Demonstration and Characterization of Specific Binding Sites for Factor VIII/von Willebrand Factor on Human Platelets KUO-JANG KAo, 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 R A C T The presence of specific Factor VIII/ von Willebrand factor (FVIII/vWF) binding sites on human platelets has been demonstrated by using 125I1 FVIII/vWF and washed human platelets. Binding is ristocetin-dependent and increases in proportion to the concentration of ristocetin from 0.2 to 1 mg/ml. Binding of '251-FVIII/vWF to platelets can be competitively inhibited by unlabeled human or bovine FVIII/vWF, but not by human thrombin, fibrinogen, a2-macroglobulin, equine collagen, or a lectin of Ricinus communis. Scatchard analysis of binding data indicated that the dissociation constant of FVIII/vWF receptors is 0.45-0.5 nM. There are 31,000 binding sites per platelet at 1 mg/ml of ristocetin concentration. The optimal pH range for binding is from 7.0 to 7.5. At a concentration of 2 mM, EGTA inhibits 86% of the binding; however, 20 mM of Ca++, Mg++, or EDTA have little effect. Binding sites for FVIII/vWF were found only on platelets, and no significant binding was detected 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 number of 200,000-dalton subunits linked covalently by disulfide bonds (1, 2). This glycoprotein has two Dr. McKee is an Investigator ofthe Howard Hughes Medical Institute. Received for publication 5 September 1978 and in revised form 15 December 1978. 'Abbreviations used in this paper: BSA, bovine serum albumin; FVIII/vWF, Factor VIII/von Willebrand Factor; PEG, polyethylene glycol; PMN, polymorphonuclear leukocyte(s); RBC, erythrocytes.
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distinct biological activities: one being procoagulant activity (Factor VIII) which can specifically correct the blood coagulation defect of classical hemophilia patients; the other being platelet-aggregating activity, also known as von Willebrand factor activity, which is important for platelet plug formation at the site of vascular injury (3, 4). In von Willebrand's disease, a marked decrease in platelet-aggregating activity results in easy bruising, hemorrhage from mucous membranes, and a prolonged bleeding time (5); these defects can usually be corrected by administration of human FVIII/vWF concentrates. In 1973, Howard et al. (6) reported that the in vitro addition of an antibiotic, ristocetin, to normal platelet-rich plasma caused platelets to clump. In contrast, the addition of ristocetin promoted little or no clumping activity in the majority of platelet-rich plasma samples from patients with von Willebrand's disease (6). These observations were used to develop methods for quantitating plasma von Willebrand factor activity by measuring the rate or degree of aggregation of washed, normal human platelets to which aliquots of test plasma and ristocetin were added (7). At present, the mechanism by which FVIII/vWF protein interacts with platelets to cause aggregate formation in vivo or in vitro is still unknown. Nevertheless, immunofluorescent techniques (8) and the recovery of FVIII/ vWF activity from platelet membrane (9) or platelet aggregates (10, 11) suggest that FVIII/vWF binds to human platelets. Such results prompted the hypothesis that binding of FVIII/vWF to specific sites on human platelets may be required for normal platelet aggregation to occur. We now report evidence for the presence of specific binding sites for FVIII/vWF on human platelets, using a competitive, radiolabeled ligand binding assay. Some of the characteristics of these binding sites are also described.
J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738179/0410656/09 $1.00 Volume 63 April 1979 656-664
METHODS Materials. Intermediate purity human FVII1/vWF concentrates (900 U/vial) and purified human thrombin (180 U/vial) were obtained from American National Red Cross, Bethesda, Md., and the Bureau of Biologics, Bethesda, Md., respectively. Bovine lactoperoxidase was obtained from Calbiochem-Behring Corp., American Hoechst Corp., San Diego, Calif. A lectin of Ricinus communis (RC-60) was from Boehringer Mannheim 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 purchased from Sigma Chemical Co., St. Louis, Mo. Ristocetin was purchased from Bio/Data Corp., Horsham, Pa. Carrier-free Na125I (20 mCi/ml) was from New England Nuclear, Boston, Mass. a-Chymotrypsin (40 U/mg) was from Worthington Biochemical Corp., Freehold, N. J. BioGel A-15m was from Bio-Rad Laboratories, Richmond, Calif. Sepharose 4B and Sephadex G-25 were from Pharmacia Fine Chemicals, Div. of Pharmacia Inc., Piscataway, N. J. Lymphocyte separation medium and Hank's balanced salt solution were purchased from Bionetics Laboratory Products, Litton Bionetics Inc., Kensington, Md. and Gibco Diagnostics, Gibco Invenex Div., Chagrin Falls, Ohio, respectively. Plasma gel was from Roger Bellon Laboratoire, France. Polyethylene glycol (Carbowax 4000) was obtained from 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 or the best grade available.
