr-Va, 8 or 16 p~ PCPs, and variable concentrations of factor Va are illustrated in Fig. 5. n both cases polariation decreased saturably with increasing Va concentrations, indicating that unmodified Va was capable of displacing Pyr-Va bound to PCPs. The lines shown in the figure were drawn according to Equation 7 (see "Data Analysis") with the fitted values for the dissociation constants listed in Table 111. These values indicate that the dissociation constants describing the separate interactions of Pyr-Va with PCPs and Va with PCPs are essentially identical. The fitted dissociation constant for the Pyr-Va-PCPs interaction obtained from this experiment is indistinguishable from the value independently obtained from titration experiments illustrated in Fig. 4, further vali-

572 Binding of Factor Vu to Phospholipid Vesicles.27 Z 2.26 c.25 E <.24 a. 23.22 1. 2. 3. 4. 5. 6. 7. 8. CV~ x17 M FG. 5. Displacement of PCPs-bound Pyr-Va by unmodified factor Va. The reaction mixtures contained 1.7 X lo" M Pyr-Va in 2 mm Hepes,.15 M NaCl, 2. mm CaCl,, ph 7.4, and 8 p~ () or 16 p~ () PCPs. Fluorescence polariation measurements were made after the incremental addition of unmodified factor Va to the reaction mixtures. The lines are drawn according to Equation 7 assuming 42 mol of PCPs combined per mol of protein at saturation, using the fitted constants listed in Table 111. TABLE 11 Dissociation constants obtained from competition experiments Dissociation constants were obtained by fitting data illustrated in Fies. 7 and 8 to Eauations 6 and 7. Competitor Measured interaction Kd * x in8 M Factor Va Component E Component Ex. Ex.-3 Pyr-Va + PCPs Va + PCPs Pyr-Va + PCPs Component E + PCPs Pyr-Va + PCPs Ex. + PCPs Pyr-Va + PCPs Ex.-3 + PCPs Standard error of the determination. 2.71 f.48 2.51 f.44 2.78 k.71 1.93 f.49 1.74 f 1.22 1.17 f.81 3.18 f 1.15 1.55 f.55 dating the assumptions used to derive Equation 7. The data indicate that the Pyr-Va-PCPS interaction is reversible and is quantitatively indistinguishable from the interaction between unmodified Va and the same vesicles. The nfluence of Phospholipid Composition on the Pyr- Va- PCPs nteraction-previous studies of the influence of vesicle composition on the binding of factor V to phospholipids studied by light scattering indicated an absolute requirement for negatively charged phospholipids (16, 21). f the Pyr-Va. PCPs interaction was purely determined by the availability of PS molecules, a systematic variation of the PS content of the vesicles would result in a change in the number of molecules of Pyr-Va bound per vesicle with no change in the affinity for the interaction. Conversely, if the Pyr-Va combining site on the vesicle surface resulted from contributions from both populations of phospholipids, one might expect a systematic change in affinity and stoichiometry as the PS content of the vesicles was systematically varied. To test these predictions, vesicles of varying PS content were used to determine their ability to support Pyr-Va binding. The equilibrium constants obtained from binding studies performed using two fixed concentrations of Pyr-Va (7.5 X and 1.5 X M) and variable concentrations of PCPs of the indicated composition are listed in Table 11. Vesicles composed of 1% phosphatidylcholine (PC) were unable to support Pyr- Va binding with no detectable increase in fluorescence polar- iation at concentrations of PC as high as 9 pm. Fitted constants from titration curves obtained using vesicles com- posed of 15,25,3, and 4% PS (Table 11) indicated that the limiting fluorescence anisotropy change at saturating lipid was independent of the PS composition of the vesicles. The dissociation constant for the Pyr-Va-phospholipid interaction was not significantly influenced by this variation in PS concentration, while the number of moles of phospholipid bound per mole of Pyr-Va at saturation decreased with the increase in PS composition. Assuming that the variation in the PS composition of the vesicles between 15 and 4% does not grossly alter vesiclesie or the overall distribution of the phospholipids (18), the data indicate that the PS composition dictates the number of Pyr-Va combining sites on the vesicle surface without significantly altering the affinity of these sites for the cofactor. The invariance in the number of moles of PS combining per mole of Pyr-Va at saturation (Table 11) further suggests that the combining site on the vesicle surface may be composed of a discrete number of PS molecules. nfluence of Calcium ons on the Binding of Pyr-Va to PCPs-Previous studies of the phospholipid binding properties of factors V and Va, indicated that these interactions did not require the presence of exogenously added calcium ions (16). The titration curves illustrated in Fig. 6 were obtained using identical concentrations of Pyr-Va with vari- able concentrations of PCPs in the presence of 2. mm CaC12 or in the absence of added calcium ion with the addition of 1 p~ EGTA. EGTA was included in the second case to deplete the low concentration (approximately 12.5 p ~ of ) free calcium carried over into the reaction mixture from the Pyr- Va stock solution. The line in the figure, drawn according to the constants obtained from