Proc. Nati. Acad. Sci. USA
Vol. 76, No. 3, pp. 1069-1073, March 1979 Biochemistry
Human platelet membrane receptor for bovine von Willebrand factor (platelet aggregating factor): An integral membrane glycoprotein (macromolecules/Factor VIII/affinity chromatography/detergents/lectins)
HERBERT A. COOPER*, KENNETH J. CLEMETSONt, AND ERNST F. LUSCHERt *Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514; and tTheodor Kocher Institute, University of Bern, Bern, Switzerland
Communicated by Kenneth M. Brinkhous, November 30, 1978
ABSTRACT The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property o a soluble glycoprotein, glycocalicin, that is loosely attached to the plate let surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose-response curve obtained with Brij 99solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or Lens culinaris lectin. By using wheat germ agglutininSepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4VC in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents. Certain animal plasmas, particularly bovine, contain a glycoprotein that directly aggregates human platelets without known cofactors (1, 2). This platelet aggregating factor (PAF) is closely associated with Factor VIII procoagulant activity (1, 3, 4) and has an apparent homology with human von Willebrand factor (vWF) (3,5,6). Both human and animal vWF have been shown to be a heterodisperse family of oligomers with a minimal molecular weight of 1 X 106 (7-10). Interaction of bovine vWF with fresh washed human platelets causes aggregation within seconds and, under optimal conditions, produces a biphasic aggregation curve with the release of adenine nucleotides (11). The initial interaction of PAF with the platelet occurs in the presence of metabolic inhibitors (12) and even after fixation of platelets in formaldehyde (13). The interaction has recently taken on new biological importance with the reports (14, 15) that it is required for the normal adhesion and spreading of platelets on exposed subendothelium. The binding of PAF to platelets as a part of the aggregation phenomenon was first shown by using fresh washed platelets
(1) and subsequently was confirmed in studies using formaldehyde-fixed washed platelets (FWP) (16). The number of high-affinity binding sites was estimated to be in the range of 1-1.25 X 103 per platelet (16). It must be stressed, however, that this calculation is probably influenced by the number of PAF molecules that can be physically accommodated on the surface of the fixed platelet. Therefore, the determination may drastically underestimate the true number of binding sites available. Adsorption of PAF from bovine plasma by FWP was dependent on the platelet concentration and the length of incubation. PAF was bound to high-affinity sites as well as to nonspecific or lower-affinity sites. The PAF bound to the low-affinity sites could be removed by washing the aggregated FWP in 1 M KC1. Such treatment, however, did not remove the PAF bound to the high-affinity sites. Proteolytic treatment with trypsin destroys the ability of FWP to bind PAF (16). Because enzymatic treatment of platelet membranes with trypsin leads to the cleavage of surface-oriented glycoproteins (17), it seems reasonable to assign receptor function to one of these components. Such a hypothesis is further substantiated by the fact that the platelets of patients with Bernard-Soulier syndrome fail to aggregate with PAF (18) and have abnormalities of their surface glycoproteins (19, 20). Recently, anti-platelet membrane antisera absorbed with chymotrypsin-treated platelets gave an antiserum highly specific for the interaction of vWF and platelets (21). Further immunological studies have demonstrated possible receptor function in the GP1 region of the polyacrylamide gel electrophoretic pattern of platelet membranes (22, 23). A glycoprotein that is thought not to be an integral membrane component has also been implicated as the possible receptor for vWF (24-28). Primarily referred to as glycocalicin or GPS, it is obtained in soluble form from whole platelets by sonication or incubation in buffers that do not contain EDTA, repeated freezing and thawing, and extraction with 3 M KCI (27, 28). Glycocalicin is water soluble and reportedly lacks the usual lipophilic amino acids essential for the interaction of integral membrane proteins with the lipid constituents of the membrane. That glycocalicin is the receptor for vWF was suggested (29) based on the fact that high concentrations of glycocalicin inhibit ristocetin-induced aggregation of twicewashed human platelets. This glycoprotein has also been reported to be the receptor for PAF because platelets that have lost GPS are unable to aggregate with bovine Factor VIII, and GPS is missing from the surface of platelets from patients with Bernard-Soulier syndrome (17). Glycocalicin has the same lectin-binding affinity and similar electrophoretic mobility in
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Abbreviations: PAF, platelet aggregating factor; vWF, von Willebrand factor; FWP, formaldehyde-fixed washed platelets; PRP, platelet-rich plasma; WGA, wheat germ agglutinin; GPS, soluble glycoprotein.
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reducing and nonreducing polyacrylamide gels as one of the integral membrane glycoproteins, GP lb (30). Thus, the question remains as to whether glycocalicin is an intact, loosely attached membrane constituent with receptor function or a secondary product derived from an integral membrane protein that is actually the bovine PAF receptor. We have purified glycocalicin and studied this purified glycoprotein for bovine von Willebrand receptor activity. Because glycocalicin concentrations of up to 10 mg/ml caused less than 5% inhibition of PAF aggregation of FWP, it seemed worthwhile to look further for other membrane components that might have higher binding affinities for PAF. The- studies reported here describe the PAF receptor activity as being associated with an integral glycoprotein that requires detergent to release it from the platelet membrane vesicle. Solubilization of the receptor in an active state can be accomplished by using Brij 99 and the solubilized receptor has been partially characterized. MATERIALS AND METHODS Isolation of Human Blood Platelets. Within 20 hr after collection, platelets were isolated from citrated blood collected for the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross (31). The buffy coats were siphoned into a buffered glucose solution to give platelet-rich plasma (PRP) containing about 20 mM glucose, 12 mM phosphate buffer (pH 6.8), and about 4 X 109 platelets per ml (32). The PRP was washed three times in 0.01 M Tris-HCl/0.8% NaCI/10 mM EDTA, pH 7.4. After the third wash, the platelet pellet was resuspended in 0.01 M Tris-HCl/0.8% NaCl/1 mM EDTA, pH 7.4, at 4VC in a ratio by volume of 1 part platelets to 3 parts buffer. The platelet suspension was then cooled in an ice bath to about 4"C for sonication. Platelet Membrane Preparation. Human platelet membranes were processed from the platelet concentrates from 50-100 units of blood. The cooled, washed, platelet suspension was sonicated by using a B-30 sonifier (Branson Sonic Power Company, Danbury, CT for 2 min (output control, 7; 50% duty cycle; pulsed mode). The membranes were obtained by differential centrifugation as described (33) but without