- Email: [email protected]
von Willebrand factor binding to platelet glycoprotein Ib complex
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subunits is centrifuged for 10 min at 4° in an Eppendorf microcentrifuge, loaded onto the column, and eluted with the 0.05% Triton X-100 buffer. Figure 5 shows a typical separation of GPIIb from GPIIIa. Ten percent of the GPIIb peak is GPIIIa; 5% of the GPIIIa peak is GPIIb. Reassociation of the isolated glycoproteins requires immediate readdition of C a 2+ (6 m M ) to the fractions obtained from the high-performance liquid chromatography column. When isolated GPIIb is mixed with isolated GPIIIa, 10 to 15% of the dissociated glycoproteins reform the heterodimeric complex. Acknowledgments This work was supported in part by Grants HL 28947 and HL 32254 form the National Institutes of Health. The authors thank Barbara Allen and Sally Gullatt Seehafer for editorial assistance, James X. Warger and Norma Jean Gargasz for graphics, and Kate Sholly, Michele Prator, Linda Harris Odumade, and Linda Parker for manuscript preparation.
[23] v o n W i l l e b r a n d F a c t o r B i n d i n g to P l a t e l e t G l y c o p r o t e i n Ib Complex B y ZAVERIO M. RUGGERI, THEODORE S. ZIMMERMAN, 1 SUSAN RUSSELL, ROSSELLA BADER, a n d LUIGI DE MARCO
von Willebrand factor (vWF) is a complex multimeric glycoprotein that plays an essential role in platelet function. ~a It is required for normal platelet adhesion to exposed subendothelium and for normal platelet plug formation at sites of vascular injury. The function of vWF is particularly important in vessels of small caliber, where conditions of high wall shear rate prevail. It is now recognized that the mechanisms underlying vWF function comprise interaction with components of the subendothelium as well as with specific receptors on the platelet membrane. Two distinct vWF-binding sites have been recognized so far on platelets, one related to the membrane glycoprotein (GP) Ib and the other to the heterodimeric GPIIb-IIIa complex. 2 Deceased. la Z.M. Ruggeri and T. S. Zimmerman, Blood 70, 895 (1970). 2 Z.M. Ruggeri, L. DeMarco, L. Gatti, R. Bader, and R. R. Montgomery, J. Clin. lnvest. 72, 1 (1983).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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This chapter deals with reviewing the methodology involved in studying the vWF interaction with GPIb. von Willebrand factor binds to GPIb in the presence of the antibiotic ristocetin. Such binding correlates with the occurrence ofplatelet aggregation induced by ristocetin in the presence of vWF. 3 The latter function of vWF is often referred to as the ristocetin cofactor activity. If platelets are metabolically inactive, as after fixation with formalin, they will nonetheless still respond to ristocetin in the presence of vWF with what is more properly called agglutination. Therefore, the ristocetin-dependent interaction of vWF with GPIb can be analyzed using either fresh, metabolically active platelets or fixed, metabolically inactive platelets. Ristocetin, however, is not the only tool with which to study vWF binding to GPIb under experimental conditions. Following removal of sialic acid residues from the vWF glycoprotein, the resulting asialo-vWF exhibits the ability to interact directly with GPIb. 4 It is, therefore, apparent that GPIb-related binding sites for vWF are exposed on the membrane of unstimulated platelets and can interact with a modified form of the molecule. The concept that a modification of the vWF molecule can be the initial event promoting its interaction with platelets is a stimulating alternative, or perhaps addition, to the concept that a modification of the platelet surface or microenvironment, as possibly brought about by ristocetin, 5'6 is necessary to promote vWF binding to GPIb. Indeed, it has also been demonstrated that another substance with the property of inducing vWF binding to GPIb, the snake protein botrocetin, 7 exerts its function by forming a bimolecular complex with vWF that in turn binds to platelets7'8; botrocetin itself does not interact with the platelet surface. 8 Using asialo-vWF, it is also possible to demonstrate that receptors on GPIIb-IIIa are exposed following vWF binding to GPIb. 4 This constitutes a potentially important link between two major binding sites for adhesive glycoproteins on the platelet membrane. The procedures described in this chapter relate to the general methodology for studying receptor-ligand interaction. The basic constituents of the experimental system are, in this case, highly purified vWF and platelets washed free, as much as possible, of other blood components. The vWF to be used as a tracer is radiolabeled with 125I. As mentioned above, 3 K.-J. Kao, S. V. Pizzo, and P. A. McKee, Proc. Natl. Acad. Sci. U.S.A. 76, 5317 (1979). 4 L. De Marco, A. Girolami, S. Russell, and Z. M. Ruggeri, J. Clin. Invest. 75, 1198 (1985). 5 S. E. Senogles and G. L. Nelsestuen, J. Biol. Chem. 258, 12327 (1983). 6 B. S. Coller, J. Clin. Invest. 61, 1168 (1978). 7 M. S. Read, S. V. Smith, M. A. Lamb, and K. M. Brinkhous, Blood 74, 1031 (1989). 8 M. Sugimoto, G. Ricca, M. E. Hrinda, A. B. Schreiber, G. H. Searfoss, E. Bottini, and Z. M. Ruggeri, Biochemistry 30, 5202 (1991).
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washed platelets can be either fresh or fixed. A detailed description of the methods used to prepare these reagents and to study ristocetin-induced binding of vWF to platelets is presented. The procedure used to prepare asialo-vWF and to study its interaction with platelets, as well as that to measure botrocetin-mediated vWF binding to platelets, will not be described here but have been the subjects of previous reports/'8'9 Botrocetin-induced platelet agglutination/aggregation is also described in [12] in Volume 169 of this series.
Purification of von Willebrand Factor
Reagents Human plasma cryoprecipitate (the source of vWF): It can be obtained as such or, if blood bank facilities are available, it can be prepared from flesh plasma using the procedure of Pool and Shanon. l0 Aluminum hydroxide (Rehsorptar from Armour Pharmaceutical Co., Kankakee, IL) Bentonite (B-3378 from Sigma Chemical Co., St. Louis, MO) Polyethylene glycol (P-2139, average M r 8000; Sigma): A 40% (w/v) solution is prepared in imidazole buffer (see below) Sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, N J) Citrate buffer: 0.02 M Tris, 0.02 M trisodium citrate, 0.02 M e-aminocaproic acid, pH 7.0 Imidazole buffer: 0.02 M imidazole, 0.01 M trisodium citrate, 0.02 M eaminocaproic acid, 0.15 M NaC1, pH 6.5; sodium azide at a concentration of 0.02% (w/v) is added as a bacteriostatic agent Tris buffer: 0.02 M Tris, 0.15 M NaC1, pH 7.35
Procedure The method is a modification of two published procedures, ~J,j2 and begins with 1000 ml of cryoprecipitate, from which a final yield of 15 to 20 mg of purified vWF is expected. The cryoprecipitate is thawed at 0-4 ° (approximately 18 hr) and then centrifuged at 9800 g (rmax) for 45 min at 4°. The supernatant is discarded and the precipitate is dissolved with several additions of citrate buffer, up to a final volume of 300 ml. The 9 L. De Marco and S. S. Shapiro, J. Clin. Invest. 168, 321 (1981). to j. G. Pool and A. E. Shanon, N. Engl. J. Med. 273, 1443 (1965). tt j. Newman, A. J. Johnson, M. H. Karpatkin, and S. Puszkin, Br. J. Haematol. 21, 1 (1971). t2 M. E. Switzer and P. A. McKee, J. Clin. Invest. 57, 925 (1976).
