Demonstration and Characterization ofSpecific Binding Sites forFactor VIII/von Willebrand Factor on Human Platelets

KUO-JANGKAo, S. V. Pizzo, and PATRICK A. MCKEE, Departments of Medicine,Biochemistry, and Pathology, Duke University Medical Center, Durham,North Carolina 27710

A B S T RA C T The presence of specific Factor VIII/von Willebrand factor (FVIII/vWF) binding sites onhuman platelets has been demonstrated by using 125I1FVIII/vWF and washed human platelets. Binding isristocetin-dependent and increases in proportion to theconcentration of ristocetin from 0.2 to 1 mg/ml. Bindingof '251-FVIII/vWF to platelets can be competitivelyinhibited by unlabeled human or bovine FVIII/vWF,but not by human thrombin, fibrinogen, a2-macro-globulin, equine collagen, or a lectin of Ricinuscommunis. Scatchard analysis of binding data indicatedthat the dissociation constant of FVIII/vWF receptorsis 0.45-0.5 nM. There are 31,000 binding sites perplatelet at 1 mg/ml of ristocetin concentration. Theoptimal pH range for binding is from 7.0 to 7.5. At aconcentration of 2 mM, EGTA inhibits 86% of thebinding; however, 20 mMof Ca++, Mg++, or EDTAhave little effect. Binding sites for FVIII/vWF werefound only on platelets, and no significant binding wasdetected with human erythrocytes or polymorpho-nuclear leukocytes.

INTRODUCTION

Human Factor VIII/von Willebrand factor (FVIII/vWF)1 is a plasma glycoprotein which has a mol wt

1.1 x 106 and is composed of an undetermined num-ber of 200,000-dalton subunits linked covalently bydisulfide bonds (1, 2). This glycoprotein has two

Dr. McKee is an Investigator ofthe Howard Hughes MedicalInstitute.

Received for publication 5 September 1978 and in revisedform 15 December 1978.

'Abbreviations used in this paper: BSA, bovine serumalbumin; FVIII/vWF, Factor VIII/von Willebrand Factor;PEG, polyethylene glycol; PMN, polymorphonuclear leu-kocyte(s); RBC, erythrocytes.

656

distinct biological activities: one being procoagulantactivity (Factor VIII) which can specifically correctthe blood coagulation defect of classical hemophiliapatients; the other being platelet-aggregating activity,also known as von Willebrand factor activity, which isimportant for platelet plug formation at the site ofvascular injury (3, 4). In von Willebrand's disease,a marked decrease in platelet-aggregating activityresults in easy bruising, hemorrhage from mucousmembranes, and a prolonged bleeding time (5); thesedefects can usually be corrected by administrationof human FVIII/vWF concentrates. In 1973, Howardet al. (6) reported that the in vitro addition of anantibiotic, ristocetin, to normal platelet-rich plasmacaused platelets to clump. In contrast, the addition ofristocetin promoted little or no clumping activity inthe majority of platelet-rich plasma samples frompatients with von Willebrand's disease (6). These ob-servations were used to develop methods for quan-titating plasma von Willebrand factor activity bymeasuring the rate or degree of aggregation ofwashed, normal human platelets to which aliquots oftest plasma and ristocetin were added (7). At present,the mechanism by which FVIII/vWF protein interactswith platelets to cause aggregate formation in vivo orin vitro is still unknown. Nevertheless, immuno-fluorescent techniques (8) and the recovery of FVIII/vWF activity from platelet membrane (9) or plateletaggregates (10, 11) suggest that FVIII/vWF binds tohuman platelets. Such results prompted the hypothesisthat binding of FVIII/vWF to specific sites on humanplatelets may be required for normal platelet aggrega-tion to occur.

Wenow report evidence for the presence of specificbinding sites for FVIII/vWF on human platelets, usinga competitive, radiolabeled ligand binding assay. Someof the characteristics of these binding sites are alsodescribed.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738179/0410656/09 $1.00Volume 63 April 1979 656-664

