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Binding characteristics of homologous plasma lipoproteins to human platelets
[32]
PLATELET
RECEPTOR FOR LIPOPROTEINS
383
[32] B i n d i n g C h a r a c t e r i s t i c s o f H o m o l o g o u s P l a s m a L i p o p r o t e i n s to H u m a n P l a t e l e t s
By ELISAaETH KOLLER and FRANZ KOLLER Assay Methods
Principle Human plasma lipoproteins of the high-density class (HDL) and the low-density class (LDL) show fast and saturable binding to human platelets. The parameters of these interactions are determined applying lipoproteins labeled covalently in their protein moieties (either with 125I o r with fluorescent dyes).
Buffers Buffer A: Tyrode's solution without Ca z+ (NaCI 137 raM, KC1 2.7 raM, NaHCO3 11.9 mM, MgCl 2 1.0 mM, NaH2PO 4 0.42 mM, D-glucose 5.5 raM, human serum albumin 3.5 g/liter, pH 6.5). To improve the pH stability, 5.0 mM HEPES was added in all experiments requiring longer incubation periods Buffer B: As above, with addition of 100 t~g/ml apyrase (Sigma, St. Louis, MO) and 10 units heparin/ml Buffer C: Buffer A, containing 2 mM CaCIz, pH adjusted to 7.35 Buffer L: Buffer C, without albumin and glucose
Procedures Preparation of Washed Human Platelets. Platelet-rich plasma (PRP) is obtained from ACD-blood of healthy donors immediately after drawing by sedimentation at 120 g at room temperature for 20 min. After addition of apyrase (50 p~g/ml) platelets are isolated either by gel filtration on Sepharose 2B 1 or by repeated centrifugations. 2 The final suspension medium in both cases is buffer C. i O. Tangen, H. J. Berman, and P. Marfey, Thromb. Diath. Haemorrh. 25, 268 (1971). 2 j. F. Mustard, D. W. Perry, N. G. Ardlie, and M. A. Packfiam, Br. J. Haematol. 22, 193 (1972). Briefly, PRP is spun twice for 5 min at 800 g in small tubes (5 ml) at room temperature. The combined pellets are resuspended in buffer B at 37 °. The same sequence of centrifugation is repeated three times, first in identical manner, then with resuspension in buffer A, and finally in buffer C.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
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Preparation o f Lipoproteins. The supernatant of the initial 800 g sedimentation of PRP, platelet poor plasma (PPP), is spun sequentially at 100,000 g and 5° following stepwise adjustment of required density values by addition of solid KBr. Prior to each flotation step the residual plasma is overlayered with KBr solutions of appropriate density to remove plasma proteins adhering to the lipoprotein fraction. The mimimal times of separation are 16 hr for very low-density lipoprotein (VLDL) floated at d = 1.006, 20 hr for LDL isolated at d = 1.006 to 1.063, and at least 36 hr each for HDL2 at d = 1.063 to 1.125 and for HDL 3 at d = 1.125 to 1.21. Finally, lipoproteins are dialyzed exhaustively against buffer L and passed through filters of 0.45-/zm pore size. The composition of each preparation is examined by determination of all components and compared with average literature values. 3 Based on this known composition, determinations of protein concentration routinely are sufficient to calculate lipoprotein concentrations. 1251 Labeling o f Lipoproteins. The method of Salacinski et al., 4 using insoluble 1,3,4,6-tetrachloro-3a,6a-diphenylglycolurii (Iodogen), is applied. Iodogen (80 tzg in 50 tzl of dry CH2CI 2) is transferred to a Beckman (Palo Alto, CA) microfuge vial and the solvent removed by aspiration with dry nitrogen. Two microliters of Na125I (3.7 MBq/ml; 62.9 MBq//zg) is added, immediately followed by addition of 200/xl of lipoprotein solution containing about 1 mg protein/ml. The reaction is allowed to proceed for 10 to 15 min at 5°. The liquid phase is then transferred to another vial containing 20/zl of 2 M aqueous KI and 800 /xl of buffer L. After mixing, the content of this vial is applied to a 10-ml column of Sephadex G-25 fine, equilibrated in the same buffer, and fractionated at room temperature, collecting l0 drops per fraction. Radioactively labeled lipoprotein appears as a very narrow peak without detectable contamination with lower molecular weight material. The radioactivity incorporated typically is about 40 kBq//zg of protein. More than 90% of the label introduced by this procedure is precipitable by trichloroacetic acid in all lipoprotein density classes. The portion of the total label associated with the lipid moiety was found to be -<8% (HDL3) by delipidation following Folch et al. 5 Fluorescence Labeling of Lipoproteins. Standard procedures are applied to achieve covalent modification of different lipoprotein fractions 3 S. Eisenberg, Ann. N. Y. Acad. Sci. 348, 30 (1980); J. C. Osborne, Jr., and H. B. Brewer, Jr., Adv. Prot. Chem. 31, 253 (1977). 4 p. R. P. Salacinski, C. H. McLean, J. E. C. Sykes, V. V. Clement-Jones, and P. J. Lowry, Anal. Biochem. 117, 139 (1981). 5 j. Folch, M. Lees, and G. H. S. Stanley, J. Biol. Chem. 226, 497 (1957).
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PLATELET RECEPTOR FOR LIPOPROTE|NS
385
with fluorescein isothiocyanate (FITC) at pH 8.5, and with N-iodoacetylN'-(S-sulfo- 1-naphthyl)ethylenediamine (1,5-I-AEDANS) at pH 6.5.6 Purification of the labeled lipoprotein is achieved by centrifugation and repeated gel filtration at high and low salt concentrations, finally with buffer L. The amounts of covalently attached dyes are calculated spectroscopically using/3495 37,500 for FITC, and e350 = 6600 for 1,5-I-AEDANS, respectively. 7 Radioactive assay method: A series of incubations in conical microfuge tubes is carried out at 37° for 15 min. Each vial contains 0.2 ml of washed platelets suspended in buffer C (7-30 x 108 cells/ml), varying amounts of labeled lipoprotein, and buffer L up to a total volume of 0.5 ml. To determine the amount of unspecific, nonsaturable binding, otherwise identical incubations were performed with the addition of unlabeled lipoprotein. The amounts of added unlabeled lipoprotein were sufficient to yield total lipoprotein concentrations at least 100 times higher than those required for half-maximal saturation of the highaffinity sites. After the desired incubation times the incubation mixtures are layered on top of 0.5 ml 20% sucrose solution in buffer L and spun in a Beckman Microfuge at 8740 x g for 45 sec. The tips of the tubes containing the platelets are amputated after careful aspiration of the supernatant and radioactivity is determined in both tips and supernatants. Almost identical results were obtained with the following procedure formerly in use, which, however, is not fast enough to be applied to large series of determinations. 8 The platelets are sedimented by centrifugation of the incubation mixtures (8740 x g, 45 sec). The pellets are resuspended three times in 150 tzl of ice-cold buffer C by repeated agitation of the medium against the platelet sediment by use of an appropriately sized plastic tip attached to a hand-operated automatic pipette and immediate centrifugation for 30 sec, the whole washing procedure being accomplished within less than 5 min. Finally the platelet pellet is resuspended in another 150 tzl of buffer C and transferred to a new plastic tube. Radioactivity in the latter as well as in the original supernatant solution and in the three washing solutions is determined in a 3' counter. The concentration of the fraction of lipoprotein remaining unbound at equilibrium is derived from the sum of counts of all supernatant solutions; =
6 K. G. Mann and W. W. Fish, this series, Vol. 26, p. 28; E. N. Hudson and G. Weber, Biochemistry 12, 4154 (1973). 7 C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry." Freeman, New York, 1980. 8 E. Koller, F. Koller, and W. Doleschel, Hoppe-Seyler's Z. Physiol. Chem. 363,395 (1982).