binding studies conducted in the presence of 2. mm CaC12, adequately describes the interaction of Pyr-Va with PCPs at low micromolar concentrations of calcium ion. The equilibrium constants for the Pyr-Va-PCPS interaction are therefore not influenced by changes in the calcium ion concentration over the range described above. Factor V possesses two types of binding sites for calcium ions with dissociation constants of lo-' and M, with the tightly bound metal ion contributing to the functional and structural integrity of the cofactor (46). The observations described above and those made in other studies indicate that loosely bound calcium ions contribute little if anything to the ability of the cofactor to bind to phospholipid vesicles. The addition of high concentrations (>2 pm) of EDTA or EGTA to reaction mixtures containing PCPS-bound Pyr-Va in the absence of exogenously added calcium ion, resulted in.27 L ' J.21 2 4 6 E 1 12 14 16 CPCPS (pm) FG. 6. Effect of calcium ion on the Pyr-Va-PCPs interaction. Reaction mixtures contained 1.7 X 1" M Pyr-Va in 2 mm Hepes,.15 M NaCl, ph 7.4, with 2. mm CaC12 () or 1 p~ EGTA (). Fluorescence polariation was recorded after incremental additions of PCPs. The line is drawn according to Equation 1 using the fitted constants obtained for the data illustrated in Fig. 4.

Binding of Factor Va to Phospholipid Vesicles 5721 a rapid decrease of the polariation signal to values lower than that observed for Pyr-Va alone, indicating that high concentrations of chelators could disrupt the reaction as detected by this fluorescent probe. As the pyrene moiety is present in non-lipid-binding component D, the disruption of the polariation signal by high concentrations of EDTA probably results from the dissociation of component D from lipidbound component E (18,47). Displacement of PCPs-bound Pyr-Va by solated Subunits of the Cofactor-Competition experiments indicated that isolated component D (heavy chain) was unable to reduce the polariation signal associated with the Pyr-Va. PCPs complex, consistent with the interpretation that this subunit contains little or no lipid-binding capability. Displacement curves obtained by systematically varying the concentration of isolated component E (light chain) are illustrated in Fig. 7, and the fitted equilibrium constants are listed in Table 111. The dissociation constant calculated for the interaction between component E and PCPs is essentially indistinguishable from the values obtained for Pyr-Va or factor Va, indicating that this subunit competes with factor Va for lipid-binding sites with identity. Taken together, these observations confirm previous conclusions (18) and indicate that component E is quantitatively responsible for the phospholipid binding properties of the cofactor. Displacement of PCPs-bound Pyr-Va by Component Exa and Deriuatiue Peptides-solated component E is cleaved by activated protein C, factor Xa, and by plasmin into the NH2- terminal-derived Exa-3 peptide (M, = 3,) and the COOH-terminal derived Ex.-48 peptide (M, = 48,) which remain tightly associated (27, 28). The phospholipid binding properties of component Ex. were examined in competition studies to evaluate the consequences of cleavage of component E on its ability to bind PCPs. Displacement curves obtained using 1.5 X M Pyr-Va, 8 or 16 FM PCPs, and variable concentrations of Ex* are illustrated in Fig. 8A with the dissociation constants listed in Table 111. The data indicate that cleavage of component E by factor Xa has no detectable effect on the ability of this subunit to bind to the vesicle surface. The curves illustrated in Fig. 8B were obtained using the purified Ex.-3 peptide was the varied competitor. The dissociation constants obtained for this peptide (Table 111) indicate that essentially all the lipid-binding properties of component E can be accounted for by the lipid-binding capacity of this amino-terminal portion of the subunit. Similar experiments using purified Exa-48 could not be undertaken.28 1 ' 4.27 E.26 L \ a,.23 -.22 1. 2. 3. 4. 5. 6. 7. 8. 9. CCOMPONENT E (x 1' M) FG. 7. Displacement of PCPs-bound Pyr-Va by isolated component E. Experimental conditions were identical to those described for Fig. 5, except that isolated component E was the varied species. The lines are drawn using the fitted constants listed in Table 111..26 c < E.25 < d.24 a.23 1 2 3 4 5 6 7 8 9 [E,, -3kl (x 1 O7 M) FG. 8. Competitive displacement of PCPs-bound Pyr-Va by component Ex. and the isolated peptide Ex.-3. Reaction mixtures contained 1.5 X M Pyr-Va in 2 mm Hepes,.15 M NaCl, 2. mm CaCl,, ph 7.4, and 8. () or 16. pm () PCPs with variable concentrations of component EX. (A) or 8. () or 16. pm () PCPs with variable concentrations of Ex.-3 (B). The lines are drawn according to the fitted values listed in Table 111. because all attempts to remove acetonitrile from the peptide preparations resulted in irreversible insolubility. The data cannot be used to discount participation of the COOH-terminal-derived half of component E (Ex,-48) in the lipid- binding process but are consistent with the interpretation that the Ex.