METHODS

Materials. Intermediate purity human FVII1/vWF con-centrates (900 U/vial) and purified human thrombin (180U/vial) were obtained from American National Red Cross, Be-thesda, Md., and the Bureau of Biologics, Bethesda, Md., re-spectively. Bovine lactoperoxidase was obtained from Cal-biochem-Behring Corp., American Hoechst Corp., San Diego,Calif. A lectin of Ricinus communis (RC-60) was from BoehringerMannheim Biochemicals, Indianapolis, Ind. Human fibrinogen(grade L) was purchased from A. B. Kabi, Stockholm, Sweden.Equine collagen was purchased from Hormon-Chemie,Munich, West Germany. Bovine serum albumin (BSA),EGTA, EDTA, and diisopropylfluorophosphate were pur-chased from Sigma Chemical Co., St. Louis, Mo. Risto-cetin was purchased from Bio/Data Corp., Horsham, Pa.Carrier-free Na125I (20 mCi/ml) was from New EnglandNuclear, Boston, Mass. a-Chymotrypsin (40 U/mg) wasfrom Worthington Biochemical Corp., Freehold, N. J. Bio-Gel A-15m was from Bio-Rad Laboratories, Richmond,Calif. Sepharose 4B and Sephadex G-25 were from PharmaciaFine Chemicals, Div. of Pharmacia Inc., Piscataway, N. J.Lymphocyte separation medium and Hank's balanced saltsolution were purchased from Bionetics Laboratory Prod-ucts, Litton Bionetics Inc., Kensington, Md. and GibcoDiagnostics, Gibco Invenex Div., Chagrin Falls, Ohio,respectively. Plasma gel was from Roger Bellon Laboratoire,France. Polyethylene glycol (Carbowax 4000) was obtainedfrom Union Carbide Corp., Carbon Products Div., New York.Highly purified bovine FVIII/vWF was a gift from Dr. E. W.Davie, Department of Biochemistry, University of Washington,Seattle, Wash. All other reagents were of analytical grade orthe best grade available.

Purification of FVIII/cWIF protein. The purification pro-cedures for human FVIII/vWF protein from intermediatepurity concentrates were the same as previously describedfrom this laboratory (12) with some slight modifications.Polyethylene containers were used throughout, exceptwhere it is specifically stated that siliconized glasswarewas used. Lyophilized, intermediate purity FVIII/vWF con-centrate, containing 1,800 U of Factor VIII procoagulantactivity, was dissolved in 150 ml of distilled water. ThepH of the solution was adjusted to 6.1 with 0.02 M citricacid and then diluted with 2 vol 0.02 M sodium citrate(pH 6.1). Polyethylene glycol (PEG), 40% (wt/vol) in H2O,was added dropwise to this solution to give a final PEGconcentration of 5%. After 15 min, the solution was cen-trifuged at 7,000g for 10 min. The supernate was removed,and 40% PEG was added to bring the final PEG concen-tration to 12%. The solution was left at room temperaturefor 30 min and then centrifuged at 7,000 g for 10 min. Theprecipitate was placed on ice; gently washed once with ice-cold 0.02 M Tris-HCl buffer (pH 7.4); made 8% inethanol, to remove residual PEG; and then dissolved in 25ml of a 0.05-M Tris-HCl, 0.15-M NaCl, 1 mM-diisopropyl-fluorophosphate, and 0.02% NaN3 solution (pH 7.4). Thissolution was applied to 4% agarose (Bio-Gel A-15m)packed in a 5 x 50-cm siliconized glass column. Thecolumn was eluted with a solution of 0.05 M Tris-HCI, 0.15 M NaCl, and 0.02% of NaN3 (pH 7.4) at a flowrate of 25 ml/h. The void-volume fractions, which containedthe protein peak having FVIII/vWF activity, were pooledand precipitated at 4°C by adding solid ammonium sulfateuntil 35% saturation was attained. After 8 h at 4°C theprecipitate was collected by centrifugation at 12,000g for10 min and dissolved in 0.025 M cacodylate, 0.15 M NaClbuffer (pH 6.8). Residual ammonium sulfate in the precipi-tate was removed by dialysis at 4°C against the same

buffer. The purified FVIII/vWF was stored at 40C in a finalconceentration of 1.5-2.5 mg/ml. The purity of the final prodluetwas anialyzed by electrophoresis on sodium dodecyl sulfate-6M urea-5% polyacrylamide gels before and after reductionby 83-mercaptoethanol (12, 13).