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PLATELET RECEPTORS: ASSAYS AND PURIFICATION
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the washed platelet suspension yields the bound fraction of radioactive ligand. At low total lipoprotein concentrations (final concentrations in the vials in the nanomolar range) at least triplicate determinations should be performed; at micromolar total concentrations of ligand two series of experiments will be sufficient. Fluorescence method: To minimize disturbation by scattering, all determinations of fluorescence intensities are performed with plane polarized light. The excitation bandpass should be adjusted to less than 4 nm, that in the emitted light beam to about 5 nm. The cell holder should be thermostarted to about 25 °. The fluorescence intensities (either indicated directly or calculated as I = ~1 + 21±o-) are determined at 350/475 nm when working with 1,5-I-AEDANS-labeled lipoproteins and at 495/520 nm for FITC-modified ligands. Stock solutions of labeled lipoprotein are prepared by dilution with buffer L. Undiluted lipoprotein together with dilutions to one-third and one-fifth usually will suffice to obtain a complete binding isotherm. Platelet suspension (0.1 ml; 10 to 20 x I08 cells/ml) is then pipetted into a 1 x I cm standard fluorescence cuvette and 1.9 ml of buffer L is added. After mixing and determination of the background fluorescence intensity the chromophore-labeled lipoprotein is added stepwise (in portions of I0 or 20 td) to this suspension. After each addition the system is allowed to equilibrate (with occasional mixing) for 15 min at 25°, 9 then the fluorescence intensities are determined as above. Five (or 10) consecutive additions of lipoprotein are recommended for each of the 3 prepared stock solutions. The same sequence of additions of ligand is repeated with mixtures containing (I) 50/~1 of the original platelet suspension per 2 ml, (2) 200 /~1/2 ml, and (3) no platelets at all, otherwise proceeding in an identical manner. All measured fluorescence intensities are corrected for inner filter effect and scattering. The following procedure is both simple and reliable: A high-purity, reasonably water-soluble fluorescence standard with high quantum yield in aqueous solution and a sufficiently large absorption coefficient in the spectral range of interest is appropriately diluted with buffer L and fluorescence is determined under the conditions described above. The same is done for equally concentrated solutions of this standard chromophore, but in the presence of 0.05, 0.1, and 0.2 ml of the platelet suspension under investigation. Comparison of these 9 Fifteen minutes might be too short to allow equilibration with very low concentrations of ligand. In these rare cases no further increase in fluorescence intensities was detectable after incubation periods of 30 min.
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PLATELET RECEPTOR FOR LIPOPROTEINS
387
relative fluorescence intensities yields empirical factors correcting for optical effects related to the platelets in suspension. Effects associated with the added lipoprotein normally are negligible, since the latter will add very little to the total absorption at the wavelength of excitation. Possible complications due to interactions of the standard dye with platelets can be excluded by measuring at two temperatures separated by about 10 to 15°. In the absence of interactions the correction factors for optical effects should show no detectable temperature dependence.I° The (corrected) values of relative fluorescence intensity are increased in the presence of human platelets when compared with the series in plain buffer. The reciprocals of these changes of fluorescence intensity (l/A/) are plotted against the reciprocal concentration of lipoprotein as shown in Fig. 1. Slightly curved binding isotherms are obtained, which approach linearity with decreasing concentration of platelets. The left part of the plot can be linearly extrapolated to infinite ligand concentration with proper accuracy. Based on the assumption of kinetically indistinguishable receptor sites the intercept on the vertical axis is 1/nPb, that with the abscissa corresponds to K. P represents the concentration of platelets (in molarities, treating platelets as molecules), n is the average number of receptor sites per single platelet, K stands for the thermodynamic association constant for binding to the receptors, and b is the incremental change of fluorescence intensity of the average chromophore molecule on binding, depending on instrumental parameters and to some extent on the degree of lipoprotein labeling. This value must be known to get access to the number of sites/platelets, which can be achieved without any additional measurement according to Fig. 2. For different (preferentially low) concentrations of total lipoprotein (T) kept constant, the corresponding values of T/A1 are plotted against the reciprocals of platelet concentration. Extrapolation of these more or less linear plots to the ordinate should yield a common intercept, its numerical value being 1/b. Again the accuracy of the individual determinations is crucial for L0The deviation of the apparent fluorescence intensities observed in the presence of platelets from the corrected values is less pronounced than might be anticipated. As typical examples from our experiments the fluorescence intensities measured at 520 nm had to be corrected by factors of 0.979 and 0.919 for 100 and 300 t~l of original platelet suspension (16.2 × 108/ml), respectively. We used lysine derivatives of both 1,5-I-AEDANS and FITC as standard dyes to determine these values (adding 40 ~1 of a solution in buffer L, containing about 5 mg of dye/ml). Reaction of either fluorescent derivative with the platelet plasma membrane proceeds only very slowly (if at all) and can easily be corrected for by extrapolating back to mixing time.
388
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[32]
5O
3O
I
0.5 1.0 1.5 ( Concenfrn'rion of LDL)-I
2.0xl 0 9
FIG. 1. Double-reciprocal plot illustrating L D L binding to blood platelets at 37 °. Lowdensity lipoprotein was labeled with FITC (44: 1), binding leading to increased fluorescence at 495/520 nm. The final concentrations of platelets were 0.425 (~I,), 0.85 (IlL 1.7 ( * ) , 2.95 (T), and 4.25 × 108 cells/ml ((3). Error bars are shown for the most diluted series.
the reliability of the final results. Therefore the reference series (in the absence of platelets) should be repeated twice. The experiments in the presence of platelets should be performed in duplicate. Quite similar results have been obtained with the two types of chromophores described. The use of FITC is to be preferred, however, because of its markedly higher fluorescence yield and its absorption at longer wavelengths. Unnecessary illumination of samples should be avoided. The serum albumin included in the platelet suspension buffer systems should be of the highest available degree of purity (delipidated if possible) to minimize background emission close to the wavelengths applied in this assay. Partial peroxidation of lipoprotein lipids can produce similar complications. Although several methods are known to inhibit lipid peroxidation, the best means to avoid this interference is to use only freshly prepared lipoproteins, since plasma lipoproteins in any case in vitro show a distinct tendency to change structure, size, and composition with time.
[32]
PLATELET
RECEPTOR FOR LIPOPROTEINS
389
~c~ 5 x
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I
I
I
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I
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FIG. 2. Extrapolation to infinite platelet concentration of the reciprocal changes of fluorescence (495/520 nm) of FITC-labeled LDL. The individual series were determined at constant total concentrations of LDL (LDLt): 0.35 (©), 0.52 (*), 1.04 (A), 3.1 (11), and 6.7 × 10 - 9 M (A). The data are obtained from the series of experiments illustrated in Fig. 1.