Purification of FVIII/cWIF protein. The purification procedures for human FVIII/vWF protein from intermediate purity concentrates were the same as previously described from this laboratory (12) with some slight modifications. Polyethylene containers were used throughout, except where it is specifically stated that siliconized glassware was used. Lyophilized, intermediate purity FVIII/vWF concentrate, containing 1,800 U of Factor VIII procoagulant activity, was dissolved in 150 ml of distilled water. The pH of the solution was adjusted to 6.1 with 0.02 M citric acid 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 PEG concentration of 5%. After 15 min, the solution was centrifuged at 7,000g for 10 min. The supernate was removed, and 40% PEG was added to bring the final PEG concentration to 12%. The solution was left at room temperature for 30 min and then centrifuged at 7,000 g for 10 min. The precipitate was placed on ice; gently washed once with icecold 0.02 M Tris-HCl buffer (pH 7.4); made 8% in ethanol, to remove residual PEG; and then dissolved in 25 ml of a 0.05-M Tris-HCl, 0.15-M NaCl, 1 mM-diisopropylfluorophosphate, and 0.02% NaN3 solution (pH 7.4). This solution was applied to 4% agarose (Bio-Gel A-15m) packed in a 5 x 50-cm siliconized glass column. The column was eluted with a solution of 0.05 M TrisHCI, 0.15 M NaCl, and 0.02% of NaN3 (pH 7.4) at a flow rate of 25 ml/h. The void-volume fractions, which contained the protein peak having FVIII/vWF activity, were pooled and precipitated at 4°C by adding solid ammonium sulfate until 35% saturation was attained. After 8 h at 4°C the precipitate was collected by centrifugation at 12,000g for 10 min and dissolved in 0.025 M cacodylate, 0.15 M NaCl buffer (pH 6.8). Residual ammonium sulfate in the precipitate was removed by dialysis at 4°C against the same
buffer. The purified FVIII/vWF was stored at 40C in a final conceentration of 1.5-2.5 mg/ml. The purity ofthe final prodluet was anialyzed by electrophoresis on sodium dodecyl sulfate6M urea-5% polyacrylamide gels before and after reduction by 83-mercaptoethanol (12, 13). Radioioditnaition of FVIII/vcW'F. Highly purified FVIII/ vWF was labeled with 125j by the Sepharose 4B-lactoperoxidase method of David and Reisfeld (14). The iodination was performed by mixing the solutions in the following sequence: 50 plI of Na125I (1 mCi/0.1 ml) in 0.1 N NaOH; 50 ,ul of 0.1 N HCl; 10 pAl of 1.1 mM KI; 100 ,lA of 50% (vol/vol) Sepharose 4B-lactoperoxidase suspended in 0.01 M sodium phosphate, 0.1 M NaCl buffer (pH 7.0); and 0.5 ml of' FVIII/vWF (2 mg/ml) dissolved in 0.01 M sodiuim phosphate, 0.1 M NaCl buffer (pH 7.0). The reaction was initiated by adding 5 ,ul of 6.7 mM H202; the solution was then left at room temperature and stirred frequently for the next 30 min. The reaction was quenched by adding 15 ,lI of 2.5 M NaN3. The solution was centrifuged, and the supernate was applied to a 1 x 15-cm Sephadex G-25 column which had been equilibrated with 0.025 M cacodylate, 0.15 M NaCl buffer (pH 6.8), and pretreated with 2 ml of 1% BSA dissolved in the same buffer. The column was eluted with 0.025 M cacodylate, 0.15 M NaCl buffer, and 0.5-ml aliquots were collected and counted in a gamma couinter (model 4000, Beckman Instruments, Inc., Fullerton, Calif.). Usually, two peaks were obtained: the first peak was iodinated FVIII/vWF, and the second was free 1251. The fractions of the first peak which contained the iodinated FVIII/vWF were pooled, andl the specific activity of the preparation was determined by measuring the radioactivity and the protein concentration (15). This iodination procedure routinely yielded 125I-FVIII/vWF with a specific radioactivity of 0.3-0.4 ,Cilt,g. The plateletaggregating activity of the iodinated FVIII/vWF was assayed by the ristocetin-induced platelet aggregation method as described previously (16). Preparation of wvashed platelets, erythrocytes (RBC), and polyrnorphonuclear letukocyte (PMN). With a two-syringe technique, 18 ml of venous blood were drawn into 2 ml of 3.8% sodium citrate. After immediate mixing, the blood sample was centrifuged at 250 g for 10 min at room temperature to obtain platelet-rich plasma and sedimented RBC. The RBC were saved while the plasma was further centrifuged at 6,000 g for 10 min at room temperature to sediment the platelets. The platelet pellet was washed five times by resuspending it in 16 ml of' 0.025 M Tris-HCl, 0.15 M NaCl, 0.01 M EDTA (pH 7.4), and then centrifuging at 6,000 g for 10 min at room temperature. After the last wash, the platelets were resuspended in 0.025 M Tris-HCl, 0.15 M NaCl, 0.1% BSA (pH 7.4). The RBC were washed in a similar fashion except that the centrifugation was carried out at 300g. To each milliliter of packed RBC, 8 ml of washing buffer was added. PMN were prepared according to the method of B0yum (17). 