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solution is transferred to a plastic beaker and Rehsorptar is added dropwise (5 ml/100 ml of redissolved precipitate) while stirring. The mixture is further stirred for 10 min at room temperature (22-25°), then transferred into plastic bottles and centrifuged at 7500 g (rmax) for 30 min at 20°. In the meantime, bentonite (2.5 mg/ml of supernatant) is placed in clean plastic centrifuge bottles and the powder is mixed with a small amount of citrate buffer to obtain a slurry. At the end of the centrifugation, the supernatant is poured directly into the bottles containing the bentonite, quickly mixed, and the mixtrue rapidly centrifuged at 3800 g (rmax) for 5 min at 20°. The supernatant from the last centrifugation is transferred to a plastic beaker and the pH is adjusted to 6.5 by slow addition of 0.02 M citric acid. A volume of the 40% polyethylene glycol solution is added dropwise, under continuous stirring, to give a final concentration of 3% (w/v) polyethylene glycol. The mixture is stirred for an additional 10 min at room temperature and then centrifuged at 3800 g (rmax) for 15 min at 20°. The supernatant is recovered and transferred to a plastic beaker and additional polyethylene glycol is added following the same procedure to give a final concentration of 15%. After stirring for an additional 20 min at room temperature, the mixture is centrifuged at 9800 g (rmax) for 30 min at room temperature. At the end of centrifugation, the supernatant is discarded and the precipitate is redissolved in the smallest possible volume of imidazole buffer. The precipitate obtained from 1 liter of cryoprecipitate can usually be redissolved in a final volume of approximately 30-35 ml. Complete dissolution usually takes 90-180 min with continuous, but gentle, agitation on a shaker. The dissolved precipitate is ultracentrifuged at 113,000 g (/'max) for 45 min at 20°, after which the lipids floating on top are discarded and the clear solution is separated from the insoluble material at the bottom of the tube. The clear solution is then applied onto a Sepharose CL-4B column, equilibrated with imidazole buffer. A 100-cm-long siliconized glass column, 5 cm in diameter and with a bed volume of approximately 1700 ml, is used (the actual agarose column is approximately 80 cm long). The column is run at a flow rate of 60 ml/hr at room temperature, usually overnight for convenience. The optical density of the effluent is continuously monitored at 280 nm and 10-ml fractions are collected. The asymmetric peak appearing at the void volume contains the purified vWF. The column is developed until all proteins are eluted. It is then washed with at least three bed volumes of buffer and finally stored with buffer containing 0.1% (w/v) sodium azide. von Willebrand factor is a multimeric protein and consists of molecular forms of varying molecular weight. Figure 1 shows the distribution of
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PLATELET GPIb COMPLEX--vWF INTERACTION
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I
I
I
267
I
0.2-
~
o.1
o
:
;
5b Effluent, ml
FZG. 1. Multimeric structure of vWF eluting from the gel-filtrationcolumn. Lower: Elution profile of the protein peak appearing at the void volume, detected by light absorbance at 280 nm. Upper: SDS-agarose gel electrophoresis of samples corresponding to different positions of the elution profile, as indicated by black bars. Note that the largest multimers appear first. Cathode at the top. Electrophoresis was performed as described in [21] in Volume 169 of this series.
multimers of different size across the v W F peak eluting from the Sepharose column. Usually, all the fractions corresponding to the ascending part and the first half of the descending part of the p e a k are pooled. Although the v W F p e a k is well separated f r o m the subsequent one, it is preferable to discard the later eluting fractions to decrease the likelihood of contaminating proteins in the v W F preparation. The protein concentration in the v W F pool obtained from the gelfiltration column is usually b e t w e e n 0.1 and 0.2 mg/ml. Because a higher concentration usually is needed, particularly for labeling the protein, the v W F must be concentrated. The m o s t effective method is to transfer the v W F solution into regular dialysis tubing and then surround it with the
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hygroscopic agent Aquacide II, obtained from Calbiochem-Behring Corporation (La Jolla, CA). When sufficient water is removed to achieve the desired concentration, the dialysis tubing is rinsed with distilled water and the vWF is dialyzed against Tris buffer. The vWF preparation is then aliquoted and stored at - 7 0 °. Comments
According to individual needs, smaller amounts of starting cryoprecipitate or plasma can be used. In that case, a gel-filtration column of smaller diameter can be employed, but its length should not be decreased. If the protein concentration in the vWF preparation is derived from the optical density (OD), the following equation, which takes into account a correction for light scattering, should be used: OD280 -
(1.7 x OD320)/0.7 = vWF concentration (mg/ml)
(1)
The method used to analyze the multimeric structure of vWF is described in detail in [21] in Volume 169 of this series. In view of the heterogeneous nature of vWF and the fact that manipulation of the molecule can lead to loss of the largest multimer, it is advisable to analyze the multimeric composition of each purified vWF preparation before use. In fact, loss of the largest multimers affects the functional properties of vWF. If multimeric analysis cannot be performed, one alternative is to measure the ristocetin cofactor activity of the purified preparation. If no contaminants are present and the preparation has a normal multimeric distribution, the specific activity should be greater than 100 units of ristocetin cofactor per milligram of v W F . 4 It is not clear whether the use of protease inhibitors during the purification procedure is of any help in achieving the goal of obtaining vWF with the full complement of multimers, including the largest. Nevertheless, protease inhibitors now are routinely added to the cryoprecipitate when it is first thawed out. The following inhibitors are used, all from CalbiochemBehring, and all at a final concentration of 10/xM in the cryoprecipitate: Dphenylalanyl-L-prolyl-L-arginine chloromethyl ketone, dansyl-L-glutamylL-glycyl-L-arginine chloromethyl ketone, and D-phenylalanyl-L-phenylalanyl-L-arginine chloromethyl ketone, as well as leupeptin (Chemicon, Los Angeles, CA), at a final concentration of 15/zg/ml. The same inhibitors, but at one-tenth the final concentration, may also be added to the imidazole buffer used to resuspend the final precipitate and to equilibrate and run the gel-filtration column.