General Considerations and Problems in Receptor Binding Studies Involving I_,ipoproteins
Both size and surface structure make plasma lipoproteins (especially of the lower b u o y a n t density classes) highly adherent to most c o m m o n laboratory equipment materials, including glass and various types of plasticware. Our attempts to find any combination of material and p r e t r e a t m e n t virtually excluding nonspecific adhesion were unsuccessful. Binding data generally are best obtained by determination of the a m o u n t of free (unbound) ligand, calculating the bound fraction as the difference b e t w e e n this concentration and the k n o w n total ligand concentration. In the case of plasma lipoproteins (especially at low degrees of r e c e p t o r saturation, i.e., at low ligand concentration), this difference represents not only ligand bound to the platelets but also
390
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ASSAYS
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Time (rain) Flo. 3. (a) Time dependence of binding of ]25I-labeled L D L to blood platelets. 1. Incubation of 4.5 × 108 platelets/ml with 1.9 x 10 7 M LDL. The solid line is calculated with kass = 6.3 x 104 M - i sec -j. 2. As above, with the addition of H D L 3 (4.4 × 10 -7 M); the dotted line was obtained with ka~ = 3.7 × 104 M -I sec -[. 3. Incubation of the same number of
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
391
adherent to the vessel wall. Therefore, the amount of platelet-bound lipoprotein must be determined directly. With radioactively labeled ligand this means separation of platelets and supernatants. As shown in Fig. 3, binding of lipoproteins to the platelet surface proceeds quite rapidly, and dissociation of this complex is still rather fast, the corresponding rate constants being 6.3 x 104 M I sec -~ and 1.27 x 10 -3 sec -j, respectively, at 37 °. Consequently, the separation must be accomplished in a rather short time. Separation by filtration was completely unsatisfactory again because of nonspecific binding of free lipoprotein to the filter matrix, irrespective of material, pretreatment, and washings. Separation by sedimentation as described obviously is fast enough not to deteriorate the results to any serious extent. The sucrose centrifugation procedure described above overcomes the problem of dissociation of bound ligand by avoiding time consuming washing steps in the separation of bound and free ligand. This method is, however, not applicable when investigating the kinetics of platelet-ligand interaction because of the delicate, rather long-lasting overlayering process. In those cases the centrifugation/resuspension procedure can be successfully applied. The platelet button obtained by the first centrifugation step may contain a rather large volume of occluded supernatant fraction, which has to be completely removed to achieve reliable results. To allow complete washing out of these contaminants, the pellet must be capable of being resuspended in rather short times. Resuspension by vortexing in our hands was far less satisfactory than the application of the method described above. Removal of the supernatant is again best done with an automatic pipette, leaving a layer of about 1 mm depth on top of the pellet. If carefully done, no loss of platelets will occur under these conditions. This volume of top layer after three washings will be sufficiently diluted to represent only negligible contami-
platelets with half the a m o u n t of [r25I]LDL as above. The same a m o u n t of ( I ) unlabeled L D L and ( , ) FITC-labeled L D L (36: 1), respectively, was added. The total concentration of L D L was thus kept u n c h a n g e d as c o m p a r e d with experiments 1 and 2. The solid line is calculated based on the a s s u m p t i o n of identical binding behavior of unmodified L D L and covalently modified ligands. The d a s h e d - d o t t e d line represents the calculated binding kinetics a s s u m i n g binding of radiolabeled ligand only. (b) Kinetics of dissociation of the ]25Ilabeled L D L - r e c e p t o r complex. Data were obtained by resuspension of 0.96 x 108 platelets incubated with L D L in 1 ml buffer C and incubation for the indicated time periods. (O) N o addition; the solid line is the theoretical behavior calculated with kas s = 6.3 x 1 0 4 M J sec i and kai~ = 1.27 x 103 s e c - I ; ( I ) addition of H D L 3 (0.76 × 10-6 M); the kinetic parameters are c h a n g e d to k ~ = 3.7 x 104 M i sec i and kdis~ = 2.2 x 10 -3 sec -I. respectively: (I') in the presence of 1800 units of heparin/ml.
392
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
[32]
nation. An alternative method, originally successfully applied by us to check our results obtained by the latter method, 8 is the separation by gel filtration on a semimicroscale. Small (5-7 ml bed volume) columns of BioGel A-150m (Bio-Rad, Richmond, CA) or similar materials, equilibrated with buffer L at 5°, are "calibrated" with respect to the elution zones of platelets and lipoproteins and then loaded with the mixtures to be separated. The platelet fraction is eluted within 3-4 min, fast enough to give correct results. With the cited column material almost complete recovery of the applied radioactivity was achieved. Since the more slowly eluting lipoprotein fraction must be collected quantitatively as well, this method is certainly too slow to be applied routinely. Aviram e t al. u and Mazurov e t al. 12developed two principally similar assay procedures to study the binding of LDL. Both methods, however, include rather slow washing steps, leading to unnecessary dilution. Significant losses by dissociation therefore can be expected, leading to nonlinear binding kinetics and isotherms. The method of radioiodination does to some extent have influence on the results. The ability of the labeled lipoprotein to bind to the platelet plasma membrane is not impaired by using either the Bolton-Hunter reagent (as originally done by us 8) or the iodine monochloride method, as for the majority of lipoprotein receptor studies in other cell systems.~3 The latter method, however, leads to some labeling of the lipid moiety (up to 25% of the total labeling), which increases the possibility of artifacts by lipid-exchange reactions between cells and ligands. The Bolton-Hunter method almost completely avoids this source of error, but the degree of labeling is markedly lower than when applying Iodogen, leading to less accuracy in the most sensitive part of the binding isotherm. Covalent labeling of the ligand with a fluorescent chromophore involves several disadvantages and two major benefits. The method principally must be expected to be less accurate than the former one, basically because of the necessity to work with solutions diluted to a higher degree. Furthermore, changes in intensity are determined rather than intensities themselves. Taking into account the slightly lower sensitivity of the method per se, this means a considerably higher demand for experimental skill and reproducibility. On the other hand this method works without separation of bound and unbound ligand and therefore allows direct obseru M. A v i r a m , J. G. Brook, A. M. Lees, and R. S. Lees, Biochem. Biophys. Res. Commun. 99, 308 (1981). i,. A. V. M a z u r o v , S. N. P r e d b r a z h e n s k y , V. L. Leytin, V. S. Repin, and V. N. Smirnov, FEBS Lett. 137, 319 (1982). t3 D. W. Bilheimer, S. Eisenberg, and R. I. L e v y , Biochirn. Biophys. Acta 260, 212 (1972).
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
393
vation of unstable (fast dissociating) complexes. In addition, changes with time can easily be observed, and it has at least the potential to provide some structural information on the receptor-ligand complex concomitantly. Covalent modification of lysine residues of lipoprotein (LDL) does not seriously affect their ability to bind to blood platelets ~H2'~3a (cf. Fig. 3). This behavior differs markedly from the results obtained with human fibroblasts. Accordingly, the interaction with the plasma membrane of platelets remains intact after incorporation of up to 50 molecules per molecule of lipoprotein (LDL) of either of the two fluorescent dyes cited. The interaction of platelets with rhodamine isothiocyanate (RITC)labeled LDL has been studied by Mazurov et al.12 The authors applied the technique of flow cytofluorimetry and found this method superior to an assay based on 125I labeling. Characteristics of Binding Specificity
As mentioned above, the major problem associated with the physicochemical characteristics of plasma lipoproteins is their tendency to associate to both polar and nonpolar surfaces. The specific nature of the observed binding to blood platelets has been demonstrated (mostly for LDL) by various methods, including replacement of bound labeled ligand by unlabeled lipoprotein, release induced by heparin of specifically bound LDL, and inhibition of binding by polyclonal and monoclonal antibodies against glycoprotein IIb and IIIa, which have been identified as lipoprotein-binding proteins, 14 and by polyclonal antibodies against apo B, 15 respectively. Binding studies with lipoprotein receptors of various types of mammalian tissue have sometimes revealed an additional nonspecific (nonsaturable, low affinity) binding. Under the experimental conditions described above, this is practically absent for lipoproteins with density > 1.006. Very low-density lipoproteins, however, show nonspecific, nonsaturable binding to the platelet surface to an extent making its elimination impossi13~The authors report a serious inhibition of binding to platelets of LDL following peracetylation. Careful examination of their results, however, indicates that the capability of LDL modified in this way to compete with t25I-labeled LDL for binding declines only after prolonged incubation at 37° (more than 60 rain). t4 E. Koller, F. Koller, and B. R. Binder, J. Biol. Chem. 248, 12412 (1989). z5 E. Koller, FEBS Lett. 200, 97 (1986).
394
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
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ble. Binding to specific sites may still occur, but under these conditions it escapes detection.
Number and Affinity of Sites The binding behavior of the different classes of lipoproteins can be interpreted by assuming one single class of macroscopically homogeneous receptor sites each. Cooperativity between sites and/or clustering following binding cannot be detected (they are ruled out by the observation that "fixation" of the platelet surface by treatment with formaldehyde prior to the addition of ligand has little influence on the binding isotherms). Quantitative description is summarized in Table I, including the data given by other authors. The conclusions drawn from preliminary investigations with respect to the assumed correlation between binding and apoprotein composition are included. As far as tested by us, every type of lipoprotein interferes with binding of any other class of lipoprotein in a mixed-type, noncompetitive way. Thus, the binding of one class of lipoprotein alters the characteristics of the remaining free binding sites for nonidentical lipoproteins, but already bound lipoproteins are also affected by subsequent binding of some different class of lipoprotein (Table II).
Temperature Dependence Within the range from 4 to 37 ° the association constant for the receptor binding of LDL shows only a slight increase, indicating an entropy-driven reaction; the rate constant of association exhibits a more pronounced temperature dependence (Table III).