20 ml of venous blood was drawn into a heparinized syringe which contained 5 ml of plasma gel. After mixing the blood with the gel, the syringe was placed vertically in an incubator at 370C. RBC sedimented in the syringe by 60 min. The PMN-rich plasma was collected from the top of the syringe and layered on top of a tube containing 12 ml of the lymphocyte separation medium. The PMN pellet was collected by centrifuging at 400 g for 30 min at room temperature, washed twice in Hank's balanced salt solution (pH 7.4), and resuspended in the same solution. The concentration of cells in the suspension was determined with a hemacytometer and phase-contrast microscopy and adjusted to the final desired concentration with an appropriate volume of the same buffer.
Factor VIII/von WVillebrand Factor Specific Binding Sites
657
FVIIIIvWF binding assay. The assay was done at room temperature in 12 x 75-mm polystyrene culture tubes in a final vol of 0.5 ml. Each incubation tube contained the following: 0.1 ,tg 125I-FVIII/vWF in 100 IlI 0.025 M TrisHCl, 0.15 M NaCl, 0.1% BSA (pH 7.4); selected amounts of unlabeled FVIII/vWF in 100 ,ul of the above buffer; 0.5 mg ristocetin in 100 ,ul of the same buffer as above, but without BSA; and 100 ,ul of the above buffer. Incubations were begun by 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-cold 0.025 M Tris, 0.15 M NaCl buffer (pH 7.4). Tubes were immediately centrifuged at 3,500 g, 4°C, for 20 min to separate the bound from the free FVIII/vWF. The supernate which contained free FVIII/vWF was aspirated, and the pellet which contained bound FVIII/vWF was counted. The assay for each point was performed in duplicate. The variation between duplicate assays was always
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 number of 200,000-dalton subunits linked covalently by disulfide bonds (1, 2). This glycoprotein has two Dr. McKee is an Investigator ofthe Howard Hughes Medical Institute. Received for publication 5 September 1978 and in revised form 15 December 1978. 'Abbreviations used in this paper: BSA, bovine serum albumin; FVIII/vWF, Factor VIII/von Willebrand Factor; PEG, polyethylene glycol; PMN, polymorphonuclear leukocyte(s); RBC, erythrocytes.
656
distinct biological activities: one being procoagulant activity (Factor VIII) which can specifically correct the blood coagulation defect of classical hemophilia patients; the other being platelet-aggregating activity, also known as von Willebrand factor activity, which is important for platelet plug formation at the site of vascular injury (3, 4). In von Willebrand's disease, a marked decrease in platelet-aggregating activity results in easy bruising, hemorrhage from mucous membranes, and a prolonged bleeding time (5); these defects can usually be corrected by administration of human FVIII/vWF concentrates. In 1973, Howard et al. (6) reported that the in vitro addition of an antibiotic, ristocetin, to normal platelet-rich plasma caused platelets to clump. In contrast, the addition of ristocetin promoted little or no clumping activity in the majority of platelet-rich plasma samples from patients with von Willebrand's disease (6). These observations were used to develop methods for quantitating plasma von Willebrand factor activity by measuring the rate or degree of aggregation of washed, normal human platelets to which aliquots of test plasma and ristocetin were added (7). At present, the mechanism by which FVIII/vWF protein interacts with platelets to cause aggregate formation in vivo or in vitro is still unknown. Nevertheless, immunofluorescent techniques (8) and the recovery of FVIII/ vWF activity from platelet membrane (9) or platelet aggregates (10, 11) suggest that FVIII/vWF binds to human platelets. Such results prompted the hypothesis that binding of FVIII/vWF to specific sites on human platelets may be required for normal platelet aggregation to occur. We now report evidence for the presence of specific binding sites for FVIII/vWF on human platelets, using a competitive, radiolabeled ligand binding assay. Some of the characteristics of these binding sites are also described.