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Labeling of yon Willebrand Factor
Reagents
1,3,4,6-Tetrachloro-3a,6a-diphenylglycouril (Iodogen; Pierce Chemical Co., Rockford, IL): A 1-mg/ml solution is prepared in dichloromethane. It can be stored sealed and protected from light for several weeks at - 20° Sephadex G-25 Medium (Pharmacia) Tris (0.02 M)-0.15 M NaC1 buffer, pH 7.35 KI: 10 mg/ml in distilled water NalZSI, carrier free (Amersham, Arlington Heights, IL)
Procedure The method described here is slightly modified from that previously published by Fraker and Speck. 13The whole procedure is performed under a fume hood in a laboratory designated for protein iodination. Between I00 and 400/xl of Iodogen solution is pipetted into a glass tube or vial, and the dichloromethane is evaporated under a stream of nitrogen. The tube is sealed, protected from light, and kept on ice until used (within a few hours). A small column (for example, 0.9-cm diameter by 15-cm length) is packed with Sephadex G-25 medium and equilibrated with Tris buffer. It is possible to use prepacked disposable columns (PD-10, Pharmacia); in any case, the column should be discarded after each use. In order to increase the recovery of radiolabeled vWF, a 1-ml volume of normal plasma is filtered through the column before its use, followed by four to five bed volumes of equilibration buffer. The flow rate should be approximately 40 ml/hr. Immediately before use, the tube coated with Iodogen is repeatedly rinsed with Tris buffer to remove excess reagent. The purified vWF solution is then pipetted into the tube (not more than 500/zl), followed by the appropriate amount of Na~25I. We routinely use 0.5 to 1 mCi/mg of vWF. The mixture is kept on ice for 10-12 min. At the end of the incubation time, 500 tzl of KI is applied onto the Sephadex column and, as soon as this solution has entered the gel, the iodination mixture is also applied. Fractions of 0.5 ml are collected and the radioactivity in a 5-~1 sample from each fraction is counted. Radioactive fractions eluting in a peak at the void volume are pooled, the radioactivity of the pool is measured accurately, and the protein concentration is measured to calculate the 13 D. J. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978).
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1
2
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3
FIG. 2. Sodium dodecyl sulfate-agarose gel electrophoresis of radiolabeled vWF. Three samples labeled separately with 125Iare shown. Lane 1 shows a sample with intact maintained multimeric structure. Lane 2 shows a sample with only moderate reduction of the largest multimers. Lane 3 shows a sample with total loss of vWF multimers. Electrophoresis was performed as described above (see Fig. 1), with the exception that the dry gel was exposed to X-ray film directly, without incubating with antibodies specific for vWF.
specific activity, which is usually between 0.2 and 0.4 mCi/mg. ~25I-Labeled vWF is stored in aliquots frozen at - 7 0 ° until used.
Comments von Willebrand factor is susceptible to degradation during and after the iodination procedure. As an example, the multimeric structure of three different preparations of lzSI-labeled vWF is shown in Fig. 2. To obtain meaningful results, only preparations containing all multimeric forms, including the largest, should be used. Therefore, it is advisable to check multimeric structure and/or ristocetin cofactor activity of lzSI-labeled vWF immediately after labeling and also at regular intervals if it is stored for prolonged periods of time. Preparation of Washed Platelets
Reagents Normal human blood: Obtain by clean venipuncture through 19-gauge needles, collect into polypropylene syringes, and transfer immediately
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into polypropylene tubes, mixing 5 vol into 1 vol of acid/citrate/ dextrose anticoagulant, which is prepared as 85 mM trisodium citrate, 65 mM citric acid, 111 mM glucose Bovine serum albumin solution (fraction V, Cat. No. 12659; Calbiochem-Behring Corp.): Aproximately 60% in distilled water, pH 6.5, osmolarity between 290 and 310 mOsm Calcium-free Tyrode's buffer: Two different buffers are used. One is composed of 137 mM NaCI, 2 mM MgCI2, 0.42 mM NaH2PO 4, 11.9 mM NaHCO3, 2.9 mM KCI, 5.5 mM glucose, 10 mM HEPES, pH 6.5. The other has the same composition, but the pH is adjusted to 7.35. Bovine serum albumin, 20 mg/ml, is added to part of the latter buffer Apyrase (grade III; Sigma Chemical Co.) Procedure
This method is a modification of that originally described by Walsh et al.14 Platelet-rich plasma is prepared from the blood as soon as possible after collection by three successive centrifugation steps at 1200 g (rma×) for 60 sec at 22-25 °. Each time the platelet-rich plasma is removed and the blood is recentrifuged without mixing. Apyrase is added to the plateletrich plasma at a final concentration of 5 ATPase units/ml. Albumin density gradients are prepared in flat-bottom polypropylene tubes (10-ml capacity). The albumin solution is used both undiluted and diluted 1:2 and 1:3 in Tyrode's buffer, pH 6.5. Aliquots of each concentration (300/zl) are pipetted into each tube and layered one over the other, starting with the most concentrated. The gradient is rendered less discontinuous by gently mixing at the interface between different layers. Platelet-rich plasma is then layered over the albumin gradient (not more than 6 ml in each tube). Platelets are then sedimented at 1200 g (rma×) for 15-20 min. At the end of the centrifugation the platelets should be collected in a narrow band that partly enters the albumin gradient and is separated from the supernatant plasma by a layer of albumin. The supernatant is discarded and most of the albumin underneath the platelets is gently removed by suction with a siliconized glass pipette. Platelets are gently resuspended in approximately half a volume of Tyrode's buffer, pH 6.5, and apyrase is added at 2 ATPase units/ml. The platelet suspension is again layered onto albumin gradients and the centrifugation repeated. The supernatant is then discarded, the albumin underneath the platelets is removed, the platelets are resuspended in 14 p. N. Walsh, D. C. B. Mills, and J. G. White, Br. J. Haematol. 36, 281 (1977).
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the same volume of Tyrode's buffer, pH 6.5, and apyrase is added at 0.2 ATPase units/ml. Again the platelet suspension is layered onto albumin gradients and the centrifugation repeated for the last time. The supernatant is discarded, albumin underneath the platelets is removed by aspiration, and the platelets are resuspended in the desired volume of Tyrode's buffer, pH 7.35. Starting from 40 to 60 ml of blood, one can expect to have 1-2 ml of washed platelet suspension with a count of 1-2 x 109/ml, depending on the initial count in the platelet-rich plasma. The albumin concentration in the final suspension varies, depending on how carefully the excess has been removed during the washing procedure, but it should be between 3 and 5 mg/ml.
Comments Platelets washed with this procedure routinely give irreversible aggregation when stimulated with ADP (4-10/zM) or epinephrine (10/xM) in the presence of fibrinogen and CaCI2. They retain the ability to respond for about 2 hr or more. Immediately after preparation, these platelets do not aggregate on addition of ristocetin, unless vWF is added to the mixture. Subsequently, some response to ristocetin may become evident even without addition of vWF, due to leakage of endogenous platelet vWF.
Fixation of Washed Platelets
Reagents Tris (0.02 M), 0.15 M NaC1 buffer, pH 7.35 Formaldehyde solution, 2% in Tris buffer, prepared from commercially available formaldehyde (which is usually a 37% solution): Dilute 2 ml of formaldehyde into 98 ml of Tris buffer
Procedure Washed platelets are mixed 1 : 1 (v/v) with formaldehyde and incubated at 37 ° for 1 hr. The mixture is then kept at 4 ° for 12-18 hr. The platelets are then sedimented by centrifugation at 2000 g (rmax) for 15 min at room temperature, and washed twice in Tris buffer. They are finally resuspended in Tris buffer containing 0.02% sodium azide and adjusted to a count of 4-6 x 108/ml. They are stored at 4°.