Uptake of Lipoproteins The probability of incorporation of surface-bound ligand into the platelet interior is rather small, taking into consideration (l) the almost complete replacement of bound labeled ligand by unlabeled ligand applied in proper excess, (2) the lack of influence of fixation with formaldehyde, and (3) (at least as far as energy-consuming processes of uptake are concerned) the almost complete lack of temperature dependence. On the other hand, since phospholipid exchange between the plasma membrane of intact platelets and HDL has been demonstrated, 16 the binding isotherms obtained by application of radiolabeled ligands could be affected by partial exchange t6 D. G. Hassall, K. Desai, J. S. Owen, and K. R. Bruckdorfer, Platelets 1, 29 (1990).
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P L A T E L E T R E C E P T O R S : ASSAYS A N D P U R I F I C A T I O N
TABLE II BINDING OF INDIVIDUAL CLASSES OF PLASMA LIPOPROTEINS IN PRESENCE OF INHIBITORY LIPOPROTEIN SUBCLASSES a
Association constants for binding of indicated class of lipoprotein b (M -t, × 10 7) Inhibitor
KBDL,
KHDL.,
KLOL
HDL 3 HDL2 LDL VLDL
(9.0) 2.3 1.6 7.5
ND a (6.1) 5.4 ND a
0.8 5.5 (5.7) 5.0
Number of LDL-binding sites c 910 1860 (1840) 1290
The pattern of inhibition in most cases is more complex than predicted by simple competition for common sites. Binding data in these cases were analyzed in analogy to noncompetitive (allotopic) enzyme inhibition schemes [cf. H. B. Halsal, Trends Biochem. Sci. 5, IX-X, and references herein (1980)]. b Binding constants as indicated represent the calculated limiting affinity at saturating concentrations of inhibitor. c The number of binding sites as a rule is also reduced in the presence of competing lipoproteins. The values as presented in the table are lower limits observed at saturating concentration of inhibitor. d ND, Not determined.
o f the (small) f r a c t i o n o f labeled lipids. As outlined elsewhere in detail, 8 the results, h o w e v e r , are barely affected b y stoichiometric e x c h a n g e . Slightly h y p e r b o l i c c u r v e s s h o u l d be e x p e c t e d in S c a t c h a r d analysis, ext r a p o l a t i o n leading to the c o r r e c t value o f n ( n u m b e r o f sites per platelet) and to o n l y slightly i n c o r r e c t values for K. A s s u m i n g n o n s t o i c h i o m e t r i c e x c h a n g e (i.e., net t r a n s f e r o f lipids f r o m lipoproteins to platelets) similar plots w o u l d be obtained. T h e a p p a r e n t binding c o n s t a n t s , h o w e v e r , w o u l d then r e p r e s e n t the affinity o f the lipid-deficient, rather than o f the native lipoprotein to the r e c e p t o r .
Identification of the Lipoprotein Binding Proteins As r e p o r t e d r e c e n t l y , TM the t w o m a j o r lipoprotein-binding m e m b r a n e proteins w e r e purified to a p p a r e n t h o m o g e n e i t y and identified as g l y c o p r o tein I I b ( G P I I b ) and I I I a ( G P I I I a ) , r e s p e c t i v e l y , by their p o l y p e p t i d e size and b y specific antibodies against these g l y c o p r o t e i n s . T h e possible exis-
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
397
T A B L E III TEMPERATURE DEPENDENCE OF L D L BINDING Temperature (°C)
K ( M -t, × 10 -7)
kass (M -I sec -t, × 10 -4)
37 25 22 12 4
5.7 5.2 4.9 4.4 3.9
6.3 5.1 3.9 3.7 2.9
tence of a further receptor protein(s) cannot be ruled out. In some of our membrane preparations a third lipoprotein binding protein was detectable, and Hassal et al. 17reported a single lipoprotein binding protein with molecular weight of 140 kDa.
Physiological Function of Blood Platelet Lipoprotein Receptors The most important role of lipoprotein receptors in a wide variety of cells is to supply them with cholesterol and to regulate the biosynthesis of cholesterol (apoB/E-receptor-mediated LDL pathway), t8 Platelets lack the ability to synthesize cholesterol and so the physiological role of lipoprotein-binding proteins can be expected to be somewhat different from that of the apoB/E receptor. This assumption is further supported by the identification of GPIIb and GPIIIa as binding proteins which are distinct from the apoB/E receptor, and by the presence of saturable LDL binding sites on platelets of patients with homozygous familial hypercholesterolaemia, lacking the classical LDL receptor. ~7 There is, however, evidence for lipid exchange between different types of lipoprotein molecules and the platelet plasma membrane. As a consequence of this exchange the functional state of the platelets may be altered. 16'~9 Most importantly, the binding of lipoproteins by platelets may be directly related to platelet activation. The long-known enhancement of aggregation by LDL and, though less unambiguous, the opposing effect
i7 D. 18 R. 19 F. C.
G. Hassall, K. Desai, J. S. Owen, and K. R. Bruckdorfer, Platelets 1, 29 (1990). W. Mahley and T. L. Innerarity, Biochim. Biophys. Acta 737, 197 (1983). Martin-Nizard, B. Richard, G. Tropier, A. Nouvelot, J. C. Fruchart, P. Duthilleul, and Delbart, Thromb. Res. 46, 811 (1987).
398
PLATELET
RECEPTORS:
ASSAYS AND PURIFICATION
[33]
of H D L support this h y p o t h e s i s . 2°-24 Furthermore, it has been demonstrated in the last few years that LDL causes inhibition of adenylate cyclase, 25 evokes protein phosphorylation and mobilization of thromboxa n e A 2 ,26 and induces the release of inositol phosphates. 27 These effects definitely bring LDL binding in close relationship with platelet reactivity, although they become significant only at high LDL concentrations, far beyond that leading to half-saturation of binding sites. 28 GPIIb-IIIa fulfills a key role in the course of platelet activation. The agonist-induced binding of fibrinogen to GPIIb-IIIa is a necessary prerequisite for aggregation. Consequently, the binding of lipoproteins to this membrane protein complex might have major effects on platelet function in vivo. In fact, we could show that fibrinogen binding to ADP or thrombinstimulated platelets is significantly enhanced in the presence of L D L . 29 2o M. Aviram and J. G. Brook, Atherosclerosis 46, 259 (1983). 21 K. Desai, K. R. Bruckdorfer, R. A. Hutton, and J. S. Owen, J. LipidRes. 30, 831 (1989). 2., E. Koller, Th. Vukovich, W. Doleschel, and W. Auerswald, Atherogenese 4 (Suppl. IV), 53 (1979), 23 D. G. Hassall, J. S. Owen, and K. R. Bruckdorfer, Biochem. J. 216, 43 (1983). 24 R. Farbiszewski, Z. Skrzydlewski, and K. Worowski, Thromb. Diath. Haemostas. 21, 89 (1963). 25 K. R. Bruckdorfer, S. Buckley, and D. G. Hassall, Biochem. J. 223, 189 (1984). 26 H. E. Andrews, J. W. Aitken, D. G. Hassall, V. O. Skinner, and K. R. Bruckdorfer, Biochem. J. 242, 559 (1987). 27 M. Knorr, R. Locher, E. Vogt, W. Vetter, L. H. Block, F. Ferracin, H. Lefkovits, and A. Pletscher, Eur. J. Biochem. 172, 753 (1988). 28 Calculations based on the in vitro interaction between platelets and LDL might, however, be misleading. The presence of additional classes of lipoproteins obviously markedly reduces the strength of this binding. The degree of saturation of sites in vivo may therefore be well below 100%. 29 E. Koller, F. Koller, and B. R. Binder, Thromb. Haemostas. 62, (abstr 830) 261 (1989).
[33] P l a t e l e t I n s u l i n R e c e p t o r By ANTHONY S. HAJEK and J. HEINRICH JOIST Introduction Platelets contain insulin receptors with characteristics that are similar to the insulin receptors found in other types of cells. These include an alkaline pH binding optimum, site-site interactions between receptors (i.e., negative cooperativity), numbers of binding sites per cell surface METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
PLATELET
RECEPTOR FOR LIPOPROTEINS
383
[32] B i n d i n g C h a r a c t e r i s t i c s o f H o m o l o g o u s P l a s m a L i p o p r o t e i n s to H u m a n P l a t e l e t s
By ELISAaETH KOLLER and FRANZ KOLLER Assay Methods
Principle Human plasma lipoproteins of the high-density class (HDL) and the low-density class (LDL) show fast and saturable binding to human platelets. The parameters of these interactions are determined applying lipoproteins labeled covalently in their protein moieties (either with 125I o r with fluorescent dyes).