the 40,000 X g centrifugation step. Membranes were used fresh or after freezing at -700C and thawing only once. For long-term experiments requiring more than 1 day, only fresh membranes were used. In various preparations, protease inhibitors (0.5 mM CBZ-a-L-glutamyl-L-tyrosine and 0.5 mM phenylmethylsulfonyl fluoride), 0.01 M N-ethylmaleimide, and 0.02% sodium azide as a general bacteriostatic agent were used, although they could not be shown to be essential requirements for reproducible results. Fixed Washed Platelets (FWP). This assay reagent was prepared from fresh human platelets washed as described above. After three washes the platelets were resuspended in 1.8% (wt/vol) formaldehyde and prepared for storage by a modification (34) of the original method (13). The FWP were stored in 0.01 M Tris-HCI/0.8% NaCI/1 mM EDTA, pH 7.4, containing 0.02% sodium azide for up to 4 months at 40C with no detectable change in their aggregability with dilutions of the standard bovine plasma. PAF Receptor Activity Assay. For the test, 0.1 ml of FWP (8 X 105/,u) and 0.1 ml of Tris/saline/EDTA buffer (pH 7.4) were stirred in an aggregometer (Agregometre, Labintec, Montpellier, France) cuvette at 1100 rpm and 370C until a stable baseline was achieved. The recorder had been previously adjusted to 100% light transmission with the buffer mixture. Bovine plasma standard diluted 1:8 with buffer was mixed 1:1 with appropriate dilutions of membranes or soluble membrane
Proc. Natl. Acad. Sci. USA 76 (1979)
preparations to give a final bovine plasma concentration of 1:16. The aggregation curves produced by adding 0.1 ml of the plasma/membrane mixture to the FWP and buffer previously equilibrated in the aggregometer could be compared to those obtained with bovine plasma diluted similarly 1:16 with the buffer. In addition, the maximal slope of the aggregation curves for serial dilutions of both intact and detergent-solubilized membranes from 1:2 to 1:64 were calculated. The maximal slopes were plotted against the logarithm of the concentration of membranes (1:2 to 1:64) in the test system. Solubilization of Platelet Membranes. This was accomplished by using Brij 99 (polyoxyethylene 20 oleyl ether, kindly donated by ICI, Switzerland). Platelet membrane suspensions containing 10-15 mg of protein per ml were adjusted to 5% Brij 99 by the addition of solid detergent. The mixture was gently mixed until the Brij 99 was completely dissolved (usually 10-15 min). The detergent/platelet membrane mixture was incubated for 1 hr at room temperature and then centrifuged at 100,000 X g for 1 hr; the supernatant was carefully removed and used for subsequent investigations. The pellet, which was usually minimal, was also resuspended to 1/1Oth the original volume in Tris/saline/EDTA buffer and assayed for receptor activity. Lectin Specificity and Affinity Chromatography. These studies were carried out essentially as described (30). Initial lectin-specificity studies were performed by using a simple batch procedure with diluted membranes solubilized to Brij 99 and incubated 1:1 with Sepharose 4B gel or Sepharose 4B gel to which Lens culinaris lectin, concanavalin A, or wheat germ agglutinin (WGA) had been coupled by cyanogen bromide. The gel suspensions were centrifuged at 1500 X g for 10 min, and the supernatant was carefully aspirated and assayed for residual receptor activity. WGA affinity column chromatography of the solubilized platelet membranes from 50-100 units of blood was carried out with a 10 X 2.5 cm column equilibrated with 4 column vol of 0.01 M Tris-HCl/0.3 M NaCl/1 mM EDTA/ 0.5% Brij 99, pH 7.4. The column was eluted with 0.01 M Tris1HCl/0. 15 M NaCl/1 mM EDTA, pH 7.4, containing 0.5% Brij 99 and 2.5% (wt/vol) N-acetylglucosamine. Fractions (2.3 ml) were collected and assayed for PAF receptor activity. Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis. The technique was performed essentially as described by Lenard (35), in 13 X 0.6 cm rod gels. Gels were fixed in 25% isopropanol/10% acetic acid and stained for carbohydrate with periodic acid-Schiff reagent or for protein with Coomassie brilliant blue.
RESULTS Sonication of Fresh Washed Platelets. Centrifugation of sonicated platelets at 9000 X g for 30 min gave a membranerich supernate and a pellet containing unbroken platelets and larger platelet fragments. The membrane-rich supernatant from six experiments was assayed (Fig. 1) and was found to have measurable PAF receptor activity at a dilution of 1:64 with Tris/saline/EDTA, pH 7.4. In addition to its high membrane content, this supernatant also contained all of the glycocalicin released from the platelet surface during sonication. Centrifugation of the Membrane/Glycocalicin Preparation. Centrifugation of the above supernatant at 100,000 X g for 1 hr resulted in a glycocalicin-rich supernatant and a pellet containing essentially all of the insoluble membrane material. Both fractions were assayed for their PAF receptor activity. The supernatant had no measurable activity (Fig. 1) even after concentration to 50% of the original volume by ultrafiltration using an XM-1OOA membrane. When the pellet, composed primarily of membrane vesicles, was resuspended to its original
Biochemistry: Cooper et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
30' C
.A 20'
E.2-
2
C
3 *4
c as
0o0r
60
10o
150
200
Time, sec
FIG. 1. Aggregation of FWP by a mixture of bovine plasma and human platelet membrane fractions. Bovine plaisma diluted 1:8 in Tris/saline/EDTA, pH 7.4, was mixed in equal volumes with the fractions or with buffer as control. This mixture ((0.1 ml) was used to aggregate FWP in an aggregometer. Curves: 1, su]pernatant (100,000 X g) containing glycocalicin; 2, buffer control (bovine plasma, 1:16); 3, membrane pellet from 100,000 X g centrifugatic)n solubilized in 5% Brij 99; 4, supernatant (9000 X g) containing bolth membranes and glycocalicin.
volume in Tris/saline/EDTA, pH 7.4, dilutesi, and assayed for receptor activity, it could inhibit bovine plasma PAF at a dilution of 1:64. The slopes of the initial aggiregation for dilutions of 1:2 through 1:64 were linear when pilotted against the logarithm of the platelet membrane concenttration. A typical dose-response curve (Fig. 2) was almost ide:ntical to that obtained with the crude 9000 X g preparation. T he dose-response curves of several membrane preparations we re found to have similar slopes. The preparations varied only in their concentration of receptor activity as demonstrated b)y the fact that the dose-response curves all were parallel to one another. For any given membrane preparation, the dose-res ponse curve remained essentially unchanged for periods of at least 6 days if the membranes were kept in EDTA-containin ig buffers at 40C. When intact membrane preparations were fr4ozen and thawed once, the dose-response curve obtained im mediately upon thawing showed no difference from the saime membranes stored briefly at 4VC. However, once frozeen and thawed, membranes lost significant receptor activity w(ithin 48 hr, even when kept at 40C in buffers containing 1 mM IEDTA. Repeated freezing and thawing or storage for more 1than 6 weeks at -200C resulted in dose-response curves with1slopes similar to those obtained with the same membranes in tihe fresh state but shifted to the right of that in Fig. 2, showing that the concentrations of PAF receptor activity had markedIly decreased.