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Comments Platelets fixed with this technique will agglutinate in response to ristocetin in the presence of vWF for at least 6-8 weeks. It is advisable to wash them once every week during storage, to change the buffer and remove possible products of bacterial contamination (proteases).
Ristocetin-Induced Binding of yon Willebrand Factor to Platelets Reagents Washed platelets, flesh or fixed tzsI-Labeled vWF and unlabeled purified vWF Ristocetin (Sigma Chemical Co.; >90% ristocetin A): Dissolve at a concentration of 15 mg/ml in 0.15 M NaCI Tyrode's buffer, pH 7.35 (as described above), containing 20 mg/ml bovine serum albumin Tris buffer, consisting of 0.02 M Tris, 0.15 M NaCI, pH 7.35 Sucrose: 20% solution in Tyrode's buffer containing albumin Procedure Platelets are used at a final count of 1 × 108/ml. A suitable volume is pipetted from the suspension of washed platelets into 1-ml plastic tubes, to give the desired final number after taking into account the addition of all other reagents. If the starting suspension must be diluted, this is done with Tyrode's buffer containing albumin. 125I-Labeled vWF is then added. In a typical experiment, six concentrations are tested, with doubling amounts of lzSI-labeled vWF from I to 32 t~g/ml (final concentration). Stock solutions are prepared in Tris buffer at 10 times the final concentration, and one-tenth the final volume is then added. Two sets of tubes are prepared. To a series of six tubes, unlabeled vWF is added at a 50-fold excess over the concentration of the 125I-labeled vWF. An equivalent volume of Tris buffer is added to the other series of six tubes. The same volume (one-tenth of final) of ristocetin solution is then pipetted into each tube, to give a final concentration of 1.5 mg/ml. The mixture is gently mixed and then left at room temperature for 30 min, without agitation. In the meantime, sucrose solution (300 t~l) is pipetted into 450-/~1 microcentrifuge tubes with capillary tips (No. 72.702, 47 x 7 mm; Sarstedt, Hayward, CA). Care must be taken that the capillary tips are filled with sucrose solution before applying the radioactive mixture; this can be
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5
~3-
72 0
I
I
5
I
1'0 115 20 :25 30 ~ZSl-vWF Added (/~glml)
FIG. 3. Ristocetin-induced binding of 12SI-labeled vWF to platelets in the presence of monoclonal antibodies. Note that the binding of vWF (at the various concentrations indicated) is blocked by an anti-GPIb monoclonal antibody (&), but not by an antibody against GPIIb-IIIa (0). Control (©).
achieved by centrifuging the tubes. At the end of the incubation, a 50-/zl aliquot of each mixture is pipetted and carefully layered on top of the sucrose solution, in duplicate. The tubes are then centrifuged at 12,000 g (rmax) for 4 min. Platelets sediment through the sucrose solution and pellet at the bottom of the tube. Soluble components of the mixture remain on top of the sucrose solution well separated from the platelets. The portion of the tube tips containing the platelet pellet is cut off with a scalpel or cutter (because the capillary tips have small diameter, there is no spilling of solution after the cut) and the radioactivity associated with the platelet pellet is counted in a y scintillation spectrometer (2 min should be sufficient). The remainder of the tube is also counted to have an objective measurement of the concentration of free vWF. Two 5-/~1 aliquots of each mixture are also counted in toto to determine the total radioactivity present. Comments
The starting solutions of 125I-labeled and unlabeled vWF should be as concentrated as possible, so that relatively small volumes can be used to achieve the final desired concentrations. A practical example is the preparation of a platelet suspension with 5 × 108 platelets/ml (25 /A is used), 125I-labeled vWF in solutions from 10 to 320 ~g/ml (12.5/~1 is used), and unlabeled vWF in solutions from 0.125 to 4 mg/ml (50/~1 is used, to give a 50-fold excess over 125I-labeled vWF). Ristocetin solution (12.5 /.d) and Tyrode's buffer with albumin (25 ~1) complete the experimental mixture. The calculation of the amount of ~25I-labeled vWF bound to platelets
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is done by subtracting the counts bound in the presence of excess unlabeled vWF (representing low-affinity, presumably nonspecific binding) from those bound in the absence of unlabeled vWF (total binding). Theoretically, a greater excess of unlabeled vWF (300-fold) should be used for the estimation of nonspecific binding; this is practically difficult because it requires large volumes of concentrated vWF solution, but can be done for the mixtures containing lower concentrations of radiolabeled vWF. From the known number of platelets in the pellet (5 × 106 if this procedure is followed) and the specific activity of 125I-labeled vWF, one can calculate the amount of vWF bound (specific binding). This result is usually expressed as micrograms per 108 platelets. The amount of free 125I-labeled vWF remaining in each mixture is calculated by determining the difference between the amount added and the amount bound to the platelets, and is also measured objectively as described above. The procedure described here can be modified to include other reagents, such as monoclonal antibodies that allow definition of the receptor specificity for ristocetin-induced binding of vWF to platelets. In Fig. 3, binding of 125I-labeled vWF to platelets has been blocked by anti-GPIb monoclonal antibody, but not by antibody to GPIIb-IIIa. This is evidence for the existence of more than one binding site for vWF on platelets, a conclusion also supported by studies with platelets deficient in GPIb or GPIIb-IIIa. 2'15 Because vWF is a multimeric glycoprotein and, therefore, a potential multivalent ligand, the interpretation of the results of binding studies using mathematical models described for monovalent ligands (Scatchard-type analysis) is potentially misleading. Nevertheless, if this type of analysis is applied to vWF-binding isotherms, the results demonstrate the existence of a single class of noninteracting binding sites with excellent linear fit. 16.17 This suggests that the binding of vWF to GPIb occurs through independent, even though potentially multiple, monovalent interactions. Scatchard-type analysis of binding isotherms can be performed with a computer-assisted program. ~8'~9 In this case, the nonsaturable (nonspecific) component of binding can be calculated from the total binding isotherm, avoiding the use of unlabeled vWF.
15 Z. M. Ruggeri, R. Bader, and L. De Marco, Proc. Natl. Acad. Sci. U.S.A. 79, 6038 (1982). ~6A. B. Federici, R. Bader, S. Pagani, M. L. Colibretti, L. De Marco, and P. M. Mannucci, Br. J. Haernatol. 73, 93 (1989). t7 L. De Marco, M. Mazzucato, D. De Roia, A. Casonato, A. B. Federici, A. Girolami, and Z. M. Ruggeri, J. Clin. Invest. 86, 785 (1990). 18 p. j. Munson and D. Rodbard, Anal. Biochem. 107, 220 (1980). 19 p. j. Munson, this series, Vol. 92, p. 542.