Radioioditnaition of FVIII/vcW'F. Highly purified FVIII/vWF was labeled with 125j by the Sepharose 4B-lacto-peroxidase method of David and Reisfeld (14). The iodina-tion was performed by mixing the solutions in the follow-ing sequence: 50 plI of Na125I (1 mCi/0.1 ml) in 0.1 NNaOH; 50 ,ul of 0.1 N HCl; 10 pAl of 1.1 mMKI; 100 ,lA of50% (vol/vol) Sepharose 4B-lactoperoxidase suspended in0.01 M sodium phosphate, 0.1 M NaCl buffer (pH 7.0); and0.5 ml of' FVIII/vWF (2 mg/ml) dissolved in 0.01 M sodiuimphosphate, 0.1 M NaCl buffer (pH 7.0). The reaction wasinitiated by adding 5 ,ul of 6.7 mMH202; the solution wasthen left at room temperature and stirred frequently for thenext 30 min. The reaction was quenched by adding 15 ,lIof 2.5 M NaN3. The solution was centrifuged, and thesupernate was applied to a 1 x 15-cm Sephadex G-25 columnwhich had been equilibrated with 0.025 Mcacodylate, 0.15 MNaCl buffer (pH 6.8), and pretreated with 2 ml of 1% BSAdissolved in the same buffer. The column was eluted with0.025 M cacodylate, 0.15 M NaCl buffer, and 0.5-ml aliquotswere collected and counted in a gammacouinter (model 4000,Beckman Instruments, Inc., Fullerton, Calif.). Usually, twopeaks were obtained: the first peak was iodinated FVIII/vWF,and the second was free 1251. The fractions of the first peakwhich contained the iodinated FVIII/vWF were pooled, andlthe specific activity of the preparation was determined bymeasuring the radioactivity and the protein concentration (15).This iodination procedure routinely yielded 125I-FVIII/vWFwith a specific radioactivity of 0.3-0.4 ,Cilt,g. The platelet-aggregating activity of the iodinated FVIII/vWF was assayedby the ristocetin-induced platelet aggregation method asdescribed previously (16).

Preparation of wvashed platelets, erythrocytes (RBC), andpolyrnorphonuclear letukocyte (PMN). With a two-syringetechnique, 18 ml of venous blood were drawn into 2 ml of 3.8%sodium citrate. After immediate mixing, the blood sample wascentrifuged at 250 g for 10 min at room temperature to ob-tain platelet-rich plasma and sedimented RBC. The RBCweresaved while the plasma was further centrifuged at 6,000 gfor 10 min at room temperature to sediment the platelets.The platelet pellet was washed five times by resuspendingit in 16 ml of' 0.025 M Tris-HCl, 0.15 M NaCl, 0.01 MEDTA (pH 7.4), and then centrifuging at 6,000 g for 10 minat room temperature. After the last wash, the platelets wereresuspended in 0.025 M Tris-HCl, 0.15 M NaCl, 0.1% BSA(pH 7.4). The RBC were washed in a similar fashionexcept that the centrifugation was carried out at 300g. Toeach milliliter of packed RBC, 8 ml of washing buffer wasadded.

PMNwere prepared according to the method of B0yum (17).20 ml of venous blood was drawn into a heparinized syringewhich contained 5 ml of plasma gel. After mixing the bloodwith the gel, the syringe was placed vertically in an incubatorat 370C. RBC sedimented in the syringe by 60 min. ThePMN-rich plasma was collected from the top of the syringeand layered on top of a tube containing 12 ml of thelymphocyte separation medium. The PMNpellet was col-lected by centrifuging at 400 g for 30 min at room tempera-ture, washed twice in Hank's balanced salt solution (pH 7.4),and resuspended in the same solution. The concentration ofcells in the suspension was determined with a hemacytometerand phase-contrast microscopy and adjusted to the final de-sired concentration with an appropriate volume of the samebuffer.