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Solubkization of the Membrane Preparation. When the tiny pellet after centrifugation of the solubilized membranes was resuspended in 1/10th the original volume and assayed, no detectable PAF receptor activity was found. The supernatant, however, had receptor activity that could be detected at a 1:64 dilution (Fig. 1). When the initial slope of the aggregation curve for each of these dilutions was calculated and a doseresponse curve was constructed as described for intact membranes, the values were linear to a 1:32 dilution (Fig. 2). At greater dilutions this curve showed a tendency to flatten out. The dose-response curves for soluble membranes were not parallel to those obtained for intact membranes and were reproducibly flatter. The differences between the slope of aggregation of the 1:2 and the 1:32 dilutions are highly significant because the rank sequence of the five points was exactly the same for all six solubilized membrane preparations studied. The point at which the two dose-response curves crossed varied slightly among the various membrane preparations but was consistently around a dilution of 1:16. The slope of the doseresponse curve for solubilized membranes was essentially the same regardless of whether dilutions were made in Tris/saline/EDTA buffer or in the same buffer but with a constant detergent concentration. When studied in parallel with human platelet membrane preparations, neither the intact nor solubilized membranes of P-815 mastocytoma cells or human erythrocyte ghosts showed any appreciable inhibitory activity. Lectin Specificity of Solubilized PAF Receptor. By using first a batch procedure, it was demonstrated that there was minimal adsorption to the uncoupled Sepharose 4B control gel. Mixtures of solubilized membranes and Sepharose 4B gel to which concanavalin A or Lens culinaris lectin had been coupled also failed to bind PAF receptor to any significant degree. WGA coupled to Sepharose 4B, on the other hand, removed more than 75% of the receptor activity from the same solubilized membrane preparation. With this information, a lectin affinity column of WGA coupled to Sepharose 4B was used to chromatograph human platelet membranes solubilized in Brij 99. The breakthrough fractions contained no measurable PAF receptor activity. The fractions eluted with N-acetylglucosamine were found to contain PAF receptor activity (Fig. 3). Because the dose-response curves of the intact and solubilized membranes were nonparallel, quantitation of receptor activity before and after solubilization could not be compared. When compared with activity of membranes solubilized in Brij 99, the activity eluted from the WGA-Sepharose 4B showed parallel
dose-response curves. Although not systematically studied, 2.51
CL"2.00
A
c
2r 1.510 0, < 1.01
1:2 1:R2 1:c6 1:t8 1:4 Receptor concentration FIG. 2. Effect of PAF receptor concentration on the slope of aggregation of FWP produced by a constant amount of bovine plasma. Bovine plasma diluted 1:8 in Tris/saline/EDTA, pH 7.4, was mixed in equal volumes with serial double dilutions of human platelet membranes () or of human platelet membranes solubilized in 5% Brij 99 (-). The mixture (0.1 ml) was used to aggregate FWP in an aggregometer. U-0
1:64
Fraction FIG. 3. Affinity chromatography on WGA-Sepharose 4B of human platelet membranes solubilized in 5% Brij 99. *, Distribution of protein (OD at 280 nm); am, fractions containing PAF receptor activity (inhibition of PAF aggregation of FWP).
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d
b
e
c
f
FIG. 4. Scans (at 550 nm) of 5% polyacrylamide gels stained for carbohydrate with periodic acid-Schiff reagent. (a) Supernatant after differential centrifugation of sonicated platelets; 500 1Ag of protein. (b) Purified glycocalicin; 20 ltg of protein. (c) Fraction of Brij 99solubilized platelet membranes, binding to WGA-Sepharose 4B; 100 1g of protein. (a-c) Nonreduced; (d-f), same samples run under reducing conditions.
quantitation of activity in those fractions that were assayed accounted for greater than 50% of the activity applied to the column.
Polyacrylamide Gel Electrophoresis. Various fractions obtained during the preparation of the platelet membrane receptor were studied on both nonreducing and reducing 5% gels. The gels shown in Fig. 4 show the results obtained when the gels were stained for carbohydrate and scanned at 550 nm. The supernatant obtained after sonication and differential centrifugation of platelets demonstrated primarily a single band in both the unreduced (Fig. 4a) and reduced (Fig. 4d) states. The band was essentially the same as that obtained for unreduced and reduced purified glycocalicin (Fig. 4 b and e). The supernatant (Fig. 4 a and d) and purified glycocalicin (Fig. 4 b and e) had no PAF receptor activity. Fractions with receptor activity, eluting from WGA-Sepharose 4B, gave two major bands of glycoprotein (Fig. 4c); interestingly, upon reduction, these gave a band (Fig. 4f) similar in electrophoretic mobility to purified glycocalicin (Fig. 4 b and e). DISCUSSION Conclusions that glycocalicin is the receptor for bovine vWF (26) have been primarily based on circumstantial evidence. These reports have not shown directly that glycocalicin interacts with PAF to inhibit its aggregating properties. It is known that sonication of washed human platelets releases glycocalicin into the supernatant as a soluble glycoprotein. Using the supernatant obtained by centrifugation of sonicated human platelets, we demonstrate in this report that the glycocalicin-rich solution (see Fig. 4) has no detectable inhibitory effect on PAF-induced aggregation of FWP (Fig. 1) and therefore contains virtually no PAF receptor. Even after a 50% increase in the concentration by ultrafiltration, the supernatant would not inhibit PAF aggregation of FWP. These results are consistent with our other
findings (unpublished data) that glycocalicin purified from such supernatant material, at up to 10 mg/ml in the assay system, had no significant PAF receptor activity. Further search for the PAF receptor showed that all of the measurable activity was located in the resuspended pellet which contained the platelet membrane vesicles. Most membrane preparations when diluted 1:64 in Tris/saline/EDTA buffer still showed some ability to inhibit PAF aggregation of FWP. When the slopes of the aggregation curves obtained with serial dilutions of membranes and diluted bovine plasma were plotted against the logarithm of the concentration of membranes, the values fell on a straight line (Fig. 2). This allowed at least semiquantitative estimations of receptor activity, and comparison of various membrane preparations showed parallel dose-re-
Proc. Natl. Acad. Sci. USA 76 (1979)
sponse curves. Thus, among preparations, the major difference appeared to be only the concentration of receptor activity. The PAF receptor activity was solubilized by treatment of the membrane preparations with detergents. We report here the use of a nonionic detergent, Brij 99. This detergent at the concentrations used had no effect on the assay system and demonstrated no significant effect on the receptor activity. Upon incubation of human platelet membranes in Brij 99, all of the receptor activity was released into the supernatant (Fig. 1). The pellet obtained by centrifugation of the membrane/ detergent mixture had no residual receptor activity when reconstituted to 1/10th the original volume. The release of receptor activity from the membrane by detergent suggests that the molecule has a significant hydrophobic domain that inserts into the lipid matrix of the membrane. This is in marked contrast to purified glycocalicin which has been reported to lack any significant hydrophobic domain and therefore to be only loosely attached to the platelet surface (28). The dose-response curves of membranes solubilized in Brij 99 were not parallel to those of intact membranes (Fig. 2). This suggests that the binding parameters