PLATELET GPIb COMPLEX-vWFINTERACTION
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subunits is centrifuged for 10 min at 4° in an Eppendorf microcentrifuge, loaded onto the column, and eluted with the 0.05% Triton X-100 buffer. Figure 5 shows a typical separation of GPIIb from GPIIIa. Ten percent of the GPIIb peak is GPIIIa; 5% of the GPIIIa peak is GPIIb. Reassociation of the isolated glycoproteins requires immediate readdition of C a 2+ (6 m M ) to the fractions obtained from the high-performance liquid chromatography column. When isolated GPIIb is mixed with isolated GPIIIa, 10 to 15% of the dissociated glycoproteins reform the heterodimeric complex. Acknowledgments This work was supported in part by Grants HL 28947 and HL 32254 form the National Institutes of Health. The authors thank Barbara Allen and Sally Gullatt Seehafer for editorial assistance, James X. Warger and Norma Jean Gargasz for graphics, and Kate Sholly, Michele Prator, Linda Harris Odumade, and Linda Parker for manuscript preparation.
[23] v o n W i l l e b r a n d F a c t o r B i n d i n g to P l a t e l e t G l y c o p r o t e i n Ib Complex B y ZAVERIO M. RUGGERI, THEODORE S. ZIMMERMAN, 1 SUSAN RUSSELL, ROSSELLA BADER, a n d LUIGI DE MARCO
von Willebrand factor (vWF) is a complex multimeric glycoprotein that plays an essential role in platelet function. ~a It is required for normal platelet adhesion to exposed subendothelium and for normal platelet plug formation at sites of vascular injury. The function of vWF is particularly important in vessels of small caliber, where conditions of high wall shear rate prevail. It is now recognized that the mechanisms underlying vWF function comprise interaction with components of the subendothelium as well as with specific receptors on the platelet membrane. Two distinct vWF-binding sites have been recognized so far on platelets, one related to the membrane glycoprotein (GP) Ib and the other to the heterodimeric GPIIb-IIIa complex. 2 Deceased. la Z.M. Ruggeri and T. S. Zimmerman, Blood 70, 895 (1970). 2 Z.M. Ruggeri, L. DeMarco, L. Gatti, R. Bader, and R. R. Montgomery, J. Clin. lnvest. 72, 1 (1983).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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This chapter deals with reviewing the methodology involved in studying the vWF interaction with GPIb. von Willebrand factor binds to GPIb in the presence of the antibiotic ristocetin. Such binding correlates with the occurrence ofplatelet aggregation induced by ristocetin in the presence of vWF. 3 The latter function of vWF is often referred to as the ristocetin cofactor activity. If platelets are metabolically inactive, as after fixation with formalin, they will nonetheless still respond to ristocetin in the presence of vWF with what is more properly called agglutination. Therefore, the ristocetin-dependent interaction of vWF with GPIb can be analyzed using either fresh, metabolically active platelets or fixed, metabolically inactive platelets. Ristocetin, however, is not the only tool with which to study vWF binding to GPIb under experimental conditions. Following removal of sialic acid residues from the vWF glycoprotein, the resulting asialo-vWF exhibits the ability to interact directly with GPIb. 4 It is, therefore, apparent that GPIb-related binding sites for vWF are exposed on the membrane of unstimulated platelets and can interact with a modified form of the molecule. The concept that a modification of the vWF molecule can be the initial event promoting its interaction with platelets is a stimulating alternative, or perhaps addition, to the concept that a modification of the platelet surface or microenvironment, as possibly brought about by ristocetin, 5'6 is necessary to promote vWF binding to GPIb. Indeed, it has also been demonstrated that another substance with the property of inducing vWF binding to GPIb, the snake protein botrocetin, 7 exerts its function by forming a bimolecular complex with vWF that in turn binds to platelets7'8; botrocetin itself does not interact with the platelet surface. 8 Using asialo-vWF, it is also possible to demonstrate that receptors on GPIIb-IIIa are exposed following vWF binding to GPIb. 4 This constitutes a potentially important link between two major binding sites for adhesive glycoproteins on the platelet membrane. The procedures described in this chapter relate to the general methodology for studying receptor-ligand interaction. The basic constituents of the experimental system are, in this case, highly purified vWF and platelets washed free, as much as possible, of other blood components. The vWF to be used as a tracer is radiolabeled with 125I. As mentioned above, 3 K.-J. Kao, S. V. Pizzo, and P. A. McKee, Proc. Natl. Acad. Sci. U.S.A. 76, 5317 (1979). 4 L. De Marco, A. Girolami, S. Russell, and Z. M. Ruggeri, J. Clin. Invest. 75, 1198 (1985). 5 S. E. Senogles and G. L. Nelsestuen, J. Biol. Chem. 258, 12327 (1983). 6 B. S. Coller, J. Clin. Invest. 61, 1168 (1978). 7 M. S. Read, S. V. Smith, M. A. Lamb, and K. M. Brinkhous, Blood 74, 1031 (1989). 8 M. Sugimoto, G. Ricca, M. E. Hrinda, A. B. Schreiber, G. H. Searfoss, E. Bottini, and Z. M. Ruggeri, Biochemistry 30, 5202 (1991).
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washed platelets can be either fresh or fixed. A detailed description of the methods used to prepare these reagents and to study ristocetin-induced binding of vWF to platelets is presented. The procedure used to prepare asialo-vWF and to study its interaction with platelets, as well as that to measure botrocetin-mediated vWF binding to platelets, will not be described here but have been the subjects of previous reports/'8'9 Botrocetin-induced platelet agglutination/aggregation is also described in [12] in Volume 169 of this series.
Purification of von Willebrand Factor
Reagents Human plasma cryoprecipitate (the source of vWF): It can be obtained as such or, if blood bank facilities are available, it can be prepared from flesh plasma using the procedure of Pool and Shanon. l0 Aluminum hydroxide (Rehsorptar from Armour Pharmaceutical Co., Kankakee, IL) Bentonite (B-3378 from Sigma Chemical Co., St. Louis, MO) Polyethylene glycol (P-2139, average M r 8000; Sigma): A 40% (w/v) solution is prepared in imidazole buffer (see below) Sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, N J) Citrate buffer: 0.02 M Tris, 0.02 M trisodium citrate, 0.02 M e-aminocaproic acid, pH 7.0 Imidazole buffer: 0.02 M imidazole, 0.01 M trisodium citrate, 0.02 M eaminocaproic acid, 0.15 M NaC1, pH 6.5; sodium azide at a concentration of 0.02% (w/v) is added as a bacteriostatic agent Tris buffer: 0.02 M Tris, 0.15 M NaC1, pH 7.35
Procedure The method is a modification of two published procedures, ~J,j2 and begins with 1000 ml of cryoprecipitate, from which a final yield of 15 to 20 mg of purified vWF is expected. The cryoprecipitate is thawed at 0-4 ° (approximately 18 hr) and then centrifuged at 9800 g (rmax) for 45 min at 4°. The supernatant is discarded and the precipitate is dissolved with several additions of citrate buffer, up to a final volume of 300 ml. The 9 L. De Marco and S. S. Shapiro, J. Clin. Invest. 168, 321 (1981). to j. G. Pool and A. E. Shanon, N. Engl. J. Med. 273, 1443 (1965). tt j. Newman, A. J. Johnson, M. H. Karpatkin, and S. Puszkin, Br. J. Haematol. 21, 1 (1971). t2 M. E. Switzer and P. A. McKee, J. Clin. Invest. 57, 925 (1976).