Factor VIII/von WVillebrand Factor Specific Binding Sites 657

FVIIIIvWF binding assay. The assay was done at roomtemperature in 12 x 75-mm polystyrene culture tubes in afinal vol of 0.5 ml. Each incubation tube contained thefollowing: 0.1 ,tg 125I-FVIII/vWF in 100 IlI 0.025 M Tris-HCl, 0.15 MNaCl, 0.1% BSA (pH 7.4); selected amounts ofunlabeled FVIII/vWF in 100 ,ul of the above buffer; 0.5 mgristocetin in 100 ,ul of the same buffer as above, but withoutBSA; and 100 ,ul of the above buffer. Incubations were begunby adding 100 ,lI of platelet suspension (5 x 106 cells/100,ul, unless otherwise stated). After reaching equilibrium,the incubations were stopped by adding 1 ml of ice-cold0.025 M Tris, 0.15 M NaCl buffer (pH 7.4). Tubes wereimmediately centrifuged at 3,500 g, 4°C, for 20 min to sepa-rate the bound from the free FVIII/vWF. The supernatewhich contained free FVIII/vWF was aspirated, and thepellet which contained bound FVIII/vWF was counted.The assay for each point was performed in duplicate. Thevariation between duplicate assays was always <4%. Thespecific binding of 125I-FVIII/vWF to platelets was deter-mined by performing incubations in the presence and absenceof excess amounts of unlabeled FVIII/vWF (30 ,ug). Thebound 125j-FVIII/vWF which could not be displaced bythe presence of excess amounts of unlabeled FVIII/vWFrepresented nonspecific binding. The difference in radio-activity between incubations with and without excess un-labeled FVIII/vWF was defined as specific binding. Thefree 125j-FVIII/vWF is derived by subtracting the boundcounts from the total counts in each incubation mixture.In this study, the nonspecific binding was only 2-3% of thetotal 125I-FVIII/vWF contained in each incubation mixture. Itwas found that filtration methods cannot be used to separatethe bound FVIII/vWF from the free FVIII/vWF because theFVIII/vWF protein sticks to the millipore or glass fiber filter.Dissociation of 1251-FVIII/vWF binding from platelets wasstudied at room temperature by adding unlabeled FVIII/vWF(50 ,g/0.5 ml) into incubations which had reached equi-librium. Specific binding was then determined at the specifiedtime intervals. Unlabeled FVIII/vWF was not added to thecontrol tubes. The increase of nonspecific binding with timewas corrected at each point by subtracting the value ob-tained for an incubation mixture which contained 30 ,ug ofunlabeled FVIII/vWF before the chase quantity of FVIII/vWFwas added.

RESULTS

After reduction, the purified FVIII/vWF showed theusual single, -200,000-dalton protein band by sodiumdodecyl sulfate gel electrophoresis. Only one immuno-precipitin line was seen when 3 ,g of purifiedFVIII/vWF was diffused against 5 ul of unadsorbedrabbit anti-FVIII/vWF on 1% agarose slides. Afterradioiodination and reduction, sodium dodecyl sulfategel analyses of the 125I-FVIII/vWF showed a singleprotein band which corresponded to a single peak ofradioactivity when sliced and counted. No detectableloss of platelet-aggregating activity of 1251-FVIII/vWF was observed when assayed by the ristocetin-induced platelet aggregation method with unlabeledFVIII/vWF as a standard.

Effect of incubation time, numbers of platelets,and ristocetin concentration on binding. The time-course of 125I-FVIII/vWF binding to human plateletsat different concentrations of ristocetin was studied.

Fig. 1 shows that 2 h of incubation time were re-quired for the binding to reach equilibrium when3 x 106 platelets per incubation were used, regard-less of the concentration of ristocetin. Binding reachedequilibrium at 100 min when 5 x 106 platelets perincubation were used, whereas equilibrium was at-tained at 40 min when 8 x 106 platelets per incuba-tion were present. These results suggest that the timerequired to reach equilibrium depends upon plateletconcentration, but not upon ristocetin concentration.

The effect of ristocetin concentration on the bindingof 125I-FVIII/vWF when incubated with different con-centrations of platelets is shown in Fig. 2. The re-sults clearly demonstrate that if the period of incuba-tion is now held constant, the amount of 1251-FVIII/vWFwhich becomes bound to platelets depends uponthe concentration of ristocetin in the incubation mix-ture. Binding increases in a cooperative manner asthe concentration of ristocetin increases, being linearbetween ristocetin concentrations of 0.2 and 1.0 mg/ml.At ristocetin concentrations above 1 mg/ml, the amountof binding gradually approaches a plateau. Thisphenomenon was most obvious at the higher plateletconcentration, 8 x 106 cells per incubation, in whichcase binding clearly leveled off after 1.0 mglml ofristocetin.

Kinetic study of 125I-FVIII/vWF binding to platelets.The above studies indicate that the binding of 1251_FVIII/vWF to platelets is ristocetin-dependent. Tostudy how ristocetin might promote FVIII/vWF bind-ing, the following experiments were performed. Aconstant number of platelets (5 x 106 cells per mixture)

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FIGURE 1 Time-course of 1251-FVIII/vWF binding to humanplatelets at different concentrations of ristocetin. Platelets(3 x 106 cells) were incubated with 0.1 ,ug of 125I-FVIII/vWF(_15,000 cpm) at specified concentrations of ristocetin. Thekey to the concentration of ristocetin used for each incuba-tion is as follows: (0), 1.5 mg/ml; (-), 1.0 mg/ml; (E), 0.8mg/ml; (-), 0.6 mg/ml; (A), 0.3 mg/ml; (A), 0.1 mg/ml; (),0 mg/ml.