J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738179/0410656/09 $1.00 Volume 63 April 1979 656-664
METHODS Materials. Intermediate purity human FVII1/vWF concentrates (900 U/vial) and purified human thrombin (180 U/vial) were obtained from American National Red Cross, Bethesda, Md., and the Bureau of Biologics, Bethesda, Md., respectively. Bovine lactoperoxidase was obtained from Calbiochem-Behring Corp., American Hoechst Corp., San Diego, Calif. A lectin of Ricinus communis (RC-60) was from Boehringer Mannheim 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 purchased from Sigma Chemical Co., St. Louis, Mo. Ristocetin was purchased from Bio/Data Corp., Horsham, Pa. Carrier-free Na125I (20 mCi/ml) was from New England Nuclear, Boston, Mass. a-Chymotrypsin (40 U/mg) was from Worthington Biochemical Corp., Freehold, N. J. BioGel A-15m was from Bio-Rad Laboratories, Richmond, Calif. Sepharose 4B and Sephadex G-25 were from Pharmacia Fine Chemicals, Div. of Pharmacia Inc., Piscataway, N. J. Lymphocyte separation medium and Hank's balanced salt solution were purchased from Bionetics Laboratory Products, Litton Bionetics Inc., Kensington, Md. and Gibco Diagnostics, Gibco Invenex Div., Chagrin Falls, Ohio, respectively. Plasma gel was from Roger Bellon Laboratoire, France. Polyethylene glycol (Carbowax 4000) was obtained from 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 or the best grade available.
Purification of FVIII/cWIF protein. The purification procedures for human FVIII/vWF protein from intermediate purity concentrates were the same as previously described from this laboratory (12) with some slight modifications. Polyethylene containers were used throughout, except where it is specifically stated that siliconized glassware was used. Lyophilized, intermediate purity FVIII/vWF concentrate, containing 1,800 U of Factor VIII procoagulant activity, was dissolved in 150 ml of distilled water. The pH of the solution was adjusted to 6.1 with 0.02 M citric acid 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 PEG concentration of 5%. After 15 min, the solution was centrifuged at 7,000g for 10 min. The supernate was removed, and 40% PEG was added to bring the final PEG concentration to 12%. The solution was left at room temperature for 30 min and then centrifuged at 7,000 g for 10 min. The precipitate was placed on ice; gently washed once with icecold 0.02 M Tris-HCl buffer (pH 7.4); made 8% in ethanol, to remove residual PEG; and then dissolved in 25 ml of a 0.05-M Tris-HCl, 0.15-M NaCl, 1 mM-diisopropylfluorophosphate, and 0.02% NaN3 solution (pH 7.4). This solution was applied to 4% agarose (Bio-Gel A-15m) packed in a 5 x 50-cm siliconized glass column. The column was eluted with a solution of 0.05 M TrisHCI, 0.15 M NaCl, and 0.02% of NaN3 (pH 7.4) at a flow rate of 25 ml/h. The void-volume fractions, which contained the protein peak having FVIII/vWF activity, were pooled and precipitated at 4°C by adding solid ammonium sulfate until 35% saturation was attained. After 8 h at 4°C the precipitate was collected by centrifugation at 12,000g for 10 min and dissolved in 0.025 M cacodylate, 0.15 M NaCl buffer (pH 6.8). Residual ammonium sulfate in the precipitate was removed by dialysis at 4°C against the same
buffer. The purified FVIII/vWF was stored at 40C in a final conceentration of 1.5-2.5 mg/ml. The purity ofthe final prodluet was anialyzed by electrophoresis on sodium dodecyl sulfate6M urea-5% polyacrylamide gels before and after reduction by 83-mercaptoethanol (12, 13). Radioioditnaition of FVIII/vcW'F. Highly purified FVIII/ vWF was labeled with 125j by the Sepharose 4B-lactoperoxidase method of David and Reisfeld (14). The iodination was performed by mixing the solutions in the following sequence: 50 plI of Na125I (1 mCi/0.1 ml) in 0.1 N NaOH; 50 ,ul of 0.1 N HCl; 10 pAl of 1.1 mM KI; 100 ,lA of 50% (vol/vol) Sepharose 4B-lactoperoxidase suspended in 0.01 M sodium phosphate, 0.1 M NaCl buffer (pH 7.0); and 0.5 ml of' FVIII/vWF (2 mg/ml) dissolved