of PAF and receptor are different in the two solvent systems and indicates that the conformation of the receptor solubilized in Brij 99 differs from that in the intact membrane. It is also of interest that the membrane concentration at which the two lines intersect represents the conditions under which the binding parameters are most similar. Thus, in order to study the solubilized receptor under conditions that most closely reflect those of the intact membrane, this concentration of receptor would be most appropriate. In preliminary batch experiments, PAF receptor solubilized in Brij 99 was shown to bind to WGA covalently linked to Sepharose 4B. In other batch experiments, it bound only weakly or not at all to Lens culinaris lectin or concanavalin A insolubilized on the same gel matrix. It has been reported that WGA affinity chromatography of human platelet membranes solubilized in 1% sodium deoxycholate resulted in binding and then elution with N-acetylglucosamine of only two of the major platelet membrane glycoproteins, GPla and GP1b (30). Solubilization of membranes in Brij 99 and affinity chromatography on WGA-Sepharose 4B gave similar results, suggesting that the receptor is one of these two glycoproteins (Fig. 4). Although GP1b and glycocalicin show similar electrophoretic properties in the reduced and nonreduced states and comparable lectin specificity (30), it nevertheless is premature to assume that these two molecules are identical. Even if this were the case, it does not necessarily mean that GPla must be the receptor because much circumstantial evidence links glycocalicin to the receptor. For example, conditions that were reported to destroy the ability of whole platelets to aggregate with PAF and at the same time release glycocalicin (27) also destroy the receptor solubilized in Brij 99. In addition, platelets from patients with Bernard-Soulier syndrome, which lack the receptor for PAF, also lack glycocalicin. Thus, it is tempting to suggest that glycocalicin is derived from the receptor, perhaps by some hitherto unknown mechanism. It would then follow that the resulting material (glycocalicin) has lost its ability to bind PAF due to conformational change or represents a fragment that no longer contains the binding site for PAF. The physiological significance of this information is not clear but one could speculate that the ease with which glycocalicin is split off from the membrane may play a role in normal platelet function. Because adhesion of platelets to subendothelium requires interaction with vWF it could be that the subsequent release of the platelet from the subendothelium is through the cleavage of the receptor, leaving glycocalicin at-
Biochemistry: Cooper et al. tached to the subendothelium. This type of hypothesis fo-W patible with studies of membrane glycoproteins and at' senescence. In those studies, platelets were shown to lose membrane glycoproteins during normal circulation (36) and the loss was accelerated by intravascular coagulation and diminished by the administration of aspirin and dipyridamole (37), suggesting that perhaps platelet activation favors conditions for shedding of glycoproteins. In other studies, platelets whose surface glycoproteins were radiolabeled became attached to damaged autologous aorta (38); after thorough washing to remove platelets, it could be demonstrated that significant amounts of labeled membrane glycoproteins were bound to the aorta. Because both PAF and human vWF have antigenic and structural similarities (5-10), it seems reasonable to speculate that they might share a common receptor on the human platelet. However, it was impossible to make direct assays of human vWF receptor activity because our membrane preparations, solubilized membranes, and fractions from WGA affinity chromatography all aggregated FWP in the presence of ristocetin. This demonstrates the presence of human platelet vWF in these preparations. It may be that other detergents, such as sodium dodecyl sulfate (23), destroy vWF activity and thus eliminate the problem of platelet vWF interference in the assay. The presence of platelet vWF in our preparations is not responsible for any inhibition in the PAF receptor assay because human vWF, in the absence of ristocetin, does not appear to compete with PAF. For instance, the latter aggregates human platelets in PRP, in which there is an excess of human vWF. The authors thank Miss S. Weber for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research, Grant 3.238.077. H.A.C. is supported by a Research Career Development Award HL-00081 from the National Heart, Lung and Blood Institute of the National Institutes of Health. 1. Griggs, T. R., Cooper, H. A., Webster, W. P., Wagner, R. H. & Brinkhous, K. M. (1973) Proc. Nati. Acad. Sci. USA 70,28142818. 2. Forbes, C. D. & Prentice, C. R. M. (1973) Nature (London) New Biol. 241, 149-150. 3. Griggs, T. R., Webster, W. P., Cooper, H. A., Wagner, R. H. & Brinkhous, K. M. (1974) Proc. Nati. Acad. Sci. USA 71, 20872090. 4. Donati, M. B., de Gaetano, G. & Vermylen, J. (1973) Thromb. Res. 2, 97-104. 5. Sarji, K. E., Stratton, R. D., Wagner, R. H. & Brinkhous, K. M. (1974) Proc. Nati. Acad. Sci. USA 71,2937-2941. 6. Brinkhous, K. M. (1975) Am. J. Clin. Pathol. 63,609-617. 7. Zimmerman, T. S., Roberts, J. & Edgington, T. S. (1975) Proc. Nati. Acad. Sci. USA 72,5121-5125. 8. Griggs, T. R., Potter, J., McClanahan, S. B., Webster, W. P. & Brinkhous, K. M. (1977) Proc. Nati. Acad. Sci. USA 74, 759763.
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10. Van Mourik, J. A., Bouma, B. N., La Bruyere, W. T., de Graaf, S. & Mochtar, J. A. (1974) Thromb. Res. 4, 155-164. 11. Kirby, E. P. (1977) Thromb. Haemostas. (Stuttgart) 38, 1054-1072. 12. Kirby, E. P. & Mills, D. C. B. (1975) J. Clin. Invest. 56, 491502. 13. Allain, J. P., Cooper, H. A., Wagner, R. H. & Brinkhous, K. M. (1975) J. Lab. Clin. Med. 85,318-328. 14. Tschopp, T., Weiss, H. J. & Boumgartner, H. R. (1974) J. Lab. Clin. Med. 83,296-300. 15. Weiss, H. J., Boumgartner, H. R., Tschopp, T. B., Juritto, V. T. & Cohen, D. (1978) Blood 51, 267-279. 16. Cooper, H. A., Wilkins, K. W., Jr., Johnson, P. R., Jr. & Wagner, R. H. (1977) J. Lab. Clin. Med. 90,512-521. 17. Phillips, D. A. (1972) Biochemistry 11, 4582-4588. 18. Bithell, T. C., Parekh, S. J. & Strong, R. R. (1972) Ann. N. Y. Acad. Sci. 201, 145-160. 19. Jenkins, C. S. P., Phillips, D. R., Clemetson, K. J., Meyer, D., Larrieu, M. J. & Luischer, E. F. (1976) J. Clin. Invest. 57, 112124. 20. Caen, J. P., Nurden, A. T., Jeanneau, C., Michel, H., Tobelem, G., Levy-Toledano, S., Sultan, Y., Valensi, F. & Bernard, J. (1976) J. Lab. Clin. Med. 87,586-596. 21. Nachman, R. L., Jaffe, E. A. & Weksler, B. B. (1977) J. Clin. Invest. 59, 143-148. 22. Tobelem, G., Levy-Toledano, S., Bredoux, R., Michel, H., Nurden, A., Caen, J. & Degos, L. (1976) Nature (London) 263, 427-429. 23. Nachman, R. L., Tarasov, E., Weksler, B. B. & Ferris, B. (1977) Thromb. Res. 12,91-104. 24. Okumura, T. & Jamieson, G. A. (1976) J. Biol. Chem. 251, 5944-5949. 25. Nurden, A. T. (1977) Experientia 35,331-332. 26. Solum, N. O., Hagen, I. & Peterka, M. (1977) Thromb. Res. 10, 71-82. 27. Solum, N. O., Hagen, I. & Gjemdal, T. (1977) Thromb. Haemostas. (Stuttgart) 38,914-923. 28. Okumura, T., Lombart, C. & Jamieson, G. A. (1976) J. Biol. Chem. 251, 5950-5955. 29. Okumura, T. & Jamieson, G. A. (1976) Thromb. Res. 8, 701706. 30. Clemetson, K. J., Pfueller, S., Luischer, E. F. & Jenkins, C. P. S. (1977) Biochim. Biophys. Acta 464, 493-508. 31. Bettex-Galland, M. & Luscher, E. F. (1960) Thromb. Diath. Haemorrh. 4, 178-195. 32. Massini, P. & Luscher, E. F. (1974) Biochim. Biophys. Acta 372, 109-121. 33. Kaser-Glanzmann, R., Jakabova, M., George, J. N. & Luscher, E. F. (1977) Biochim. Biophys. Acta 466,429-440. 34. Cooper, H. A., Reisner, F. F., Hall, M. & Wagner, R. H. (1975) J. Clin. Invest. 56,751-760. 35. Lenard, J. (1970) Biochemistry 9, 1129-1132. 36. George, J. N., Lewis, P. C. & Sears, D. A. (1976) J. Lab. Clin. Med.