658 K-J. Kao, S. V. Pizzo, and P. A. McKee

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FIGURE 2 Effect of different concentrations of platelets andristocetin on the specific binding of 1251-FVIII/vWF. Thespecific binding of 8 x 106 platelets per incubation (0);6 x 106 platelets per incubation (0); 5 x 106 platelets perincubation (0); 4 x 106 platelets per incubation (U); or2 x 106 platelets per incubation (A) was examined at thedifferent concentrations of ristocetin shown on the abscissa.All incubations were done at 24°C for 120 min. The 1251_FVIII/vWF added in each incubation is 0.1 ,g (-65,000 cpm).

was incubated with increasing amounts of 125I-FVIII/vWF at two concentrations of ristocetin (1 or 0.5 mg/ml). The results are illustrated in Fig. 3. The specificbinding of 1251-FVIII/vWF initially increased as theconcentration of 1251-FVIII/vWF increased, but even-tually a plateau was reached. This result indicatesthat the binding of 1251-FVIII/vWF to platelets is asaturable process, and that the specific binding at 1mg/ml of ristocetin is greater than at 0.5 mg/ml ofristocetin. Nonspecific binding, which represents non-displaceable, bound 1251-FVIII/vWF, was determinedby adding excessive amounts of unlabeled FVIII/vWF(300 times the concentration of 1251-FVIII/vWF). Fig.3 shows that nonspecific binding increased linearlywith increasing concentrations of 1251-FVIII/vWF andwas therefore nonsaturable. It is noteworthy thatnonspecific binding is the same at either concentra-tion of ristocetin. This result suggests that nonspecificbinding is not affected by different concentrations ofristocetin, at least up to 1 mg/ml.

The binding data were also analyzed by Scatchardplot (18) to determine the dissociation constant ofbinding and the total number of binding sites. TheScatchard analysis in Fig. 4A shows that the apparentdissociation constant (Kd) is 0.46 and 0.5 nM at con-centrations of ristocetin of 1.0 and 0.5 mg/ml, respec-tively. The total concentrations of binding sites deter-

9"5I-F=vm/vWF Added per Incubation (XIO-5cpm)

FIGURE 3 Saturable binding of 1251-FVIII/vWF to washed,human platelets. Incubations were performed at 24°C with aconstant amount of platelets (5 x 106 cells per incubation) andincreasing concentrations of '251-FVIII/vWF at two differentconcentrations of ristocetin: (0), 1 mg/ml of ristocetin and(O), 0.5 mg/ml of ristocetin. The solid lines representspecific binding; the broken lines represent nonspecificbinding.

mined from the intercepts of the abscissa were 0.52nM (31,000 sites per platelet) and 0.23 nM (13,700 sitesper platelet), respectively; these results are calculatedassuming univalency for the FVIII/vWF molecule of1.1 x 106 daltons. In Fig. 4B, these values for thebinding affinity and receptor concentration were vali-dated by Scatchard analysis of the data obtained fromthe competition study illustrated in Fig. 5. Theseresults confirmed that a significantly greater number oftotal specific binding sites are present at the higherristocetin concentration, but that the binding affinitiesare about the same at either concentration of ristocetin.

Specificity of the 1251-FVIIIIvWF binding to plate-lets. The specificity of FVIII/vWF binding sites onplatelets was studied by incubating 0.1 ,ug of 1251_FVIII/vWF with various concentrations of unlabeledhuman FVIII/vWF, thrombin, fibrinogen, a2-macro-globulin, bovine FVIII/vWF, equine collagen, orRC-60. Fig. 5 shows that only unlabeled human orbovine FVIII/vWF can compete with 125I-FVIII/vWFfor the binding, and that bovine FVIII/vWF is some-what less potent in competing for binding than humanFVIII/vWF. Human thrombin, fibrinogen, a2-macro-globulin, equine collagen, and RC-60, even at veryhigh concentrations (at least 2-800-fold molar excessof 1251-FVIII/vWF added), could not compete, despitethe fact that most of these proteins are either knownor believed to interact with platelets; some induce ag-gregation and(or) release cytoplasmic granules (20, 21).