Buffers Buffer A: Tyrode's solution without Ca z+ (NaCI 137 raM, KC1 2.7 raM, NaHCO3 11.9 mM, MgCl 2 1.0 mM, NaH2PO 4 0.42 mM, D-glucose 5.5 raM, human serum albumin 3.5 g/liter, pH 6.5). To improve the pH stability, 5.0 mM HEPES was added in all experiments requiring longer incubation periods Buffer B: As above, with addition of 100 t~g/ml apyrase (Sigma, St. Louis, MO) and 10 units heparin/ml Buffer C: Buffer A, containing 2 mM CaCIz, pH adjusted to 7.35 Buffer L: Buffer C, without albumin and glucose
Procedures Preparation of Washed Human Platelets. Platelet-rich plasma (PRP) is obtained from ACD-blood of healthy donors immediately after drawing by sedimentation at 120 g at room temperature for 20 min. After addition of apyrase (50 p~g/ml) platelets are isolated either by gel filtration on Sepharose 2B 1 or by repeated centrifugations. 2 The final suspension medium in both cases is buffer C. i O. Tangen, H. J. Berman, and P. Marfey, Thromb. Diath. Haemorrh. 25, 268 (1971). 2 j. F. Mustard, D. W. Perry, N. G. Ardlie, and M. A. Packfiam, Br. J. Haematol. 22, 193 (1972). Briefly, PRP is spun twice for 5 min at 800 g in small tubes (5 ml) at room temperature. The combined pellets are resuspended in buffer B at 37 °. The same sequence of centrifugation is repeated three times, first in identical manner, then with resuspension in buffer A, and finally in buffer C.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[32]
Preparation o f Lipoproteins. The supernatant of the initial 800 g sedimentation of PRP, platelet poor plasma (PPP), is spun sequentially at 100,000 g and 5° following stepwise adjustment of required density values by addition of solid KBr. Prior to each flotation step the residual plasma is overlayered with KBr solutions of appropriate density to remove plasma proteins adhering to the lipoprotein fraction. The mimimal times of separation are 16 hr for very low-density lipoprotein (VLDL) floated at d = 1.006, 20 hr for LDL isolated at d = 1.006 to 1.063, and at least 36 hr each for HDL2 at d = 1.063 to 1.125 and for HDL 3 at d = 1.125 to 1.21. Finally, lipoproteins are dialyzed exhaustively against buffer L and passed through filters of 0.45-/zm pore size. The composition of each preparation is examined by determination of all components and compared with average literature values. 3 Based on this known composition, determinations of protein concentration routinely are sufficient to calculate lipoprotein concentrations. 1251 Labeling o f Lipoproteins. The method of Salacinski et al., 4 using insoluble 1,3,4,6-tetrachloro-3a,6a-diphenylglycolurii (Iodogen), is applied. Iodogen (80 tzg in 50 tzl of dry CH2CI 2) is transferred to a Beckman (Palo Alto, CA) microfuge vial and the solvent removed by aspiration with dry nitrogen. Two microliters of Na125I (3.7 MBq/ml; 62.9 MBq//zg) is added, immediately followed by addition of 200/xl of lipoprotein solution containing about 1 mg protein/ml. The reaction is allowed to proceed for 10 to 15 min at 5°. The liquid phase is then transferred to another vial containing 20/zl of 2 M aqueous KI and 800 /xl of buffer L. After mixing, the content of this vial is applied to a 10-ml column of Sephadex G-25 fine, equilibrated in the same buffer, and fractionated at room temperature, collecting l0 drops per fraction. Radioactively labeled lipoprotein appears as a very narrow peak without detectable contamination with lower molecular weight material. The radioactivity incorporated typically is about 40 kBq//zg of protein. More than 90% of the label introduced by this procedure is precipitable by trichloroacetic acid in all lipoprotein density classes. The portion of the total label associated with the lipid moiety was found to be -<8% (HDL3) by delipidation following Folch et al. 5 Fluorescence Labeling of Lipoproteins. Standard procedures are applied to achieve covalent modification of different lipoprotein fractions 3 S. Eisenberg, Ann. N. Y. Acad. Sci. 348, 30 (1980); J. C. Osborne, Jr., and H. B. Brewer, Jr., Adv. Prot. Chem. 31, 253 (1977). 4 p. R. P. Salacinski, C. H. McLean, J. E. C. Sykes, V. V. Clement-Jones, and P. J. Lowry, Anal. Biochem. 117, 139 (1981). 5 j. Folch, M. Lees, and G. H. S. Stanley, J. Biol. Chem. 226, 497 (1957).
[32]
PLATELET RECEPTOR FOR LIPOPROTE|NS
385
with fluorescein isothiocyanate (FITC) at pH 8.5, and with N-iodoacetylN'-(S-sulfo- 1-naphthyl)ethylenediamine (1,5-I-AEDANS) at pH 6.5.6 Purification of the labeled lipoprotein is achieved by centrifugation and repeated gel filtration at high and low salt concentrations, finally with buffer L. The amounts of covalently attached dyes are calculated spectroscopically using/3495 37,500 for FITC, and e350 = 6600 for 1,5-I-AEDANS, respectively. 7 Radioactive assay method: A series of incubations in conical microfuge tubes is carried out at 37° for 15 min. Each vial contains 0.2 ml of washed platelets suspended in buffer C (7-30 x 108 cells/ml), varying amounts of labeled lipoprotein, and buffer L up to a total volume of 0.5 ml. To determine the amount of unspecific, nonsaturable binding, otherwise identical incubations were performed with the addition of unlabeled lipoprotein. The amounts of added unlabeled lipoprotein were sufficient to yield total lipoprotein concentrations at least 100 times higher than those required for half-maximal saturation of the highaffinity sites. After the desired incubation times the incubation mixtures are layered on top of 0.5 ml 20% sucrose solution in buffer L and spun in a Beckman Microfuge at 8740 x g for 45 sec. The tips of the tubes containing the platelets are amputated after careful aspiration of the supernatant and radioactivity is determined in both tips and supernatants. Almost identical results were obtained with the following procedure formerly in use, which, however, is not fast enough to be applied to large series of determinations. 8 The platelets are sedimented by centrifugation of the incubation mixtures (8740 x g, 45 sec). The pellets are resuspended three times in 150 tzl of ice-cold buffer C by repeated agitation of the medium against the platelet sediment by use of an appropriately sized plastic tip attached to a hand-operated automatic pipette and immediate centrifugation for 30 sec, the whole washing procedure being accomplished within less than 5 min. Finally the platelet pellet is resuspended in another 150 tzl of buffer C and transferred to a new plastic tube. Radioactivity in the latter as well as in the original supernatant solution and in the three washing solutions is determined in a 3' counter. The concentration of the fraction of lipoprotein remaining unbound at equilibrium is derived from the sum of counts of all supernatant solutions; =
6 K. G. Mann and W. W. Fish, this series, Vol. 26, p. 28; E. N. Hudson and G. Weber, Biochemistry 12, 4154 (1973). 7 C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry." Freeman, New York, 1980. 8 E. Koller, F. Koller, and W. Doleschel, Hoppe-Seyler's Z. Physiol. Chem. 363,395 (1982).