-3 peptide contributes significantly to the lipidbinding properties of component E. Effect of Factor Xa on the Phospholipid-binding Properties of Pyr-Va-Since the assembly of the prothrombinase complex requires the interaction between factor Xa and factor Va on the phospholipid surface, experiments were undertaken to assess the effect of factor Xa on the interaction between Pyr- Va and PCPs. Factor Xa was pretreated with the active-sitedirected inhibitor isoleucylglutamylglycylarginyl chloromethyl ketone to prevent proteolysis of Pyr-Va during the course of the experiment. Factor Xa modified by this reagent competes with identity for the interactions between unmodified Xa, Va, and PCPs (data not shown). Titration curves similar to those illustrated in Fig. 4 but obtained in the presence of 4.5 x 1" M Xa (data not shown) yielded fitted values of Kd = 4.32 X lo-' M f 1.43 X lo-' and 53 * 2.5 mol of PCPs bound per mole of Pyr-Va at saturation. These data indicate that the presence of factor Xa has no detectable effect on the affinity of Pyr-Va for PCPs. Furthermore, the presence of factor Xa had no effect on the polariation values in the absence of PCPs or at saturating concentrations of PCPs, indicating that factor Xa does not perturb the fluorophore on the PCPs surface. f the fluorophore could report a direct solution-phase interaction between factor Xa and Pyr-

5722 Binding of Factor Va to Phospholipid Vesicles Va, the present data indicate that the dissociation constant for this interaction would be significantly greater than 1-~ M. By using the previously measured dissociation constant for the Xa-PCPS interaction (ll), the measured dissociation constant for the Pyr-Va-PCPS interaction, the measured overall apparent dissociation constant for prothrombinase assembly (see above), and th expressions described by Jencks (48), it is possible to calculate a Gibbs connection energy (AG") of -6.5 kcal. mol" for the interaction between factor Xa and factor Va on the phospholipid surface. This low value for AG" raises two possibilities: 1) Factor Xa is not associated with phospholipid in the ternary complex; or 2) a significant decrease in entropy accompanies the assembly of the prothrombinase complex. The loss in entropy could reflect substantial conformational changes in one or both of the proteins that may be required for their assembly on the vesicle surface. DSCUSSON The approaches described in this paper represent an extension of the initial workof saacs et al. (22) who exploited fluorescently modified Va to obtain distance measurements between the site of modification in lipid-bound Va and the phospholipid surface. n this study, a readily accessible thiol was identified in component D of factor Va and was selectively modified with N-(1-pyrene)maleimide. The increase in fluorescence polariation of Pyr-Va in the presence of PCPs was utilied to obtain quantitative information regarding the interaction between Va and the vesicle surface. Pyr-Va is reversibly bound to PCPs with a & = 2.7 X lo-' M to a site composed of a discrete number of PS molecules. The results of competition studies provide additional support for available data indicating that the binding ofva to phospholipids is mediated by component E of the cofactor. Cleavage of component E (or factor Va) by factor Xa did not alter the lipid- protein interaction and further experiments implicated the amino-terminal portion (Ex.-3) of this subunit in the PCPSbinding process. The dissociation constants obtained in this study for the lipid-binding properties of factor Va and derived peptides are significantly different from values previously reported by this laboratory (approximately M) (16, 18) and several other groups (lo-' M (21) and lo-" M (17, 2)). n light of our rigorous demonstration that Pyr-Va is indistinguishable from unmodified Va, the most likely explanation for these discrepancies lie in the methodologies used for the various binding studies. Binding measurements utiliing light scattering have been criticied by Pusey et al. (17) because of problems arising from less than quantitative binding of added protein to the membrane. The light scattering techniques developed by Nelsestuen and Lim (49) further assume that the dn/dc of the protein-lipid complex is equal to the weighted average of the dn/dc values for the protein and phospholipid. Reversibility of the protein-lipid interaction cannot be demonstrated by light scattering, therefore preventing the technique from distinguishing between protein-lipid interactions and secondary protein-protein aggregation phenomena. The dissociation constant for the Va-phospholipid interaction reported by Pusey et al. (lo-" M) (17) was obtained by kinetic studies of the rates of association and dissociation obtained by two independent techniques. Association rates were obtained by stopped-flow light scattering and dissociation rates were obtained by energy transfer techniques between Va and dansylated phospholipids. The result of both independent techniques was not verified by measurements at equilibrium and required substantial assumptions that could have contributed to the extremely low dissociation constant reported in that study. Stopped-flow kinetic studies of the Va-PCPs interaction undertaken in our laboratory (5) have permitted a calculation of the dissociation constant on the