The binding of 125I-FVIII/vWF to human RBC orPMN in the presence of ristocetin was also tested.Table I shows that only insignificant amounts of1251-FVIII/vWF can bind to these two types of blood

Factor VIll/vot XVillebrand Factor Specific Binding Sites 659

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FIGURE 4 Scatchard plots of '251-FVIII/vWF binding to re-ceptors at two concentrations of ristocetin: (0), 1 mg/ml or(0), 0.5 mg/ml. (A) Scatchard plot of the data from Fig. 3.The concentration of free FVIII/vWF is the molar concentra-tion of free '25I-FVIII/vWF in each incubation. Kd is the appar-ent dissociation constant. (B) Scatchard plots of the data fromFig. 5. [FVIII/vWF bound] is the molar concentration oftotal specifically bound FVIII/vWF, including both iodinatedand noniodinated species in each incubation. [FVIII/vWFfree] is the molar concentration of total free FVIII/vWFin each incubation. All of the Scatchard plots were drawnusing the least squares method (19). The assumption of aone-to-one binding ratio between FVIII/vWF molecule andreceptor is made for the Scatchard analysis. The molecularweight of FVIII/vWF is taken as 1.1 x 106.

cells. We found that small numbers of plateletscontaminated the preparations of RBCand PMN(2.5and 10.6%, respectively). When the number of washesfor the RBC was increased from 5 to 12 times, theamount of specifically bound 1251-FVIII/vWF was re-duced to 2.5%. Increasing the number of times theplatelets were washed had no effect on the amount ofFVIII/vWF which became bound to them. Hence, it ismost likely that the 125I-FVIII/vWF which bound to theRBCor PMNresulted from the contamination of thesecells by small numbers of platelets. The sum of thesestudies supports the notion that the binding sites forFVIII/vWF are unique to platelets.

Effect of Mg++, Ca++, EDTA, EGTA, and nucleotideson the binding of 125I-FVIII/vWF to platelets. Theimportance of Mg++, Ca++, and different nucleotideson the binding of drug and hormone receptors is wellknown (22-24). The results shown in Table II indi-cate that Ca++ and Mg++ are not important for thebinding of 125I-FVIII/vWF to platelets. Some inhibitoryeffects were observed at relatively high concentrations(20 mM) of Ca++, Mg+', or EDTA; however, noeffect on binding was observed when 2 or 20 AMofATP, ADP, AMP, adenosine, guanosine triphosphate,guanosine diphosphate, or guanosine monophosphate

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FIGURE 5 Competition curve of '251-FVIII/vWF binding towashed, human platelets. The experiments were performed byincubating a constant amount of platelets (5 x 106) and125I-FVIII/vWF (0.1 .g/incubation) with specified concentra-tions of unlabeled proteins (0-50 ,g); B defines the amountof '251-FVIII/vWF which specifically binds to platelets andcannot be displaced by amounts of unlabeled FVIII/vWFas specified on the abscissa. Bo is the total specific bindingwhich is the difference in counts per minute betweenplatelet pellets from incubations with and without 50 ,ugof unlabeled FVIII/vWF. B/Bo gives the ratio of total specificbinding which remains. Two concentrations of ristocetin wereused: (0), 0.5 mg/ml and (0, A), 1.0 mg/ml.

were used. In contrast, EGTAwas highly inhibitory.At a concentration of 2 mM, EGTA inhibited 86%of 125I-FVIII/vWF binding. The inhibitory effect ofEGTA cannot be attributed to its Ca++ chelatingability because EDTA, Ca++, and Mg++ do not affectbinding. Thus, the inhibitory effect of EGTAmust bea result of other of its physiochemical properties.

Effect of pH on the binding of 125I-FVIII/vWF toplatelets. This study was performed in 0.025 MTris-maleate-NaOH, 0.15 MNaCl buffers ranging from pH

TABLE IBinding of 125I-FVIIIIvWF to Platelets, RBC, and PMN

Type of cells Specific binding

cpm/5 x 106 cells

Platelet 36,058RBC 2,736PMN 5,165

125I-FVIII/vWF (0.1 ,ug) and 5 x 106 cells (platelets, RBC, orPMN) were incubated with and without 30 ug of unlabeledFVIII/vWF for 100 min at 240C, and the specific binding wasthen calculated as described in Methods. Each value is themean of duplicate experiments and has a variation of <4%.

660 K-J. Kao, S. V. Pizzo, and P. A. McKee

TABLE IIEffects of Ca++, Mg++, EDTA, and EGTAon the Binding of

125I-FVIIIIvWF to Washed, Human Platelets

Finial concentration Percentage ofin incubate Specific binding control

m.M cpm/5 x 10' cells S

Control 33,154 100CaCl2 2 31,052 94

10 26,831 8120 22,603 68

MgCl2 2 32,059 9710 30,173 9120 25,279 76

EDTA 2 32,044 9710 30,735 9320 31,647 87

EGTA 2 4,771 1410 1,679 520 1,463 4.4

The technique for determining specific binding is describedin Methods. The pH of all incubations was 7.4. Each specific-binding value is the mean of duplicate experiments, thevariation between duplicates is <4%.