386
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[32]
the washed platelet suspension yields the bound fraction of radioactive ligand. At low total lipoprotein concentrations (final concentrations in the vials in the nanomolar range) at least triplicate determinations should be performed; at micromolar total concentrations of ligand two series of experiments will be sufficient. Fluorescence method: To minimize disturbation by scattering, all determinations of fluorescence intensities are performed with plane polarized light. The excitation bandpass should be adjusted to less than 4 nm, that in the emitted light beam to about 5 nm. The cell holder should be thermostarted to about 25 °. The fluorescence intensities (either indicated directly or calculated as I = ~1 + 21±o-) are determined at 350/475 nm when working with 1,5-I-AEDANS-labeled lipoproteins and at 495/520 nm for FITC-modified ligands. Stock solutions of labeled lipoprotein are prepared by dilution with buffer L. Undiluted lipoprotein together with dilutions to one-third and one-fifth usually will suffice to obtain a complete binding isotherm. Platelet suspension (0.1 ml; 10 to 20 x I08 cells/ml) is then pipetted into a 1 x I cm standard fluorescence cuvette and 1.9 ml of buffer L is added. After mixing and determination of the background fluorescence intensity the chromophore-labeled lipoprotein is added stepwise (in portions of I0 or 20 td) to this suspension. After each addition the system is allowed to equilibrate (with occasional mixing) for 15 min at 25°, 9 then the fluorescence intensities are determined as above. Five (or 10) consecutive additions of lipoprotein are recommended for each of the 3 prepared stock solutions. The same sequence of additions of ligand is repeated with mixtures containing (I) 50/~1 of the original platelet suspension per 2 ml, (2) 200 /~1/2 ml, and (3) no platelets at all, otherwise proceeding in an identical manner. All measured fluorescence intensities are corrected for inner filter effect and scattering. The following procedure is both simple and reliable: A high-purity, reasonably water-soluble fluorescence standard with high quantum yield in aqueous solution and a sufficiently large absorption coefficient in the spectral range of interest is appropriately diluted with buffer L and fluorescence is determined under the conditions described above. The same is done for equally concentrated solutions of this standard chromophore, but in the presence of 0.05, 0.1, and 0.2 ml of the platelet suspension under investigation. Comparison of these 9 Fifteen minutes might be too short to allow equilibration with very low concentrations of ligand. In these rare cases no further increase in fluorescence intensities was detectable after incubation periods of 30 min.
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
387
relative fluorescence intensities yields empirical factors correcting for optical effects related to the platelets in suspension. Effects associated with the added lipoprotein normally are negligible, since the latter will add very little to the total absorption at the wavelength of excitation. Possible complications due to interactions of the standard dye with platelets can be excluded by measuring at two temperatures separated by about 10 to 15°. In the absence of interactions the correction factors for optical effects should show no detectable temperature dependence.I° The (corrected) values of relative fluorescence intensity are increased in the presence of human platelets when compared with the series in plain buffer. The reciprocals of these changes of fluorescence intensity (l/A/) are plotted against the reciprocal concentration of lipoprotein as shown in Fig. 1. Slightly curved binding isotherms are obtained, which approach linearity with decreasing concentration of platelets. The left part of the plot can be linearly extrapolated to infinite ligand concentration with proper accuracy. Based on the assumption of kinetically indistinguishable receptor sites the intercept on the vertical axis is 1/nPb, that with the abscissa corresponds to K. P represents the concentration of platelets (in molarities, treating platelets as molecules), n is the average number of receptor sites per single platelet, K stands for the thermodynamic association constant for binding to the receptors, and b is the incremental change of fluorescence intensity of the average chromophore molecule on binding, depending on instrumental parameters and to some extent on the degree of lipoprotein labeling. This value must be known to get access to the number of sites/platelets, which can be achieved without any additional measurement according to Fig. 2. For different (preferentially low) concentrations of total lipoprotein (T) kept constant, the corresponding values of T/A1 are plotted against the reciprocals of platelet concentration. Extrapolation of these more or less linear plots to the ordinate should yield a common intercept, its numerical value being 1/b. Again the accuracy of the individual determinations is crucial for L0The deviation of the apparent fluorescence intensities observed in the presence of platelets from the corrected values is less pronounced than might be anticipated. As typical examples from our experiments the fluorescence intensities measured at 520 nm had to be corrected by factors of 0.979 and 0.919 for 100 and 300 t~l of original platelet suspension (16.2 × 108/ml), respectively. We used lysine derivatives of both 1,5-I-AEDANS and FITC as standard dyes to determine these values (adding 40 ~1 of a solution in buffer L, containing about 5 mg of dye/ml). Reaction of either fluorescent derivative with the platelet plasma membrane proceeds only very slowly (if at all) and can easily be corrected for by extrapolating back to mixing time.
388
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[32]
5O
3O
I
0.5 1.0 1.5 ( Concenfrn'rion of LDL)-I
2.0xl 0 9
FIG. 1. Double-reciprocal plot illustrating L D L binding to blood platelets at 37 °. Lowdensity lipoprotein was labeled with FITC (44: 1), binding leading to increased fluorescence at 495/520 nm. The final concentrations of platelets were 0.425 (~I,), 0.85 (IlL 1.7 ( * ) , 2.95 (T), and 4.25 × 108 cells/ml ((3). Error bars are shown for the most diluted series.
the reliability of the final results. Therefore the reference series (in the absence of platelets) should be repeated twice. The experiments in the presence of platelets should be performed in duplicate. Quite similar results have been obtained with the two types of chromophores described. The use of FITC is to be preferred, however, because of its markedly higher fluorescence yield and its absorption at longer wavelengths. Unnecessary illumination of samples should be avoided. The serum albumin included in the platelet suspension buffer systems should be of the highest available degree of purity (delipidated if possible) to minimize background emission close to the wavelengths applied in this assay. Partial peroxidation of lipoprotein lipids can produce similar complications. Although several methods are known to inhibit lipid peroxidation, the best means to avoid this interference is to use only freshly prepared lipoproteins, since plasma lipoproteins in any case in vitro show a distinct tendency to change structure, size, and composition with time.
[32]
PLATELET
RECEPTOR FOR LIPOPROTEINS
389
~c~ 5 x
.w-
oJ
3 i/] oJ
,-r 2 -4--
I
I
I
0.5
I
1.0x10-13
(Concentration of Ptatelets)-I
FIG. 2. Extrapolation to infinite platelet concentration of the reciprocal changes of fluorescence (495/520 nm) of FITC-labeled LDL. The individual series were determined at constant total concentrations of LDL (LDLt): 0.35 (©), 0.52 (*), 1.04 (A), 3.1 (11), and 6.7 × 10 - 9 M (A). The data are obtained from the series of experiments illustrated in Fig. 1.