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solution is transferred to a plastic beaker and Rehsorptar is added dropwise (5 ml/100 ml of redissolved precipitate) while stirring. The mixture is further stirred for 10 min at room temperature (22-25°), then transferred into plastic bottles and centrifuged at 7500 g (rmax) for 30 min at 20°. In the meantime, bentonite (2.5 mg/ml of supernatant) is placed in clean plastic centrifuge bottles and the powder is mixed with a small amount of citrate buffer to obtain a slurry. At the end of the centrifugation, the supernatant is poured directly into the bottles containing the bentonite, quickly mixed, and the mixtrue rapidly centrifuged at 3800 g (rmax) for 5 min at 20°. The supernatant from the last centrifugation is transferred to a plastic beaker and the pH is adjusted to 6.5 by slow addition of 0.02 M citric acid. A volume of the 40% polyethylene glycol solution is added dropwise, under continuous stirring, to give a final concentration of 3% (w/v) polyethylene glycol. The mixture is stirred for an additional 10 min at room temperature and then centrifuged at 3800 g (rmax) for 15 min at 20°. The supernatant is recovered and transferred to a plastic beaker and additional polyethylene glycol is added following the same procedure to give a final concentration of 15%. After stirring for an additional 20 min at room temperature, the mixture is centrifuged at 9800 g (rmax) for 30 min at room temperature. At the end of centrifugation, the supernatant is discarded and the precipitate is redissolved in the smallest possible volume of imidazole buffer. The precipitate obtained from 1 liter of cryoprecipitate can usually be redissolved in a final volume of approximately 30-35 ml. Complete dissolution usually takes 90-180 min with continuous, but gentle, agitation on a shaker. The dissolved precipitate is ultracentrifuged at 113,000 g (/'max) for 45 min at 20°, after which the lipids floating on top are discarded and the clear solution is separated from the insoluble material at the bottom of the tube. The clear solution is then applied onto a Sepharose CL-4B column, equilibrated with imidazole buffer. A 100-cm-long siliconized glass column, 5 cm in diameter and with a bed volume of approximately 1700 ml, is used (the actual agarose column is approximately 80 cm long). The column is run at a flow rate of 60 ml/hr at room temperature, usually overnight for convenience. The optical density of the effluent is continuously monitored at 280 nm and 10-ml fractions are collected. The asymmetric peak appearing at the void volume contains the purified vWF. The column is developed until all proteins are eluted. It is then washed with at least three bed volumes of buffer and finally stored with buffer containing 0.1% (w/v) sodium azide. von Willebrand factor is a multimeric protein and consists of molecular forms of varying molecular weight. Figure 1 shows the distribution of
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PLATELET GPIb COMPLEX--vWF INTERACTION
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I
I
I
267
I
0.2-
~
o.1
o
:
;
5b Effluent, ml
FZG. 1. Multimeric structure of vWF eluting from the gel-filtrationcolumn. Lower: Elution profile of the protein peak appearing at the void volume, detected by light absorbance at 280 nm. Upper: SDS-agarose gel electrophoresis of samples corresponding to different positions of the elution profile, as indicated by black bars. Note that the largest multimers appear first. Cathode at the top. Electrophoresis was performed as described in [21] in Volume 169 of this series.
multimers of different size across the v W F peak eluting from the Sepharose column. Usually, all the fractions corresponding to the ascending part and the first half of the descending part of the p e a k are pooled. Although the v W F p e a k is well separated f r o m the subsequent one, it is preferable to discard the later eluting fractions to decrease the likelihood of contaminating proteins in the v W F preparation. The protein concentration in the v W F pool obtained from the gelfiltration column is usually b e t w e e n 0.1 and 0.2 mg/ml. Because a higher concentration usually is needed, particularly for labeling the protein, the v W F must be concentrated. The m o s t effective method is to transfer the v W F solution into regular dialysis tubing and then surround it with the
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hygroscopic agent Aquacide II, obtained from Calbiochem-Behring Corporation (La Jolla, CA). When sufficient water is removed to achieve the desired concentration, the dialysis tubing is rinsed with distilled water and the vWF is dialyzed against Tris buffer. The vWF preparation is then aliquoted and stored at - 7 0 °. Comments
According to individual needs, smaller amounts of starting cryoprecipitate or plasma can be used. In that case, a gel-filtration column of smaller diameter can be employed, but its length should not be decreased. If the protein concentration in the vWF preparation is derived from the optical density (OD), the following equation, which takes into account a correction for light scattering, should be used: OD280 -
(1.7 x OD320)/0.7 = vWF concentration (mg/ml)
(1)
The method used to analyze the multimeric structure of vWF is described in detail in [21] in Volume 169 of this series. In view of the heterogeneous nature of vWF and the fact that manipulation of the molecule can lead to loss of the largest multimer, it is advisable to analyze the multimeric composition of each purified vWF preparation before use. In fact, loss of the largest multimers affects the functional properties of vWF. If multimeric analysis cannot be performed, one alternative is to measure the ristocetin cofactor activity of the purified preparation. If no contaminants are present and the preparation has a normal multimeric distribution, the specific activity should be greater than 100 units of ristocetin cofactor per milligram of v W F . 4 It is not clear whether the use of protease inhibitors during the purification procedure is of any help in achieving the goal of obtaining vWF with the full complement of multimers, including the largest. Nevertheless, protease inhibitors now are routinely added to the cryoprecipitate when it is first thawed out. The following inhibitors are used, all from CalbiochemBehring, and all at a final concentration of 10/xM in the cryoprecipitate: Dphenylalanyl-L-prolyl-L-arginine chloromethyl ketone, dansyl-L-glutamylL-glycyl-L-arginine chloromethyl ketone, and D-phenylalanyl-L-phenylalanyl-L-arginine chloromethyl ketone, as well as leupeptin (Chemicon, Los Angeles, CA), at a final concentration of 15/zg/ml. The same inhibitors, but at one-tenth the final concentration, may also be added to the imidazole buffer used to resuspend the final precipitate and to equilibrate and run the gel-filtration column.