basis of rate data (data not shown) which is within 3% of the values determined in the present work. A Kd of lo-' M was reported by van de Waart et al. (21) for the interaction between Va and large volume vesicles using nonequilibrium sedimentation techniques to separate and quantitate free Va and lipid-bound protein. These studies assumed that the binding of Va to phospholipids was independent of vesicle sie and that labeling factor Va with 1251 did not alter its lipid-binding properties. These workers also reported a dependence of the dissociation constant for the Va-lipid interaction on the concentration of calcium ion, which could have resulted from calcium-induced vesicle aggregation. The dissociation constants described in the present study were obtained by equilibrium binding measurements using well described techniques that eliminate the need for any nonverifiable assumptions. The properties of Pyr-Va were rigorously characteried to reduce the likelihood of erroneous conclusions due to the unforseen behavior of the modified protein and the equilibrium constants reported are internally consistent with ongoing studies in this laboratory. The interaction between factor Va and platelets is characteried by two types of binding sites with dissociation constants of 1"O and lo-' M (23). Based on the similarities between these values and the dissociation constants obtained for the Va-PCPs interaction, it is conceivable that phospholipids contribute significantly to one or both classes of Va binding sites on the platelet surface. Recently available amino acid sequence data for human factor V inferred from the cdna sequence (51) have led to speculation that the carboxyl terminus of component E, which shares homology with the phospholipid-binding protein discoidin, is involved in phospholipid binding. The observation that the peptide derived from the amino terminus of component E (Ex.-3) is capable of displacing phospholipid-bound Pyr-Va is therefore somewhat surprising. The present data cannot be used as a basis for discounting any lipid-binding activity of the Ex.-48 peptide but are consistent with results of other studies (52) that have indicated that only a small domain of component E is associated with the lipid bilayer. The results obtained with vesicles containing varying amounts of PS are consistent with most other studies but contradict the results of van de Waart et al. who found that varying PS composition of their vesicles led to a systematic change in the dissociation constant for Va binding with very little change in the stoichiometry for the reaction (21). The interpretation that the Va combining site on the vesicle surface may be composed of a discrete number of PS molecules raises the possibility that the binding event may result in the enrichment of PS molecules in the vicinity of factor Va. The enrichment of negatively charged head groups in the vicinity of lipid-bound prothrombin has been reported (53). The requirement for PS to support the Va-PCPS interaction raises the possibility of a significant contribution due to electrostatic interactions between the protein and the phospholipid surface. Substantial evidence indicates that hydrophobic interactions are involved in the binding of factors V and Va to PCPs (18, 54). However, the literature contains conflicting reports regarding the effects of ionic strength on the factor Va-PCPS interaction (18, 2, 21). Attempts to examine the ionic strength dependence of the Pyr-Va-PCPS interaction were unsuccessful, due to the extensive decrease in fluores-

cence intensity of the pyrene probe at high ionic strengths. The independence of the Pyr-Va-PCPs interaction on exogenously added calcium ions is consistent with the results of previous studies of the factor V-phospholipid interaction (16, 18) and with the results obtained with activated protein C (15). n the latter case, fluorescence studies indicated that the activatedprotein C-factor V-PCPs complex was not disrupted by the addition of EDTA to chelate exogenously added calcium ions. The work described in this paper has permitted the development of a relatively well defined fluorescent probe for factor Va and has led to the determination of the quantitative aspects of the interaction between factor Va and PCPs. The equilibrium constants determined by this approach suffer from few of the problems that have presumably influenced the results of previous studies and have provided information that is necessary and invaluable to further modeling of the assembly of the prothrombinase complex. Acknowledgments-We are grateful to Dr. Pete Lollar for his assistance in the determination of the extinction coefficients reported in this study, for his assistance with some of the computer programs, and for useful discussions. We are also grateful to Dr. Franklyn G. Prendergast of the Mayo Clinic for graciously permitting the use of his multifrequency phase-modulated fluorescence spectrophotometer. Note Added n Proof-Analysis of the free sulfiydryl content of factor Va denatured with sodium dodecyl sulfate rather than 4. M guanidine hydrochloride indicates that each subunit of the cofactor contains two free thiols. 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