in 0.01 M sodiuim phosphate, 0.1 M NaCl buffer (pH 7.0). The reaction was initiated by adding 5 ,ul of 6.7 mM H202; the solution was then left at room temperature and stirred frequently for the next 30 min. The reaction was quenched by adding 15 ,lI of 2.5 M NaN3. The solution was centrifuged, and the supernate was applied to a 1 x 15-cm Sephadex G-25 column which had been equilibrated with 0.025 M cacodylate, 0.15 M NaCl buffer (pH 6.8), and pretreated with 2 ml of 1% BSA dissolved in the same buffer. The column was eluted with 0.025 M cacodylate, 0.15 M NaCl buffer, and 0.5-ml aliquots were collected and counted in a gamma couinter (model 4000, Beckman Instruments, Inc., Fullerton, Calif.). Usually, two peaks were obtained: the first peak was iodinated FVIII/vWF, and the second was free 1251. The fractions of the first peak which contained the iodinated FVIII/vWF were pooled, andl the specific activity of the preparation was determined by measuring the radioactivity and the protein concentration (15). This iodination procedure routinely yielded 125I-FVIII/vWF with a specific radioactivity of 0.3-0.4 ,Cilt,g. The plateletaggregating activity of the iodinated FVIII/vWF was assayed by the ristocetin-induced platelet aggregation method as described previously (16). Preparation of wvashed platelets, erythrocytes (RBC), and polyrnorphonuclear letukocyte (PMN). With a two-syringe technique, 18 ml of venous blood were drawn into 2 ml of 3.8% sodium citrate. After immediate mixing, the blood sample was centrifuged at 250 g for 10 min at room temperature to obtain platelet-rich plasma and sedimented RBC. The RBC were saved while the plasma was further centrifuged at 6,000 g for 10 min at room temperature to sediment the platelets. The platelet pellet was washed five times by resuspending it in 16 ml of' 0.025 M Tris-HCl, 0.15 M NaCl, 0.01 M EDTA (pH 7.4), and then centrifuging at 6,000 g for 10 min at room temperature. After the last wash, the platelets were resuspended in 0.025 M Tris-HCl, 0.15 M NaCl, 0.1% BSA (pH 7.4). The RBC were washed in a similar fashion except that the centrifugation was carried out at 300g. To each milliliter of packed RBC, 8 ml of washing buffer was added. PMN were prepared according to the method of B0yum (17). 20 ml of venous blood was drawn into a heparinized syringe which contained 5 ml of plasma gel. After mixing the blood with the gel, the syringe was placed vertically in an incubator at 370C. RBC sedimented in the syringe by 60 min. The PMN-rich plasma was collected from the top of the syringe and layered on top of a tube containing 12 ml of the lymphocyte separation medium. The PMN pellet was collected by centrifuging at 400 g for 30 min at room temperature, washed twice in Hank's balanced salt solution (pH 7.4), and resuspended in the same solution. The concentration of cells in the suspension was determined with a hemacytometer and phase-contrast microscopy and adjusted to the final desired concentration with an appropriate volume of the same buffer.
Factor VIII/von WVillebrand Factor Specific Binding Sites
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FVIIIIvWF binding assay. The assay was done at room temperature in 12 x 75-mm polystyrene culture tubes in a final vol of 0.5 ml. Each incubation tube contained the following: 0.1 ,tg 125I-FVIII/vWF in 100 IlI 0.025 M TrisHCl, 0.15 M NaCl, 0.1% BSA (pH 7.4); selected amounts of unlabeled FVIII/vWF in 100 ,ul of the above buffer; 0.5 mg ristocetin in 100 ,ul of the same buffer as above, but without BSA; and 100 ,ul of the above buffer. Incubations were begun by 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-cold 0.025 M Tris, 0.15 M NaCl buffer (pH 7.4). Tubes were immediately centrifuged at 3,500 g, 4°C, for 20 min to separate the bound from the free FVIII/vWF. The supernate which contained free FVIII/vWF was aspirated, and the pellet which contained bound FVIII/vWF was counted. The assay for each point was performed in duplicate. The variation between duplicate assays was always