5.3 to pH 9.0. Fig. 6 shows that maximum bindingoccurs between pH 7.0 and pH 7.5. On either side ofthis range, binding is reduced drastically. These valuesof optimal pH for the binding of 1251-FVIII/vWF toplatelets in the presence of ristocetin are essentiallythe same as reported for FVIII/vWF-dependent, risto-cetin-induced platelet aggregation (25, 26).

Dissociation of specifically bound 125I-FVIII/vIVFfrom platelets. The dissociation of specifically bound1251-FVIII/vWF from platelets was investigated at roomtemperature by adding a chase quantity of unlabeledFVIII/vWF into incubations in which binding hadreached equilibrium. Fig. 7 shows that the dissocia-tion of bound 1251-FVIII/vWF is very slow. Initiallya relatively rapid dissociation is observed, but thenthe dissociation gradually ceases at longer incubationtimes. Because of the loss of reversibility of thebinding after long-term incubation, kinetic analysis ofthe dissociation of bound 1251-FVIII/vWF was not prac-tical. Similar phenomena have been observed pre-viously with peptide hormone receptors for prolactin(27) and gonadotropin (28). Fig. 7 shows that if theristocetin concentration is reduced to 0.1 mg/ml at zerotime, the amount of 1251-FVIII/vWF becoming disso-ciated is increased. At 6 h only 45% of that boundat zero time remained on fresh, washed platelets;beyond 6 h, no further dissociation occurred. Fig. 7also shows that if formalin-fixed platelets are usedand the ristocetin concentration is reduced to 0.1

mg/ml at zero time, -90% of the bound 1251-FVIII/vWFbecomes dissociated by 6 h.

Binding of 125I-FVIIIIvWF to platelets treated withchymotrypsin. It has been demonstrated that plateletmembrane proteins are hydrolyzed during incubationwith trypsin (29) or chymotrypsin (30). The bindingsites of FVIII/vWF are presumed to be on the mem-brane of platelets because of the high molecularweight of FVIII/vWF. If the membrane binding sitesfor FVIII/vWF are protein, they might be susceptibleto enzymatic digestion and, therefore, the specificbinding of 1251-FVIII/vWF should become diminished.This hypothesis is supported by this study. Table IIIshows that the specific binding of 1251-FVIII/vWF toplatelets did decrease progressively as platelets werepreincubated with increasing concentrations of a-chymotrypsin.

DISCUSSION

Our studies demonstrate that high affinity specificbinding sites for FVIII/vWF exist on human plate-

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Factor VIIIvotin XVillebrand Factor Specific Binding Sites

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0 2 4 6 8 10 12 14 16 18 20Hours

FIGURE 7 Dissociation of specifically bound 125I-FVIII/vWFfrom washed (0, 0, A) and formalin-fixed (x) platelets. Theclosed circles give the control curve, and the open circlesgive the amount of dissociation of '251-FVIII/vWF in thepresence of' 1.0 mg/ml of ristocetin. The triangles representthe degree of dissociation when the ristocetin is diluted to0.1 mg/ml. The curve given by the (x) indicates theamount of dissociation which occurs when formalin-fixedplatelets are used and the ristocetin concentration is re-duced to 0;1 mg/ml.

lets. Uniquely, ristocetin, an antibiotic with a mol wt-5,000 (31), is essential for the binding of FVIII/vWFto the platelet membranes. Two possible mechanisms,either of which could account for the promotion ofspecific binding by ristocetin, were considered in ourstudies: (a) ristocetin enhances the binding affinity andtherefore shifts the equilibrium toward binding; and(b) ristocetin somehow increases the number of ac-cessible binding sites for FVIII/vWF on platelets. Ourresults clearly demonstrate that ristocetin increasesthe number of accessible binding sites on platelets;however, the exact mechanism by which this is ac-complished is still unclear. By using highly negativelycharged molecules (11, 25) or chemically modified

ristocetin and electrometric titration (32), other inves-tigators have suggested that ristocetin neutralizes nega-tive charges on the platelet surface and that this effectsomehow promotes' the interaction of platelets withFVIII/vWF and, subsequently, platelet aggregation.This hypothesis is also supported by the report that theelectrophoretic mobility of platelets becomes alteredwhen exposed to ristocetin, presumably by ristocetinneutralizing the surface negative charges of platelets(33). Yet, Baugh et al. (34) could not show that 125I1labeled ristocetin became bound to platelets duringristocetin-induced platelet aggregation, suggestingperhaps that charge-neutralizing effects may not be thefundamental explanation for the role of ristocetin as acofactor for von Willebrand activity.

