Biochimica et Biophysica Acta, 439 (1976) 339-348
¢~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 37412 PLATELET LIPOPROTEINS A COMPARATIVE STUDY WITH SERUM LIPOPROTEINS*
MARIA FERNANDA LOPES-VIRELLA a, K. SARJP, G. VIRELLA b and J. A. COLWELL a ~ Endocrinology, Metabolism and Nutrition Division, Department o f Medicine, Medical University o f South Carolina and Veterans Administration Hospital, ~ Department o f Basic and Clinical hnmunology and Microbiology, Medical University o f South Carolina, Charleston, S.C. (U.S.A.)
(Received December 23rd, 1975)
SUMMARY The nature of human platelet lipoproteins was studied in two series of experiments. In the first series, whole platelets were utilized for extraction of lipoproteins by three different methods : chloroform/methanol/phenol; saline; or sucrose-gradient ultracentrifugation of platelet homogenates. By polyacrylamide gel electrophoresis we were able to demonstrate the existence of lipoprotein in the extracts obtained by the last two methods. These lipoproteins were found not to share antigenic determinants with alpha and beta serum lipoproteins. The second series of experiments utilized platelets solubilized either in sodium deoxycholate or sodium dodecyl sulfate. The solubilized product was characterized by double immunodiffusion and polyacrylamide gel electrophoresis. The nonidentity between plasma and platelet lipoproteins previously demonstrated in the first series of experiments was confirmed. This nonidentity was also supported by a comparison between the apoproteins of purified serum lipoproteins and platelet proteins released after solubilization with sodium dodecyl sulfate. No identical protein fractions were found. Our results suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoproteins are structurally different from plasma lipoproteins. _
_
_
INTRODUCTION In recent years, several reports have been published concerning the lipid and protein composition of cell membranes and tissues [1-9]. Most authors have been concerned with the study of the lipid composition of cell membranes, while lipoproteins have usually not been characterized. This is particularly true in the case of platelets. Recently, Langdon [9] published an interesting report suggesting that the apoproteins of the serum lipoproteins are immunologically and biochemically similar Abbreviations: LDL, low density lipoprotein (d ~ 1.006-1.063 g/ml); VLDL, very low density lipoprotein (d < 1.006 g/ml); HDL, high density lipoprotein (d ~ 1.063-1.21 g/ml); SDS, sodium dodecyl sulfate. * Publication No. 34 of the Department of Basic and Clinical Immunology and Microbiology.
340 to thoe sof the erythrocyte membrane. In platelets, however, the relation between plasma lipoproteins and platelet lipoproteins is not known [5]. In view of these observations and because of the importance of platelet lipoproteins in platelet function and coagulation [10, I1], we have undertaken a study to compare serum and platelet lipoproteins of normal individuals. MATERIALS AND METHODS Platelets. Human platelets were obtained from freshly drawn blood of healthy donors whose serum lipoproteins had been characterized as normal. The platelet suspensions were prepared according to the method described by Sarji et al. [12], exhaustively washed to avoid any contamination with plasma, and finally resuspended in sodium phosphate buffer 0.5 mM, pH 8.0. The final suspension contained 1 000 000 platelets/mm 3. Platelet lipoprotein extraction. I_ipoproteins were extracted from platelets by three methods. The first was based on a modification by Lacave [I] of the methods described by Ames [8], Westphal [13] and Okuda [14]. Free lipids of whole platelets were removed according to Ames [8]. The remaining residue was then extracted with phenol [13] and phenol was finally removed by dialysis against glycerol [14]. The fraction obtained in the phenolic phase was dialyzed against distilled water for three days and then was tested by conventional lipoprotein polyacrylamide gel electrophoresis [15-18]. Secondly, we used a modification of the method described by Srinivasan for the extraction of lipoproteins from fatty streaks of human aorta [2, 6]. This consisted of extracting water-soluble material from a platelet suspension (109 platelets) with 4 volumes of 0.15 M sodium chloride by gentle shaking at 4 °C for 24 h, followed by low speed centrifugation (2000 rev./min) for 30 min. Finally, we used the method described by Marcus [7], consisting of homogenizing a platelet suspension with a Teflon pestle rotated at 1700 rev./min for 5 min. After washing the tube and plunger with sodium phosphate buffer and mixing homogenate and washes, the product was centrifuged for 30 min at 2000 ~ g. The