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Vol. 76, No. 3, pp. 1069-1073, March 1979 Biochemistry
Human platelet membrane receptor for bovine von Willebrand factor (platelet aggregating factor): An integral membrane glycoprotein (macromolecules/Factor VIII/affinity chromatography/detergents/lectins)
HERBERT A. COOPER*, KENNETH J. CLEMETSONt, AND ERNST F. LUSCHERt *Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514; and tTheodor Kocher Institute, University of Bern, Bern, Switzerland
Communicated by Kenneth M. Brinkhous, November 30, 1978
ABSTRACT The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property o a soluble glycoprotein, glycocalicin, that is loosely attached to the plate let surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose-response curve obtained with Brij 99solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or Lens culinaris lectin. By using wheat germ agglutininSepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4VC in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents. Certain animal plasmas, particularly bovine, contain a glycoprotein that directly aggregates human platelets without known cofactors (1, 2). This platelet aggregating factor (PAF) is closely associated with Factor VIII procoagulant activity (1, 3, 4) and has an apparent homology with human von Willebrand factor (vWF) (3,5,6). Both human and animal vWF have been shown to be a heterodisperse family of oligomers with a minimal molecular weight of 1 X 106 (7-10). Interaction of bovine vWF with fresh washed human platelets causes aggregation within seconds and, under optimal conditions, produces a biphasic aggregation curve with the release of adenine nucleotides (11). The initial interaction of PAF with the platelet occurs in the presence of metabolic inhibitors (12) and even after fixation of platelets in formaldehyde (13). The interaction has recently taken on new biological importance with the reports (14, 15) that it is required for the normal adhesion and spreading of platelets on exposed subendothelium. The binding of PAF to platelets as a part of the aggregation phenomenon was first shown by using fresh washed platelets
(1) and subsequently was confirmed in studies using formaldehyde-fixed washed platelets (FWP) (16). The number of high-affinity binding sites was estimated to be in the range of 1-1.25 X 103 per platelet (16). It must be stressed, however, that this calculation is probably influenced by the number of PAF molecules that can be physically accommodated on the surface of the fixed platelet. Therefore, the determination may drastically underestimate the true number of binding sites available. Adsorption of PAF from bovine plasma by FWP was dependent on the platelet concentration and the length of incubation. PAF was bound to high-affinity sites as well as to nonspecific or lower-affinity sites. The PAF bound to the low-affinity sites could be removed by washing the aggregated FWP in 1 M KC1. Such treatment, however, did not remove the PAF bound to the high-affinity sites. Proteolytic treatment with trypsin destroys the ability of FWP to bind PAF (16). Because enzymatic treatment of platelet membranes with trypsin leads to the cleavage of surface-oriented glycoproteins (17), it seems reasonable to assign receptor function to one of these components. Such a hypothesis is further substantiated by the fact that the platelets of patients with Bernard-Soulier syndrome fail to aggregate with PAF (18) and have abnormalities of their surface glycoproteins (19, 20). Recently, anti-platelet membrane antisera absorbed with chymotrypsin-treated platelets gave an antiserum highly specific for the interaction of vWF and platelets (21). Further immunological studies have demonstrated possible receptor function in the GP1 region of the polyacrylamide gel electrophoretic pattern of platelet membranes (22, 23). A glycoprotein that is thought not to be an integral membrane component has also been implicated as the possible receptor for vWF (24-28). Primarily referred to as glycocalicin or GPS, it is obtained in soluble form from whole platelets by sonication or incubation in buffers that do not contain EDTA, repeated freezing and thawing, and extraction with 3 M KCI (27, 28). Glycocalicin is water soluble and reportedly lacks the usual lipophilic amino acids essential for the interaction of integral membrane proteins with the lipid constituents of the membrane. That glycocalicin is the receptor for vWF was suggested (29) based on the fact that high concentrations of glycocalicin inhibit ristocetin-induced aggregation of twicewashed human platelets. This glycoprotein has also been reported to be the receptor for PAF because platelets that have lost GPS are unable to aggregate with bovine Factor VIII, and GPS is missing from the surface of platelets from patients with Bernard-Soulier syndrome (17). Glycocalicin has the same lectin-binding affinity and similar electrophoretic mobility in
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Abbreviations: PAF, platelet aggregating factor; vWF, von Willebrand factor; FWP, formaldehyde-fixed washed platelets; PRP, platelet-rich plasma; WGA, wheat germ agglutinin; GPS, soluble glycoprotein.
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reducing and nonreducing polyacrylamide gels as one of the integral membrane glycoproteins, GP lb (30). Thus, the question remains as to whether glycocalicin is an intact, loosely attached membrane constituent with receptor function or a secondary product derived from an integral membrane protein that is actually the bovine PAF receptor. We have purified glycocalicin and studied this purified glycoprotein for bovine von Willebrand receptor activity. Because glycocalicin concentrations of up to 10 mg/ml caused less than 5% inhibition of PAF aggregation of FWP, it seemed worthwhile to look further for other membrane components that might have higher binding affinities for PAF. The- studies reported here describe the PAF receptor activity as being associated with an integral glycoprotein that requires detergent to release it from the platelet membrane vesicle. Solubilization of the receptor in an active state can be accomplished by using Brij 99 and the solubilized receptor has been partially characterized. MATERIALS AND METHODS Isolation of Human Blood Platelets. Within 20 hr after collection, platelets were isolated from citrated blood collected for the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross (31). The buffy coats were siphoned into a buffered glucose solution to give platelet-rich plasma (PRP) containing about 20 mM glucose, 12 mM phosphate buffer (pH 6.8), and about 4 X 109 platelets per ml (32). The PRP was washed three times in 0.01 M Tris-HCl/0.8% NaCI/10 mM EDTA, pH 7.4. After the third wash, the platelet pellet was resuspended in 0.01 M Tris-HCl/0.8% NaCl/1 mM EDTA, pH 7.4, at 4VC in a ratio by volume of 1 part platelets to 3 parts buffer. The platelet suspension was then cooled in an ice bath to about 4"C for sonication. Platelet Membrane Preparation. Human platelet membranes were processed from the platelet concentrates from 50-100 units of blood. The cooled, washed, platelet suspension was sonicated by using a B-30 sonifier (Branson Sonic Power Company, Danbury, CT for 2 min (output control, 7; 50% duty cycle; pulsed mode). The membranes were obtained by differential centrifugation as described (33) but without the 40,000 X g centrifugation step. Membranes were used fresh or after freezing at -700C and thawing only once. For long-term experiments requiring more than 1 day, only fresh membranes were used. In various preparations, protease inhibitors (0.5 mM CBZ-a-L-glutamyl-L-tyrosine and 0.5 mM phenylmethylsulfonyl fluoride), 0.01 M N-ethylmaleimide, and 0.02% sodium azide as a general bacteriostatic agent were used, although they could not be shown to be essential requirements for reproducible results. Fixed Washed Platelets (FWP). This assay reagent was prepared from fresh human platelets washed as described above. After three washes the platelets were resuspended in 1.8% (wt/vol) formaldehyde and prepared for storage by a modification (34) of the original method (13). The FWP were stored in 0.01 M Tris-HCI/0.8% NaCI/1 mM EDTA, pH 7.4, containing 0.02% sodium azide for up to 4 months at 40C with no detectable change in their aggregability with dilutions of the standard bovine plasma. PAF Receptor Activity Assay. For the test, 0.1 ml of FWP (8 X 105/,u) and 0.1 ml of Tris/saline/EDTA buffer (pH 7.4) were stirred in an aggregometer (Agregometre, Labintec, Montpellier, France) cuvette at 1100 rpm and 370C until a stable baseline was achieved. The recorder had been previously adjusted to 100% light transmission with the buffer mixture. Bovine plasma standard diluted 1:8 with buffer was mixed 1:1 with appropriate dilutions of membranes or soluble membrane
Proc. Natl. Acad. Sci. USA 76 (1979)
preparations to give a final bovine plasma concentration of 1:16. The aggregation curves produced by adding 0.1 ml of the plasma/membrane mixture to the FWP and buffer previously equilibrated in the aggregometer could be compared to those obtained with bovine plasma diluted similarly 1:16 with the buffer. In addition, the maximal slope of the aggregation curves for serial dilutions of both intact and detergent-solubilized membranes from 1:2 to 1:64 were calculated. The maximal slopes were plotted against the logarithm of the concentration of membranes (1:2 to 1:64) in the test system. Solubilization of Platelet Membranes. This was accomplished by using Brij 99 (polyoxyethylene 20 oleyl ether, kindly donated by ICI, Switzerland). Platelet membrane suspensions containing 10-15 mg of protein per ml were adjusted to 5% Brij 99 by the addition of solid detergent. The mixture was gently mixed until the Brij 99 was completely dissolved (usually 10-15 min). The detergent/platelet membrane mixture was incubated for 1 hr at room temperature and then centrifuged at 100,000 X g for 1 hr; the supernatant was carefully removed and used for subsequent investigations. The pellet, which was usually minimal, was also resuspended to 1/1Oth the original volume in Tris/saline/EDTA buffer and assayed for receptor activity. Lectin Specificity and Affinity Chromatography. These studies were carried out essentially as described (30). Initial lectin-specificity studies were performed by using a simple batch procedure with diluted membranes solubilized to Brij 99 and incubated 1:1 with Sepharose 4B gel or Sepharose 4B gel to which Lens culinaris lectin, concanavalin A, or wheat germ agglutinin (WGA) had been coupled by cyanogen bromide. The gel suspensions were centrifuged at 1500 X g for 10 min, and the supernatant was carefully aspirated and assayed for residual receptor activity. WGA affinity column chromatography of the solubilized platelet membranes from 50-100 units of blood was carried out with a 10 X 2.5 cm column equilibrated with 4 column vol of 0.01 M Tris-HCl/0.3 M NaCl/1 mM EDTA/ 0.5% Brij 99, pH 7.4. The column was eluted with 0.01 M Tris1HCl/0. 15 M NaCl/1 mM EDTA, pH 7.4, containing 0.5% Brij 99 and 2.5% (wt/vol) N-acetylglucosamine. Fractions (2.3 ml) were collected and assayed for PAF receptor activity. Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis. The technique was performed essentially as described by Lenard (35), in 13 X 0.6 cm rod gels. Gels were fixed in 25% isopropanol/10% acetic acid and stained for carbohydrate with periodic acid-Schiff reagent or for protein with Coomassie brilliant blue.
RESULTS Sonication of Fresh Washed Platelets. Centrifugation of sonicated platelets at 9000 X g for 30 min gave a membranerich supernate and a pellet containing unbroken platelets and larger platelet fragments. The membrane-rich supernatant from six experiments was assayed (Fig. 1) and was found to have measurable PAF receptor activity at a dilution of 1:64 with Tris/saline/EDTA, pH 7.4. In addition to its high membrane content, this supernatant also contained all of the glycocalicin released from the platelet surface during sonication. Centrifugation of the Membrane/Glycocalicin Preparation. Centrifugation of the above supernatant at 100,000 X g for 1 hr resulted in a glycocalicin-rich supernatant and a pellet containing essentially all of the insoluble membrane material. Both fractions were assayed for their PAF receptor activity. The supernatant had no measurable activity (Fig. 1) even after concentration to 50% of the original volume by ultrafiltration using an XM-1OOA membrane. When the pellet, composed primarily of membrane vesicles, was resuspended to its original
Biochemistry: Cooper et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
30' C
.A 20'
E.2-
2
C
3 *4
c as
0o0r
60
10o
150
200
Time, sec
FIG. 1. Aggregation of FWP by a mixture of bovine plasma and human platelet membrane fractions. Bovine plaisma diluted 1:8 in Tris/saline/EDTA, pH 7.4, was mixed in equal volumes with the fractions or with buffer as control. This mixture ((0.1 ml) was used to aggregate FWP in an aggregometer. Curves: 1, su]pernatant (100,000 X g) containing glycocalicin; 2, buffer control (bovine plasma, 1:16); 3, membrane pellet from 100,000 X g centrifugatic)n solubilized in 5% Brij 99; 4, supernatant (9000 X g) containing bolth membranes and glycocalicin.
volume in Tris/saline/EDTA, pH 7.4, dilutesi, and assayed for receptor activity, it could inhibit bovine plasma PAF at a dilution of 1:64. The slopes of the initial aggiregation for dilutions of 1:2 through 1:64 were linear when pilotted against the logarithm of the platelet membrane concenttration. A typical dose-response curve (Fig. 2) was almost ide:ntical to that obtained with the crude 9000 X g preparation. T he dose-response curves of several membrane preparations we re found to have similar slopes. The preparations varied only in their concentration of receptor activity as demonstrated b)y the fact that the dose-response curves all were parallel to one another. For any given membrane preparation, the dose-res ponse curve remained essentially unchanged for periods of at least 6 days if the membranes were kept in EDTA-containin ig buffers at 40C. When intact membrane preparations were fr4ozen and thawed once, the dose-response curve obtained im mediately upon thawing showed no difference from the saime membranes stored briefly at 4VC. However, once frozeen and thawed, membranes lost significant receptor activity w(ithin 48 hr, even when kept at 40C in buffers containing 1 mM IEDTA. Repeated freezing and thawing or storage for more 1than 6 weeks at -200C resulted in dose-response curves with1slopes similar to those obtained with the same membranes in tihe fresh state but shifted to the right of that in Fig. 2, showing that the concentrations of PAF receptor activity had markedIly decreased.