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Labeling of yon Willebrand Factor
Reagents
1,3,4,6-Tetrachloro-3a,6a-diphenylglycouril (Iodogen; Pierce Chemical Co., Rockford, IL): A 1-mg/ml solution is prepared in dichloromethane. It can be stored sealed and protected from light for several weeks at - 20° Sephadex G-25 Medium (Pharmacia) Tris (0.02 M)-0.15 M NaC1 buffer, pH 7.35 KI: 10 mg/ml in distilled water NalZSI, carrier free (Amersham, Arlington Heights, IL)
Procedure The method described here is slightly modified from that previously published by Fraker and Speck. 13The whole procedure is performed under a fume hood in a laboratory designated for protein iodination. Between I00 and 400/xl of Iodogen solution is pipetted into a glass tube or vial, and the dichloromethane is evaporated under a stream of nitrogen. The tube is sealed, protected from light, and kept on ice until used (within a few hours). A small column (for example, 0.9-cm diameter by 15-cm length) is packed with Sephadex G-25 medium and equilibrated with Tris buffer. It is possible to use prepacked disposable columns (PD-10, Pharmacia); in any case, the column should be discarded after each use. In order to increase the recovery of radiolabeled vWF, a 1-ml volume of normal plasma is filtered through the column before its use, followed by four to five bed volumes of equilibration buffer. The flow rate should be approximately 40 ml/hr. Immediately before use, the tube coated with Iodogen is repeatedly rinsed with Tris buffer to remove excess reagent. The purified vWF solution is then pipetted into the tube (not more than 500/zl), followed by the appropriate amount of Na~25I. We routinely use 0.5 to 1 mCi/mg of vWF. The mixture is kept on ice for 10-12 min. At the end of the incubation time, 500 tzl of KI is applied onto the Sephadex column and, as soon as this solution has entered the gel, the iodination mixture is also applied. Fractions of 0.5 ml are collected and the radioactivity in a 5-~1 sample from each fraction is counted. Radioactive fractions eluting in a peak at the void volume are pooled, the radioactivity of the pool is measured accurately, and the protein concentration is measured to calculate the 13 D. J. Fraker and J. C. Speck, Biochem. Biophys. Res. Commun. 80, 849 (1978).
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1
2
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3
FIG. 2. Sodium dodecyl sulfate-agarose gel electrophoresis of radiolabeled vWF. Three samples labeled separately with 125Iare shown. Lane 1 shows a sample with intact maintained multimeric structure. Lane 2 shows a sample with only moderate reduction of the largest multimers. Lane 3 shows a sample with total loss of vWF multimers. Electrophoresis was performed as described above (see Fig. 1), with the exception that the dry gel was exposed to X-ray film directly, without incubating with antibodies specific for vWF.
specific activity, which is usually between 0.2 and 0.4 mCi/mg. ~25I-Labeled vWF is stored in aliquots frozen at - 7 0 ° until used.
Comments von Willebrand factor is susceptible to degradation during and after the iodination procedure. As an example, the multimeric structure of three different preparations of lzSI-labeled vWF is shown in Fig. 2. To obtain meaningful results, only preparations containing all multimeric forms, including the largest, should be used. Therefore, it is advisable to check multimeric structure and/or ristocetin cofactor activity of lzSI-labeled vWF immediately after labeling and also at regular intervals if it is stored for prolonged periods of time. Preparation of Washed Platelets
Reagents Normal human blood: Obtain by clean venipuncture through 19-gauge needles, collect into polypropylene syringes, and transfer immediately
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into polypropylene tubes, mixing 5 vol into 1 vol of acid/citrate/ dextrose anticoagulant, which is prepared as 85 mM trisodium citrate, 65 mM citric acid, 111 mM glucose Bovine serum albumin solution (fraction V, Cat. No. 12659; Calbiochem-Behring Corp.): Aproximately 60% in distilled water, pH 6.5, osmolarity between 290 and 310 mOsm Calcium-free Tyrode's buffer: Two different buffers are used. One is composed of 137 mM NaCI, 2 mM MgCI2, 0.42 mM NaH2PO 4, 11.9 mM NaHCO3, 2.9 mM KCI, 5.5 mM glucose, 10 mM HEPES, pH 6.5. The other has the same composition, but the pH is adjusted to 7.35. Bovine serum albumin, 20 mg/ml, is added to part of the latter buffer Apyrase (grade III; Sigma Chemical Co.) Procedure
This method is a modification of that originally described by Walsh et al.14 Platelet-rich plasma is prepared from the blood as soon as possible after collection by three successive centrifugation steps at 1200 g (rma×) for 60 sec at 22-25 °. Each time the platelet-rich plasma is removed and the blood is recentrifuged without mixing. Apyrase is added to the plateletrich plasma at a final concentration of 5 ATPase units/ml. Albumin density gradients are prepared in flat-bottom polypropylene tubes (10-ml capacity). The albumin solution is used both undiluted and diluted 1:2 and 1:3 in Tyrode's buffer, pH 6.5. Aliquots of each concentration (300/zl) are pipetted into each tube and layered one over the other, starting with the most concentrated. The gradient is rendered less discontinuous by gently mixing at the interface between different layers. Platelet-rich plasma is then layered over the albumin gradient (not more than 6 ml in each tube). Platelets are then sedimented at 1200 g (rma×) for 15-20 min. At the end of the centrifugation the platelets should be collected in a narrow band that partly enters the albumin gradient and is separated from the supernatant plasma by a layer of albumin. The supernatant is discarded and most of the albumin underneath the platelets is gently removed by suction with a siliconized glass pipette. Platelets are gently resuspended in approximately half a volume of Tyrode's buffer, pH 6.5, and apyrase is added at 2 ATPase units/ml. The platelet suspension is again layered onto albumin gradients and the centrifugation repeated. The supernatant is then discarded, the albumin underneath the platelets is removed, the platelets are resuspended in 14 p. N. Walsh, D. C. B. Mills, and J. G. White, Br. J. Haematol. 36, 281 (1977).
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the same volume of Tyrode's buffer, pH 6.5, and apyrase is added at 0.2 ATPase units/ml. Again the platelet suspension is layered onto albumin gradients and the centrifugation repeated for the last time. The supernatant is discarded, albumin underneath the platelets is removed by aspiration, and the platelets are resuspended in the desired volume of Tyrode's buffer, pH 7.35. Starting from 40 to 60 ml of blood, one can expect to have 1-2 ml of washed platelet suspension with a count of 1-2 x 109/ml, depending on the initial count in the platelet-rich plasma. The albumin concentration in the final suspension varies, depending on how carefully the excess has been removed during the washing procedure, but it should be between 3 and 5 mg/ml.
Comments Platelets washed with this procedure routinely give irreversible aggregation when stimulated with ADP (4-10/zM) or epinephrine (10/xM) in the presence of fibrinogen and CaCI2. They retain the ability to respond for about 2 hr or more. Immediately after preparation, these platelets do not aggregate on addition of ristocetin, unless vWF is added to the mixture. Subsequently, some response to ristocetin may become evident even without addition of vWF, due to leakage of endogenous platelet vWF.
Fixation of Washed Platelets
Reagents Tris (0.02 M), 0.15 M NaC1 buffer, pH 7.35 Formaldehyde solution, 2% in Tris buffer, prepared from commercially available formaldehyde (which is usually a 37% solution): Dilute 2 ml of formaldehyde into 98 ml of Tris buffer
Procedure Washed platelets are mixed 1 : 1 (v/v) with formaldehyde and incubated at 37 ° for 1 hr. The mixture is then kept at 4 ° for 12-18 hr. The platelets are then sedimented by centrifugation at 2000 g (rmax) for 15 min at room temperature, and washed twice in Tris buffer. They are finally resuspended in Tris buffer containing 0.02% sodium azide and adjusted to a count of 4-6 x 108/ml. They are stored at 4°.