supernatant obtained after centrifugation was layered on 30 to 60~o sucrose gradients, which were prepared at 4 °C immediately before use. Ultracentrifugation was carried out in a Beckman L5-75 ultracentrifuge with SW 50.1 swinging bucket rotor at 130 000 g at 4 °C for 2 h. After ultracentrifugation, 0.5 ml samples were collected. Platelet solubilization. In the second series of experiments, whole platelets were solubilized using sodium deoxycholate and sodium dodecyl sulfate. Solubilization with sodium deoxycholate was accomplished by mixing equal quantities of platelet suspension containing 1.106 cells/mm 3 and 0.1 M sodium deoxycholate solution. The solubilization with sodium dodecyl sulfate (SDS) was carried out as described by Langdon [9] for erythrocyte membrane. To 1 ml o f a platelet suspension (109 platelets) prepared as described above, 0.1 ml of 20 ~ SDS solution was added. The solution obtained was heated in a steam bath for 1.5 rain. When the resulting extract was to be tested immunochemically or by SDS-polyacrylamide electrophoresis, it was dialyzed for 48 h against 0.1 M sodium phosphate buffer pH 7.2, containing 0.1 ~,/, (w/v) of SDS. When the extract was to be submitted to gel chromatography, after it had
341 cooled to room temperature, we added 50 #1 of N-ethyl morpholine and 100 #1 of dimethylformamide containing 2 mg of dansyl chloride. This was used as a fluorescent protein marker. The solution was allowed to stand 30 minutes at room temperature and was then layered on the top o f a 1 x 30 cm Sephadex G-200 column equilibrated and eluted with 0.1 ~o SDS in pH 7.2, 0.1 M sodium phosphate buffer. 0.5 ml fractions were collected and the fluorescence of the fractions was detected by illuminating the test tubes with a 366 nm light source. All the fluorescent fractions were tested by double diffusion in agar gel [19] against antisera to human alpha and beta serum lipoproteins and studied by SDS-polyacrylamide gel electrophoresis [20-22]. Isolation of serum lipoproteins. Pooled fresh sera from fasting healthy donors was used. Platelet donors were represented in this pool. The isolation of serum lipoproteins was done according to the methods described by Burnstein [23-24]. Low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were isolated by precipitation with heparin and MgCI2 in presence of sucrose, followed by ultracentrifugation for 24 h at 100 000 ;~, g. High density lipoprotein (HDL) was isolated by precipitation with sodium phosphotungstate and MgClz. The purity of isolated serum lipoproteins was verified by conventional lipoprotein polyacrylamide electrophoresis, immunodiffusion and immunoelectrophoresis. Some albumin was found as a contaminant of beta lipoprotein. Quantitative studies on platelet extracts and homogenates and purified lipoprotein fractions. Total protein concentration was determined by the method of Lowry et al. [25]. Total cholesterol was determined by a two-step method involving an isopropanol extract (1 :I0) of the preparations to study and a colorimetric determination of cholesterol based on the reaction of concentrated sulfuric acid and ferric chloride in acetic acid with steroids having the 5-erie 3 beta-01 grouping [26]. Electrophoretic studies. The l ipoproteins of serum, platelet extracts and platelet homogenates were separated by conventional polyacrylamide gel electrophoresis as described for the separation of lipoproteins [15-18]. The protein content of the samples applied in each gel varied from 300-700 /~g of total protein. The total cholesterol content in the samples varied from 7 to 12/~g. SDS-polyacrylamide electrophoresis in 5~o (monomer w/v) gel [20-22] was used to compare the mobility of the apolipoproteins of alpha, beta and pre-beta lipoproteins, proteins contained in a SDS solubilized platelet suspension, whole serum proteins and several isolated proteins used as controls. The protein concentration of the platelet suspension, purified lipoproteins, normal serum and each control protein was adjusted to approximately 4 mg/ml prior to incubation with 0.1 ml of 20 ~o SDS and subsequent dialysis against 0.1 M phosphate buffer, pH 7.2, containing 0.1 ~ SDS (w/v). Approximately 200/~g of protein were applied to each gel. hnmunochemical studies. The purity of isolated serum lipoproteins was tested immunoelectrophoretically [27] using goat anti-human beta lipoprotein anti-~erum (Lot No. 220 s2, 9.1 mg of antibody protein per ml), obtained from Miles Laboratories, and rabbit anti-human alpha lipoprotein antiserum (Lot No. 2348 T, 0.1 units of antibody per ml), obtained from Behring Diagnostics. Antigenic analysis of serum and platelet lipoprotein extracts was carried out by double-immunodiffusion using the same antisera.