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Solubkization of the Membrane Preparation. When the tiny pellet after centrifugation of the solubilized membranes was resuspended in 1/10th the original volume and assayed, no detectable PAF receptor activity was found. The supernatant, however, had receptor activity that could be detected at a 1:64 dilution (Fig. 1). When the initial slope of the aggregation curve for each of these dilutions was calculated and a doseresponse curve was constructed as described for intact membranes, the values were linear to a 1:32 dilution (Fig. 2). At greater dilutions this curve showed a tendency to flatten out. The dose-response curves for soluble membranes were not parallel to those obtained for intact membranes and were reproducibly flatter. The differences between the slope of aggregation of the 1:2 and the 1:32 dilutions are highly significant because the rank sequence of the five points was exactly the same for all six solubilized membrane preparations studied. The point at which the two dose-response curves crossed varied slightly among the various membrane preparations but was consistently around a dilution of 1:16. The slope of the doseresponse curve for solubilized membranes was essentially the same regardless of whether dilutions were made in Tris/saline/EDTA buffer or in the same buffer but with a constant detergent concentration. When studied in parallel with human platelet membrane preparations, neither the intact nor solubilized membranes of P-815 mastocytoma cells or human erythrocyte ghosts showed any appreciable inhibitory activity. Lectin Specificity of Solubilized PAF Receptor. By using first a batch procedure, it was demonstrated that there was minimal adsorption to the uncoupled Sepharose 4B control gel. Mixtures of solubilized membranes and Sepharose 4B gel to which concanavalin A or Lens culinaris lectin had been coupled also failed to bind PAF receptor to any significant degree. WGA coupled to Sepharose 4B, on the other hand, removed more than 75% of the receptor activity from the same solubilized membrane preparation. With this information, a lectin affinity column of WGA coupled to Sepharose 4B was used to chromatograph human platelet membranes solubilized in Brij 99. The breakthrough fractions contained no measurable PAF receptor activity. The fractions eluted with N-acetylglucosamine were found to contain PAF receptor activity (Fig. 3). Because the dose-response curves of the intact and solubilized membranes were nonparallel, quantitation of receptor activity before and after solubilization could not be compared. When compared with activity of membranes solubilized in Brij 99, the activity eluted from the WGA-Sepharose 4B showed parallel
dose-response curves. Although not systematically studied, 2.51
CL"2.00
A
c
2r 1.510 0, < 1.01
1:2 1:R2 1:c6 1:t8 1:4 Receptor concentration FIG. 2. Effect of PAF receptor concentration on the slope of aggregation of FWP produced by a constant amount of bovine plasma. Bovine plasma diluted 1:8 in Tris/saline/EDTA, pH 7.4, was mixed in equal volumes with serial double dilutions of human platelet membranes () or of human platelet membranes solubilized in 5% Brij 99 (-). The mixture (0.1 ml) was used to aggregate FWP in an aggregometer. U-0
1:64
Fraction FIG. 3. Affinity chromatography on WGA-Sepharose 4B of human platelet membranes solubilized in 5% Brij 99. *, Distribution of protein (OD at 280 nm); am, fractions containing PAF receptor activity (inhibition of PAF aggregation of FWP).
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d
b
e
c
f
FIG. 4. Scans (at 550 nm) of 5% polyacrylamide gels stained for carbohydrate with periodic acid-Schiff reagent. (a) Supernatant after differential centrifugation of sonicated platelets; 500 1Ag of protein. (b) Purified glycocalicin; 20 ltg of protein. (c) Fraction of Brij 99solubilized platelet membranes, binding to WGA-Sepharose 4B; 100 1g of protein. (a-c) Nonreduced; (d-f), same samples run under reducing conditions.
quantitation of activity in those fractions that were assayed accounted for greater than 50% of the activity applied to the column.
Polyacrylamide Gel Electrophoresis. Various fractions obtained during the preparation of the platelet membrane receptor were studied on both nonreducing and reducing 5% gels. The gels shown in Fig. 4 show the results obtained when the gels were stained for carbohydrate and scanned at 550 nm. The supernatant obtained after sonication and differential centrifugation of platelets demonstrated primarily a single band in both the unreduced (Fig. 4a) and reduced (Fig. 4d) states. The band was essentially the same as that obtained for unreduced and reduced purified glycocalicin (Fig. 4 b and e). The supernatant (Fig. 4 a and d) and purified glycocalicin (Fig. 4 b and e) had no PAF receptor activity. Fractions with receptor activity, eluting from WGA-Sepharose 4B, gave two major bands of glycoprotein (Fig. 4c); interestingly, upon reduction, these gave a band (Fig. 4f) similar in electrophoretic mobility to purified glycocalicin (Fig. 4 b and e). DISCUSSION Conclusions that glycocalicin is the receptor for bovine vWF (26) have been primarily based on circumstantial evidence. These reports have not shown directly that glycocalicin interacts with PAF to inhibit its aggregating properties. It is known that sonication of washed human platelets releases glycocalicin into the supernatant as a soluble glycoprotein. Using the supernatant obtained by centrifugation of sonicated human platelets, we demonstrate in this report that the glycocalicin-rich solution (see Fig. 4) has no detectable inhibitory effect on PAF-induced aggregation of FWP (Fig. 1) and therefore contains virtually no PAF receptor. Even after a 50% increase in the concentration by ultrafiltration, the supernatant would not inhibit PAF aggregation of FWP. These results are consistent with our other
findings (unpublished data) that glycocalicin purified from such supernatant material, at up to 10 mg/ml in the assay system, had no significant PAF receptor activity. Further search for the PAF receptor showed that all of the measurable activity was located in the resuspended pellet which contained the platelet membrane vesicles. Most membrane preparations when diluted 1:64 in Tris/saline/EDTA buffer still showed some ability to inhibit PAF aggregation of FWP. When the slopes of the aggregation curves obtained with serial dilutions of membranes and diluted bovine plasma were plotted against the logarithm of the concentration of membranes, the values fell on a straight line (Fig. 2). This allowed at least semiquantitative estimations of receptor activity, and comparison of various membrane preparations showed parallel dose-re-
Proc. Natl. Acad. Sci. USA 76 (1979)
sponse curves. Thus, among preparations, the major difference appeared to be only the concentration of receptor activity. The PAF receptor activity was solubilized by treatment of the membrane preparations with detergents. We report here the use of a nonionic detergent, Brij 99. This detergent at the concentrations used had no effect on the assay system and demonstrated no significant effect on the receptor activity. Upon incubation of human platelet membranes in Brij 99, all of the receptor activity was released into the supernatant (Fig. 1). The pellet obtained by centrifugation of the membrane/ detergent mixture had no residual receptor activity when reconstituted to 1/10th the original volume. The release of receptor activity from the membrane by detergent suggests that the molecule has a significant hydrophobic domain that inserts into the lipid matrix of the membrane. This is in marked contrast to purified glycocalicin which has been reported to lack any significant hydrophobic domain and therefore to be only loosely attached to the platelet surface (28). The dose-response curves of membranes solubilized in Brij 99 were not parallel to those of intact membranes (Fig. 2). This suggests that the binding parameters of PAF and receptor