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Comments Platelets fixed with this technique will agglutinate in response to ristocetin in the presence of vWF for at least 6-8 weeks. It is advisable to wash them once every week during storage, to change the buffer and remove possible products of bacterial contamination (proteases).
Ristocetin-Induced Binding of yon Willebrand Factor to Platelets Reagents Washed platelets, flesh or fixed tzsI-Labeled vWF and unlabeled purified vWF Ristocetin (Sigma Chemical Co.; >90% ristocetin A): Dissolve at a concentration of 15 mg/ml in 0.15 M NaCI Tyrode's buffer, pH 7.35 (as described above), containing 20 mg/ml bovine serum albumin Tris buffer, consisting of 0.02 M Tris, 0.15 M NaCI, pH 7.35 Sucrose: 20% solution in Tyrode's buffer containing albumin Procedure Platelets are used at a final count of 1 × 108/ml. A suitable volume is pipetted from the suspension of washed platelets into 1-ml plastic tubes, to give the desired final number after taking into account the addition of all other reagents. If the starting suspension must be diluted, this is done with Tyrode's buffer containing albumin. 125I-Labeled vWF is then added. In a typical experiment, six concentrations are tested, with doubling amounts of lzSI-labeled vWF from I to 32 t~g/ml (final concentration). Stock solutions are prepared in Tris buffer at 10 times the final concentration, and one-tenth the final volume is then added. Two sets of tubes are prepared. To a series of six tubes, unlabeled vWF is added at a 50-fold excess over the concentration of the 125I-labeled vWF. An equivalent volume of Tris buffer is added to the other series of six tubes. The same volume (one-tenth of final) of ristocetin solution is then pipetted into each tube, to give a final concentration of 1.5 mg/ml. The mixture is gently mixed and then left at room temperature for 30 min, without agitation. In the meantime, sucrose solution (300 t~l) is pipetted into 450-/~1 microcentrifuge tubes with capillary tips (No. 72.702, 47 x 7 mm; Sarstedt, Hayward, CA). Care must be taken that the capillary tips are filled with sucrose solution before applying the radioactive mixture; this can be
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5
~3-
72 0
I
I
5
I
1'0 115 20 :25 30 ~ZSl-vWF Added (/~glml)
FIG. 3. Ristocetin-induced binding of 12SI-labeled vWF to platelets in the presence of monoclonal antibodies. Note that the binding of vWF (at the various concentrations indicated) is blocked by an anti-GPIb monoclonal antibody (&), but not by an antibody against GPIIb-IIIa (0). Control (©).
achieved by centrifuging the tubes. At the end of the incubation, a 50-/zl aliquot of each mixture is pipetted and carefully layered on top of the sucrose solution, in duplicate. The tubes are then centrifuged at 12,000 g (rmax) for 4 min. Platelets sediment through the sucrose solution and pellet at the bottom of the tube. Soluble components of the mixture remain on top of the sucrose solution well separated from the platelets. The portion of the tube tips containing the platelet pellet is cut off with a scalpel or cutter (because the capillary tips have small diameter, there is no spilling of solution after the cut) and the radioactivity associated with the platelet pellet is counted in a y scintillation spectrometer (2 min should be sufficient). The remainder of the tube is also counted to have an objective measurement of the concentration of free vWF. Two 5-/~1 aliquots of each mixture are also counted in toto to determine the total radioactivity present. Comments
The starting solutions of 125I-labeled and unlabeled vWF should be as concentrated as possible, so that relatively small volumes can be used to achieve the final desired concentrations. A practical example is the preparation of a platelet suspension with 5 × 108 platelets/ml (25 /A is used), 125I-labeled vWF in solutions from 10 to 320 ~g/ml (12.5/~1 is used), and unlabeled vWF in solutions from 0.125 to 4 mg/ml (50/~1 is used, to give a 50-fold excess over 125I-labeled vWF). Ristocetin solution (12.5 /.d) and Tyrode's buffer with albumin (25 ~1) complete the experimental mixture. The calculation of the amount of ~25I-labeled vWF bound to platelets
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is done by subtracting the counts bound in the presence of excess unlabeled vWF (representing low-affinity, presumably nonspecific binding) from those bound in the absence of unlabeled vWF (total binding). Theoretically, a greater excess of unlabeled vWF (300-fold) should be used for the estimation of nonspecific binding; this is practically difficult because it requires large volumes of concentrated vWF solution, but can be done for the mixtures containing lower concentrations of radiolabeled vWF. From the known number of platelets in the pellet (5 × 106 if this procedure is followed) and the specific activity of 125I-labeled vWF, one can calculate the amount of vWF bound (specific binding). This result is usually expressed as micrograms per 108 platelets. The amount of free 125I-labeled vWF remaining in each mixture is calculated by determining the difference between the amount added and the amount bound to the platelets, and is also measured objectively as described above. The procedure described here can be modified to include other reagents, such as monoclonal antibodies that allow definition of the receptor specificity for ristocetin-induced binding of vWF to platelets. In Fig. 3, binding of 125I-labeled vWF to platelets has been blocked by anti-GPIb monoclonal antibody, but not by antibody to GPIIb-IIIa. This is evidence for the existence of more than one binding site for vWF on platelets, a conclusion also supported by studies with platelets deficient in GPIb or GPIIb-IIIa. 2'15 Because vWF is a multimeric glycoprotein and, therefore, a potential multivalent ligand, the interpretation of the results of binding studies using mathematical models described for monovalent ligands (Scatchard-type analysis) is potentially misleading. Nevertheless, if this type of analysis is applied to vWF-binding isotherms, the results demonstrate the existence of a single class of noninteracting binding sites with excellent linear fit. 16.17 This suggests that the binding of vWF to GPIb occurs through independent, even though potentially multiple, monovalent interactions. Scatchard-type analysis of binding isotherms can be performed with a computer-assisted program. ~8'~9 In this case, the nonsaturable (nonspecific) component of binding can be calculated from the total binding isotherm, avoiding the use of unlabeled vWF.
15 Z. M. Ruggeri, R. Bader, and L. De Marco, Proc. Natl. Acad. Sci. U.S.A. 79, 6038 (1982). ~6A. B. Federici, R. Bader, S. Pagani, M. L. Colibretti, L. De Marco, and P. M. Mannucci, Br. J. Haernatol. 73, 93 (1989). t7 L. De Marco, M. Mazzucato, D. De Roia, A. Casonato, A. B. Federici, A. Girolami, and Z. M. Ruggeri, J. Clin. Invest. 86, 785 (1990). 18 p. j. Munson and D. Rodbard, Anal. Biochem. 107, 220 (1980). 19 p. j. Munson, this series, Vol. 92, p. 542.