General Considerations and Problems in Receptor Binding Studies Involving I_,ipoproteins
Both size and surface structure make plasma lipoproteins (especially of the lower b u o y a n t density classes) highly adherent to most c o m m o n laboratory equipment materials, including glass and various types of plasticware. Our attempts to find any combination of material and p r e t r e a t m e n t virtually excluding nonspecific adhesion were unsuccessful. Binding data generally are best obtained by determination of the a m o u n t of free (unbound) ligand, calculating the bound fraction as the difference b e t w e e n this concentration and the k n o w n total ligand concentration. In the case of plasma lipoproteins (especially at low degrees of r e c e p t o r saturation, i.e., at low ligand concentration), this difference represents not only ligand bound to the platelets but also
390
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ASSAYS
AND
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a ml ---7 2 '~o
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Time (rain) Flo. 3. (a) Time dependence of binding of ]25I-labeled L D L to blood platelets. 1. Incubation of 4.5 × 108 platelets/ml with 1.9 x 10 7 M LDL. The solid line is calculated with kass = 6.3 x 104 M - i sec -j. 2. As above, with the addition of H D L 3 (4.4 × 10 -7 M); the dotted line was obtained with ka~ = 3.7 × 104 M -I sec -[. 3. Incubation of the same number of
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
391
adherent to the vessel wall. Therefore, the amount of platelet-bound lipoprotein must be determined directly. With radioactively labeled ligand this means separation of platelets and supernatants. As shown in Fig. 3, binding of lipoproteins to the platelet surface proceeds quite rapidly, and dissociation of this complex is still rather fast, the corresponding rate constants being 6.3 x 104 M I sec -~ and 1.27 x 10 -3 sec -j, respectively, at 37 °. Consequently, the separation must be accomplished in a rather short time. Separation by filtration was completely unsatisfactory again because of nonspecific binding of free lipoprotein to the filter matrix, irrespective of material, pretreatment, and washings. Separation by sedimentation as described obviously is fast enough not to deteriorate the results to any serious extent. The sucrose centrifugation procedure described above overcomes the problem of dissociation of bound ligand by avoiding time consuming washing steps in the separation of bound and free ligand. This method is, however, not applicable when investigating the kinetics of platelet-ligand interaction because of the delicate, rather long-lasting overlayering process. In those cases the centrifugation/resuspension procedure can be successfully applied. The platelet button obtained by the first centrifugation step may contain a rather large volume of occluded supernatant fraction, which has to be completely removed to achieve reliable results. To allow complete washing out of these contaminants, the pellet must be capable of being resuspended in rather short times. Resuspension by vortexing in our hands was far less satisfactory than the application of the method described above. Removal of the supernatant is again best done with an automatic pipette, leaving a layer of about 1 mm depth on top of the pellet. If carefully done, no loss of platelets will occur under these conditions. This volume of top layer after three washings will be sufficiently diluted to represent only negligible contami-
platelets with half the a m o u n t of [r25I]LDL as above. The same a m o u n t of ( I ) unlabeled L D L and ( , ) FITC-labeled L D L (36: 1), respectively, was added. The total concentration of L D L was thus kept u n c h a n g e d as c o m p a r e d with experiments 1 and 2. The solid line is calculated based on the a s s u m p t i o n of identical binding behavior of unmodified L D L and covalently modified ligands. The d a s h e d - d o t t e d line represents the calculated binding kinetics a s s u m i n g binding of radiolabeled ligand only. (b) Kinetics of dissociation of the ]25Ilabeled L D L - r e c e p t o r complex. Data were obtained by resuspension of 0.96 x 108 platelets incubated with L D L in 1 ml buffer C and incubation for the indicated time periods. (O) N o addition; the solid line is the theoretical behavior calculated with kas s = 6.3 x 1 0 4 M J sec i and kai~ = 1.27 x 103 s e c - I ; ( I ) addition of H D L 3 (0.76 × 10-6 M); the kinetic parameters are c h a n g e d to k ~ = 3.7 x 104 M i sec i and kdis~ = 2.2 x 10 -3 sec -I. respectively: (I') in the presence of 1800 units of heparin/ml.
392
PLATELET RECEPTORS; ASSAYS AND PURIFICATION
[32]
nation. An alternative method, originally successfully applied by us to check our results obtained by the latter method, 8 is the separation by gel filtration on a semimicroscale. Small (5-7 ml bed volume) columns of BioGel A-150m (Bio-Rad, Richmond, CA) or similar materials, equilibrated with buffer L at 5°, are "calibrated" with respect to the elution zones of platelets and lipoproteins and then loaded with the mixtures to be separated. The platelet fraction is eluted within 3-4 min, fast enough to give correct results. With the cited column material almost complete recovery of the applied radioactivity was achieved. Since the more slowly eluting lipoprotein fraction must be collected quantitatively as well, this method is certainly too slow to be applied routinely. Aviram e t al. u and Mazurov e t al. 12developed two principally similar assay procedures to study the binding of LDL. Both methods, however, include rather slow washing steps, leading to unnecessary dilution. Significant losses by dissociation therefore can be expected, leading to nonlinear binding kinetics and isotherms. The method of radioiodination does to some extent have influence on the results. The ability of the labeled lipoprotein to bind to the platelet plasma membrane is not impaired by using either the Bolton-Hunter reagent (as originally done by us 8) or the iodine monochloride method, as for the majority of lipoprotein receptor studies in other cell systems.~3 The latter method, however, leads to some labeling of the lipid moiety (up to 25% of the total labeling), which increases the possibility of artifacts by lipid-exchange reactions between cells and ligands. The Bolton-Hunter method almost completely avoids this source of error, but the degree of labeling is markedly lower than when applying Iodogen, leading to less accuracy in the most sensitive part of the binding isotherm. Covalent labeling of the ligand with a fluorescent chromophore involves several disadvantages and two major benefits. The method principally must be expected to be less accurate than the former one, basically because of the necessity to work with solutions diluted to a higher degree. Furthermore, changes in intensity are determined rather than intensities themselves. Taking into account the slightly lower sensitivity of the method per se, this means a considerably higher demand for experimental skill and reproducibility. On the other hand this method works without separation of bound and unbound ligand and therefore allows direct obseru M. A v i r a m , J. G. Brook, A. M. Lees, and R. S. Lees, Biochem. Biophys. Res. Commun. 99, 308 (1981). i,. A. V. M a z u r o v , S. N. P r e d b r a z h e n s k y , V. L. Leytin, V. S. Repin, and V. N. Smirnov, FEBS Lett. 137, 319 (1982). t3 D. W. Bilheimer, S. Eisenberg, and R. I. L e v y , Biochirn. Biophys. Acta 260, 212 (1972).
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
393
vation of unstable (fast dissociating) complexes. In addition, changes with time can easily be observed, and it has at least the potential to provide some structural information on the receptor-ligand complex concomitantly. Covalent modification of lysine residues of lipoprotein (LDL) does not seriously affect their ability to bind to blood platelets ~H2'~3a (cf. Fig. 3). This behavior differs markedly from the results obtained with human fibroblasts. Accordingly, the interaction with the plasma membrane of platelets remains intact after incorporation of up to 50 molecules per molecule of lipoprotein (LDL) of either of the two fluorescent dyes cited. The interaction of platelets with rhodamine isothiocyanate (RITC)labeled LDL has been studied by Mazurov et al.12 The authors applied the technique of flow cytofluorimetry and found this method superior to an assay based on 125I labeling. Characteristics of Binding Specificity
As mentioned above, the major problem associated with the physicochemical characteristics of plasma lipoproteins is their tendency to associate to both polar and nonpolar surfaces. The specific nature of the observed binding to blood platelets has been demonstrated (mostly for LDL) by various methods, including replacement of bound labeled ligand by unlabeled lipoprotein, release induced by heparin of specifically bound LDL, and inhibition of binding by polyclonal and monoclonal antibodies against glycoprotein IIb and IIIa, which have been identified as lipoprotein-binding proteins, 14 and by polyclonal antibodies against apo B, 15 respectively. Binding studies with lipoprotein receptors of various types of mammalian tissue have sometimes revealed an additional nonspecific (nonsaturable, low affinity) binding. Under the experimental conditions described above, this is practically absent for lipoproteins with density > 1.006. Very low-density lipoproteins, however, show nonspecific, nonsaturable binding to the platelet surface to an extent making its elimination impossi13~The authors report a serious inhibition of binding to platelets of LDL following peracetylation. Careful examination of their results, however, indicates that the capability of LDL modified in this way to compete with t25I-labeled LDL for binding declines only after prolonged incubation at 37° (more than 60 rain). t4 E. Koller, F. Koller, and B. R. Binder, J. Biol. Chem. 248, 12412 (1989). z5 E. Koller, FEBS Lett. 200, 97 (1986).
394
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[32]
ble. Binding to specific sites may still occur, but under these conditions it escapes detection.
Number and Affinity of Sites The binding behavior of the different classes of lipoproteins can be interpreted by assuming one single class of macroscopically homogeneous receptor sites each. Cooperativity between sites and/or clustering following binding cannot be detected (they are ruled out by the observation that "fixation" of the platelet surface by treatment with formaldehyde prior to the addition of ligand has little influence on the binding isotherms). Quantitative description is summarized in Table I, including the data given by other authors. The conclusions drawn from preliminary investigations with respect to the assumed correlation between binding and apoprotein composition are included. As far as tested by us, every type of lipoprotein interferes with binding of any other class of lipoprotein in a mixed-type, noncompetitive way. Thus, the binding of one class of lipoprotein alters the characteristics of the remaining free binding sites for nonidentical lipoproteins, but already bound lipoproteins are also affected by subsequent binding of some different class of lipoprotein (Table II).
Temperature Dependence Within the range from 4 to 37 ° the association constant for the receptor binding of LDL shows only a slight increase, indicating an entropy-driven reaction; the rate constant of association exhibits a more pronounced temperature dependence (Table III).