are different in the two solvent systems and indicates that the conformation of the receptor solubilized in Brij 99 differs from that in the intact membrane. It is also of interest that the membrane concentration at which the two lines intersect represents the conditions under which the binding parameters are most similar. Thus, in order to study the solubilized receptor under conditions that most closely reflect those of the intact membrane, this concentration of receptor would be most appropriate. In preliminary batch experiments, PAF receptor solubilized in Brij 99 was shown to bind to WGA covalently linked to Sepharose 4B. In other batch experiments, it bound only weakly or not at all to Lens culinaris lectin or concanavalin A insolubilized on the same gel matrix. It has been reported that WGA affinity chromatography of human platelet membranes solubilized in 1% sodium deoxycholate resulted in binding and then elution with N-acetylglucosamine of only two of the major platelet membrane glycoproteins, GPla and GP1b (30). Solubilization of membranes in Brij 99 and affinity chromatography on WGA-Sepharose 4B gave similar results, suggesting that the receptor is one of these two glycoproteins (Fig. 4). Although GP1b and glycocalicin show similar electrophoretic properties in the reduced and nonreduced states and comparable lectin specificity (30), it nevertheless is premature to assume that these two molecules are identical. Even if this were the case, it does not necessarily mean that GPla must be the receptor because much circumstantial evidence links glycocalicin to the receptor. For example, conditions that were reported to destroy the ability of whole platelets to aggregate with PAF and at the same time release glycocalicin (27) also destroy the receptor solubilized in Brij 99. In addition, platelets from patients with Bernard-Soulier syndrome, which lack the receptor for PAF, also lack glycocalicin. Thus, it is tempting to suggest that glycocalicin is derived from the receptor, perhaps by some hitherto unknown mechanism. It would then follow that the resulting material (glycocalicin) has lost its ability to bind PAF due to conformational change or represents a fragment that no longer contains the binding site for PAF. The physiological significance of this information is not clear but one could speculate that the ease with which glycocalicin is split off from the membrane may play a role in normal platelet function. Because adhesion of platelets to subendothelium requires interaction with vWF it could be that the subsequent release of the platelet from the subendothelium is through the cleavage of the receptor, leaving glycocalicin at-
Biochemistry: Cooper et al. tached to the subendothelium. This type of hypothesis fo-W patible with studies of membrane glycoproteins and at' senescence. In those studies, platelets were shown to lose membrane glycoproteins during normal circulation (36) and the loss was accelerated by intravascular coagulation and diminished by the administration of aspirin and dipyridamole (37), suggesting that perhaps platelet activation favors conditions for shedding of glycoproteins. In other studies, platelets whose surface glycoproteins were radiolabeled became attached to damaged autologous aorta (38); after thorough washing to remove platelets, it could be demonstrated that significant amounts of labeled membrane glycoproteins were bound to the aorta. Because both PAF and human vWF have antigenic and structural similarities (5-10), it seems reasonable to speculate that they might share a common receptor on the human platelet. However, it was impossible to make direct assays of human vWF receptor activity because our membrane preparations, solubilized membranes, and fractions from WGA affinity chromatography all aggregated FWP in the presence of ristocetin. This demonstrates the presence of human platelet vWF in these preparations. It may be that other detergents, such as sodium dodecyl sulfate (23), destroy vWF activity and thus eliminate the problem of platelet vWF interference in the assay. The presence of platelet vWF in our preparations is not responsible for any inhibition in the PAF receptor assay because human vWF, in the absence of ristocetin, does not appear to compete with PAF. For instance, the latter aggregates human platelets in PRP, in which there is an excess of human vWF. The authors thank Miss S. Weber for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research, Grant 3.238.077. H.A.C. is supported by a Research Career Development Award HL-00081 from the National Heart, Lung and Blood Institute of the National Institutes of Health. 1. Griggs, T. R., Cooper, H. A., Webster, W. P., Wagner, R. H. & Brinkhous, K. M. (1973) Proc. Nati. Acad. Sci. USA 70,28142818. 2. Forbes, C. D. & Prentice, C. R. M. (1973) Nature (London) New Biol. 241, 149-150. 3. Griggs, T. R., Webster, W. P., Cooper, H. A., Wagner, R. H. & Brinkhous, K. M. (1974) Proc. Nati. Acad. Sci. USA 71, 20872090. 4. Donati, M. B., de Gaetano, G. & Vermylen, J. (1973) Thromb. Res. 2, 97-104. 5. Sarji, K. E., Stratton, R. D., Wagner, R. H. & Brinkhous, K. M. (1974) Proc. Nati. Acad. Sci. USA 71,2937-2941. 6. Brinkhous, K. M. (1975) Am. J. Clin. Pathol. 63,609-617. 7. Zimmerman, T. S., Roberts, J. & Edgington, T. S. (1975) Proc. Nati. Acad. Sci. USA 72,5121-5125. 8. Griggs, T. R., Potter, J., McClanahan, S. B., Webster, W. P. & Brinkhous, K. M. (1977) Proc. Nati. Acad. Sci. USA 74, 759763.
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10. Van Mourik, J. A., Bouma, B. N., La Bruyere, W. T., de Graaf, S. & Mochtar, J. A. (1974) Thromb. Res. 4, 155-164. 11. Kirby, E. P. (1977) Thromb. Haemostas. (Stuttgart) 38, 1054-1072. 12. Kirby, E. P. & Mills, D. C. B. (1975) J. Clin. Invest. 56, 491502. 13. Allain, J. P., Cooper, H. A., Wagner, R. H. & Brinkhous, K. M. (1975) J. Lab. Clin. Med. 85,318-328. 14. Tschopp, T., Weiss, H. J. & Boumgartner, H. R. (1974) J. Lab. Clin. Med. 83,296-300. 15. Weiss, H. J., Boumgartner, H. R., Tschopp, T. B., Juritto, V. T. & Cohen, D. (1978) Blood 51, 267-279. 16. Cooper, H. A., Wilkins, K. W., Jr., Johnson, P. R., Jr. & Wagner, R. H. (1977) J. Lab. Clin. Med. 90,512-521. 17. Phillips, D. A. (1972) Biochemistry 11, 4582-4588. 18. Bithell, T. C., Parekh, S. J. & Strong, R. R. (1972) Ann. N. Y. Acad. Sci. 201, 145-160. 19. Jenkins, C. S. P., Phillips, D. R., Clemetson, K. J., Meyer, D., Larrieu, M. J. & Luischer, E. F. (1976) J. Clin. Invest. 57, 112124. 20. Caen, J. P., Nurden, A. T., Jeanneau, C., Michel, H., Tobelem, G., Levy-Toledano, S., Sultan, Y., Valensi, F. & Bernard, J. (1976) J. Lab. Clin. Med. 87,586-596. 21. Nachman, R. L., Jaffe, E. A. & Weksler, B. B. (1977) J. Clin. Invest. 59, 143-148. 22. Tobelem, G., Levy-Toledano, S., Bredoux, R., Michel, H., Nurden, A., Caen, J. & Degos, L. (1976) Nature (London) 263, 427-429. 23. Nachman, R. L., Tarasov, E., Weksler, B. B. & Ferris, B. (1977) Thromb. Res. 12,91-104. 24. Okumura, T. & Jamieson, G. A. (1976) J. Biol. Chem. 251, 5944-5949. 25. Nurden, A. T. (1977) Experientia 35,331-332. 26. Solum, N. O., Hagen, I. & Peterka, M. (1977) Thromb. Res. 10, 71-82. 27. Solum, N. O., Hagen, I. & Gjemdal, T. (1977) Thromb. Haemostas. (Stuttgart) 38,914-923. 28. Okumura, T., Lombart, C. & Jamieson, G. A. (1976) J. Biol. Chem. 251, 5950-5955. 29. Okumura, T. & Jamieson, G. A. (1976) Thromb. Res. 8, 701706. 30. Clemetson, K. J., Pfueller, S., Luischer, E. F. & Jenkins, C. P. S. (1977) Biochim. Biophys. Acta 464, 493-508. 31. Bettex-Galland, M. & Luscher, E. F. (1960) Thromb. Diath. Haemorrh. 4, 178-195. 32. Massini, P. & Luscher, E. F. (1974) Biochim. Biophys. Acta 372, 109-121. 33. Kaser-Glanzmann, R., Jakabova, M., George, J. N. & Luscher, E. F. (1977) Biochim. Biophys. Acta 466,429-440. 34. Cooper, H. A., Reisner, F. F., Hall, M. & Wagner, R. H. (1975) J. Clin. Invest. 56,751-760. 35. Lenard, J. (1970) Biochemistry 9, 1129-1132. 36. George, J. N., Lewis, P. C. & Sears, D. A. (1976) J. Lab. Clin. Med.
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