It is significant that bovine FVIII/vWF aggregatesand adsorbs to human platelets without ristocetin (11).Such data may indicate that bovine FVIII/vWF itselfhas a ristocetin-like activity in addition to the otheractivities usually assigned to FVIII/vWF. This possi-bility is supported by the recent finding that bovineFVIII/vWF neutralizes the surface negative charges ofplatelets in the absence of ristocetin (33). Ourstudies definitely demonstrate that bovine FVIII/vWFcompetes with '251-labeled human FVIII/vWF for thesame binding site. When considered collectively, thefindings suggest that ristocetin neutralizes negativecharges on the platelet membrane, thereby forming ormaking the FVIII/vWF binding site accessible.

Recently Jenkins et al. (30) reported that membraneglycoprotein I of human platelets is involved inFVIII/vWF-dependent, ristocetin-induced platelet ag-gregation. Furthermore, Nachman et al. (35) demon-strated that ristocetin-induced platelet aggregation wasinhibited by rabbit antiserum to glycoprotein I.Okumura and Jamieson (36) reported that anotherglycoprotein (glycocalicin), which functions as a throm-bin receptor in the exterior coat of platelets, in-hibited ristocetin-induced platelet aggregation. Theseauthors further demonstrated that glycocalicin is im-munologically and electrophoretically similar to mem-brane glycoprotein I of platelets (37, 38). Thus thepossibility that FVIII/vWF shares the same receptor

TABLE IIIBinding of 125I-FVIIIIvWF to a(-Chymotrypsin-Treated Human Platelets

Concentration of chymotrypsin,,Lg/ml 0 10 50 100

Specific binding, cpm/5 x 106 cells 15,374+353 14,171+88 11,060+135 7,372+282

3 ml of washed, human platelets were prepared from 5 ml of venous blood and incubated withthe specified concentrations of a-chymotrypsin for 15 min at 37°C. The platelets were thencollected by centrifugation, washed twice, and diluted to an appropriate concentration for theassay. Each specific-binding value is the mean+SEM for three determinations at eachchymotrypsin concentration.

662 K-J. Kao, S. V. Pizzo, and P. A. McKee

with thrombin was proposed. Results of our study,however, demonstrate that human thrombin doesnot compete with FVIII/vWF for binding sites. Wealso observed that 1 mglml of ristocetin did notinhibit platelet aggregation by human thrombin (0.05NIH U/ml).2 Thus the possibility that ristocetin in-hibits thrombin action is unlikely. Hence separatereceptors appear to exist for the binding of FVIII/vWFand thrombin. Although no direct experiments wereperformed to correlate binding of FVIII/vWF to theplatelets with platelet-aggregating activity, we foundthat the optimal pH (7.0-7.5) for binding does cor-respond to the values of optimal pH (7.0-7.5) forristocetin-induced platelet aggregation (25, 26). Thiscorrespondence suggests a parallel relationship be-tween the degree of binding and aggregating activity.Weiss et al. have also shown that the extent and rateof platelet aggregation are related to the concentra-tion of ristocetin (39). Our results coupled with theirobservations suggest that binding of FVIII/vWF can becorrelated with ristocetin-induced platelet aggregation.

In conclusion, by applying competitive protein bind-ing assay techniques with 1251-labeled FVIII/vWF as atracer, we have identified a class of FVIII/vWFbinding sites on human platelets. The binding charac-teristics fulfill the criteria for receptors in terms ofhigh affinity, saturability, and specificity; however, thefunctional correlation of binding and biological effect,i.e., platelet aggregation, is not included here. Thefact that binding depends upon the presence of risto-cetin, a glycopeptide antibiotic, is an unique featurewhich has not been observed before in other systems.The ristocetin dependency of binding suggests theexistence of such a mechanism in vivo by whichplatelet aggregation reactions are regulated to allowan innocuous coexistence of FVIII/vWF and plateletsin circulating blood. Finally, the establishment of aFVIII/vWF binding assay provides a new approach tothe accurate measurement of FVIII/vWF protein con-centration in human plasma as well as a method forstudying the biochemical interactions between FVIII/vWFand platelets.

ACKNOWLEDGMENT

This work was supported by a research grant from the Na-tional Heart, Lung, and Blood Institute, National Institutesof Health (HL 15615).

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