Uptake of Lipoproteins The probability of incorporation of surface-bound ligand into the platelet interior is rather small, taking into consideration (l) the almost complete replacement of bound labeled ligand by unlabeled ligand applied in proper excess, (2) the lack of influence of fixation with formaldehyde, and (3) (at least as far as energy-consuming processes of uptake are concerned) the almost complete lack of temperature dependence. On the other hand, since phospholipid exchange between the plasma membrane of intact platelets and HDL has been demonstrated, 16 the binding isotherms obtained by application of radiolabeled ligands could be affected by partial exchange t6 D. G. Hassall, K. Desai, J. S. Owen, and K. R. Bruckdorfer, Platelets 1, 29 (1990).
[32]
PLATELET
RECEPTOR
FOR
395
LIPOPROTEINS
<
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P L A T E L E T R E C E P T O R S : ASSAYS A N D P U R I F I C A T I O N
TABLE II BINDING OF INDIVIDUAL CLASSES OF PLASMA LIPOPROTEINS IN PRESENCE OF INHIBITORY LIPOPROTEIN SUBCLASSES a
Association constants for binding of indicated class of lipoprotein b (M -t, × 10 7) Inhibitor
KBDL,
KHDL.,
KLOL
HDL 3 HDL2 LDL VLDL
(9.0) 2.3 1.6 7.5
ND a (6.1) 5.4 ND a
0.8 5.5 (5.7) 5.0
Number of LDL-binding sites c 910 1860 (1840) 1290
The pattern of inhibition in most cases is more complex than predicted by simple competition for common sites. Binding data in these cases were analyzed in analogy to noncompetitive (allotopic) enzyme inhibition schemes [cf. H. B. Halsal, Trends Biochem. Sci. 5, IX-X, and references herein (1980)]. b Binding constants as indicated represent the calculated limiting affinity at saturating concentrations of inhibitor. c The number of binding sites as a rule is also reduced in the presence of competing lipoproteins. The values as presented in the table are lower limits observed at saturating concentration of inhibitor. d ND, Not determined.
o f the (small) f r a c t i o n o f labeled lipids. As outlined elsewhere in detail, 8 the results, h o w e v e r , are barely affected b y stoichiometric e x c h a n g e . Slightly h y p e r b o l i c c u r v e s s h o u l d be e x p e c t e d in S c a t c h a r d analysis, ext r a p o l a t i o n leading to the c o r r e c t value o f n ( n u m b e r o f sites per platelet) and to o n l y slightly i n c o r r e c t values for K. A s s u m i n g n o n s t o i c h i o m e t r i c e x c h a n g e (i.e., net t r a n s f e r o f lipids f r o m lipoproteins to platelets) similar plots w o u l d be obtained. T h e a p p a r e n t binding c o n s t a n t s , h o w e v e r , w o u l d then r e p r e s e n t the affinity o f the lipid-deficient, rather than o f the native lipoprotein to the r e c e p t o r .
Identification of the Lipoprotein Binding Proteins As r e p o r t e d r e c e n t l y , TM the t w o m a j o r lipoprotein-binding m e m b r a n e proteins w e r e purified to a p p a r e n t h o m o g e n e i t y and identified as g l y c o p r o tein I I b ( G P I I b ) and I I I a ( G P I I I a ) , r e s p e c t i v e l y , by their p o l y p e p t i d e size and b y specific antibodies against these g l y c o p r o t e i n s . T h e possible exis-
[32]
PLATELET RECEPTOR FOR LIPOPROTEINS
397
T A B L E III TEMPERATURE DEPENDENCE OF L D L BINDING Temperature (°C)
K ( M -t, × 10 -7)
kass (M -I sec -t, × 10 -4)
37 25 22 12 4
5.7 5.2 4.9 4.4 3.9
6.3 5.1 3.9 3.7 2.9
tence of a further receptor protein(s) cannot be ruled out. In some of our membrane preparations a third lipoprotein binding protein was detectable, and Hassal et al. 17reported a single lipoprotein binding protein with molecular weight of 140 kDa.
Physiological Function of Blood Platelet Lipoprotein Receptors The most important role of lipoprotein receptors in a wide variety of cells is to supply them with cholesterol and to regulate the biosynthesis of cholesterol (apoB/E-receptor-mediated LDL pathway), t8 Platelets lack the ability to synthesize cholesterol and so the physiological role of lipoprotein-binding proteins can be expected to be somewhat different from that of the apoB/E receptor. This assumption is further supported by the identification of GPIIb and GPIIIa as binding proteins which are distinct from the apoB/E receptor, and by the presence of saturable LDL binding sites on platelets of patients with homozygous familial hypercholesterolaemia, lacking the classical LDL receptor. ~7 There is, however, evidence for lipid exchange between different types of lipoprotein molecules and the platelet plasma membrane. As a consequence of this exchange the functional state of the platelets may be altered. 16'~9 Most importantly, the binding of lipoproteins by platelets may be directly related to platelet activation. The long-known enhancement of aggregation by LDL and, though less unambiguous, the opposing effect
i7 D. 18 R. 19 F. C.
G. Hassall, K. Desai, J. S. Owen, and K. R. Bruckdorfer, Platelets 1, 29 (1990). W. Mahley and T. L. Innerarity, Biochim. Biophys. Acta 737, 197 (1983). Martin-Nizard, B. Richard, G. Tropier, A. Nouvelot, J. C. Fruchart, P. Duthilleul, and Delbart, Thromb. Res. 46, 811 (1987).
398
PLATELET
RECEPTORS:
ASSAYS AND PURIFICATION
[33]
of H D L support this h y p o t h e s i s . 2°-24 Furthermore, it has been demonstrated in the last few years that LDL causes inhibition of adenylate cyclase, 25 evokes protein phosphorylation and mobilization of thromboxa n e A 2 ,26 and induces the release of inositol phosphates. 27 These effects definitely bring LDL binding in close relationship with platelet reactivity, although they become significant only at high LDL concentrations, far beyond that leading to half-saturation of binding sites. 28 GPIIb-IIIa fulfills a key role in the course of platelet activation. The agonist-induced binding of fibrinogen to GPIIb-IIIa is a necessary prerequisite for aggregation. Consequently, the binding of lipoproteins to this membrane protein complex might have major effects on platelet function in vivo. In fact, we could show that fibrinogen binding to ADP or thrombinstimulated platelets is significantly enhanced in the presence of L D L . 29 2o M. Aviram and J. G. Brook, Atherosclerosis 46, 259 (1983). 21 K. Desai, K. R. Bruckdorfer, R. A. Hutton, and J. S. Owen, J. LipidRes. 30, 831 (1989). 2., E. Koller, Th. Vukovich, W. Doleschel, and W. Auerswald, Atherogenese 4 (Suppl. IV), 53 (1979), 23 D. G. Hassall, J. S. Owen, and K. R. Bruckdorfer, Biochem. J. 216, 43 (1983). 24 R. Farbiszewski, Z. Skrzydlewski, and K. Worowski, Thromb. Diath. Haemostas. 21, 89 (1963). 25 K. R. Bruckdorfer, S. Buckley, and D. G. Hassall, Biochem. J. 223, 189 (1984). 26 H. E. Andrews, J. W. Aitken, D. G. Hassall, V. O. Skinner, and K. R. Bruckdorfer, Biochem. J. 242, 559 (1987). 27 M. Knorr, R. Locher, E. Vogt, W. Vetter, L. H. Block, F. Ferracin, H. Lefkovits, and A. Pletscher, Eur. J. Biochem. 172, 753 (1988). 28 Calculations based on the in vitro interaction between platelets and LDL might, however, be misleading. The presence of additional classes of lipoproteins obviously markedly reduces the strength of this binding. The degree of saturation of sites in vivo may therefore be well below 100%. 29 E. Koller, F. Koller, and B. R. Binder, Thromb. Haemostas. 62, (abstr 830) 261 (1989).
[33] P l a t e l e t I n s u l i n R e c e p t o r By ANTHONY S. HAJEK and J. HEINRICH JOIST Introduction Platelets contain insulin receptors with characteristics that are similar to the insulin receptors found in other types of cells. These include an alkaline pH binding optimum, site-site interactions between receptors (i.e., negative cooperativity), numbers of binding sites per cell surface METHODS IN ENZYMOLOGY, VOL. 215
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