342 In order to ensure that a negative result would not be a consequence of in,correct antigen-antibody proportions, samples to be compared were either adjusted to similar protein concentrations or the tests were repeated under different antigenantibody proportions, obtained by keeping constant the values of antigen and varying the volume of antibody from 5 #1 to 50 #1. Diffusion was prolonged to 48 hours at room temperature and the agar platelets were washed, dried and stained with amidoblack 10B to ensure maximal sensitivity. RESULTS
When platelets were extracted by the method of Lacave [1] and tested by conventional lipoprotein polyacrylamide electrophoresis, no lipid-stained fractions were obtained. However, a lipoprotein fraction remaining on the top of the separation gel was observed when water soluble material obtained by extraction with 0.15 M sodium chloride as described by Srinivasan was studied. When platelet homogenates were prepared according to the method of Marcus [7] and submitted to ultracentrifugation, polyacrylamide electrophoresis of the fractions obtained revealed a sudanophilic band at the top of the separating gel sharper than the one obtained from saline extracts. This band was particularly obvious in the upper fraction of sucrose gradients after preparative ultracentrifugation (Fig. 1). /-
t~ - " ~ "
C
1
"
•
11
Fig. 1. Conventional lipoprotein electrophoresis in polyacrylamide gel of fractions obtained from platelet homogenates through sucrose-gradient ultracentrifugation. Gel C was loaded with 30/d of normal human serum. Gels 1 to 11 were loaded with 100/~1 of sucrose gradient fractions 1 to 11, starting from the bottom (fraction 1).
343
A
B
Fig. 2. Conventional lipoprotein electrophoresis of two platelet extracts obtained respectively with sodium deoxycholate (Gel A) and sodium dodecyl sulfate (gel B). Note that the top fraction seems to be constituted by two sub-fractions, more obviously separated in the sodium dodecyl sulfate extract. Exposure o f saline extracted platelet material and platelet homogenates to sodium dodecyl sulfate and sodium deoxycholate resulted in the separation of the sudanophilic material into two fractions, one migrating deep into the gel and the other remaining at the top of the separating gel as in the original untreated extracts (Fig. 2). The platelet extracts obtained according to the different extraction methods
344
Fig. 3. Double immunodiffusion study of the antigenicity of platelet lipoproteins. Wells 1, 2, and 3 contain three normal sera, wells 4 to 14 contain sucrose gradient fractions, 1 to 11, from bottom. Well 15 and 18 contain a saline platelet extract. Well 16 contains a platelet homogenate. Well 17 contains a methanol/chloroform/phenol platelet extract. The upper three central wells were filled with antisera against human serum beta lipoprotein. The lower central wells were filled with antisera against human serum alpha lipoprotein. d e s c r i b e d were tested by d o u b l e diffusion in a g a r against a n t i s e r a to h u m a n a l p h a a n d beta s e r u m l i p o p r o t e i n s . N o p r e c i p i t a t i o n lines were d e t e c t e d (Fig. 3). E l e c t r o p h o r e t i c s e p a r a t i o n s o f e x t r a c t s o b t a i n e d after p l a t e l e t s o l u b i l i z a t i o n w i t h s o d i u m d e o x y c h o l a t e were n o t v e r y s a t i s f a c t o r y and t h e i r i n t e r p r e t a t i o n difficult. H o w e v e r , e l e c t r o p h o r e t i c s e p a r a t i o n s f r o m e x t r a c t s o b t a i n e d after s o l u b i l i z a t i o n with S D S w e r e r e p r o d u c i b l e and t h e i r i n t e r p r e t a t i o n easier. W e f o u n d n o c o r r e l a t i o n b e t w e e n the p r o t e i n f r a c t i o n s s e p a r a t e d f r o m platelets extracts a n d the a p o p r o t e i n s s e p a r a t e d f r o m i s o l a t e d s e r u m l i p o p r o t e i n s (Fig. 4).
345
M A(d) G
TR ALB OVA CHY LYS
i
2
3
4
5
6
Fig. 4. Sodium dodecyl sulfate polyacrylamide electrophoresis of different lipoprotein preparations dissolved with SDS. Purified alpha lipoprotein was separated in gel 1, purified beta lipoprotein in gel 2, and purified prebeta lipoprotein in gel 3. An SDS solubilized platelet extract was separated gel 4. Gels 5 and 6 were used to separate controls, respectively ,normal serum (gel 5) and a mixture of purified proteins (gel 6). lgM (M); lgA dimeric (A(d)) ; lgG (G) ; transferrin (TR); human albumin (ALB) ; ovalbumin (OVA); alpha-chymotrypsinogen (CHY); and eggwhite lysozyme (LYS). The fluorescent fractions obtained after gel c h r o m a t o g r a p h y in Sephadex G-200 o f a SDS platelet extract treated with dansyl chloride were also studied by SDS polyacrylamide electrophoresis and again no correspondence was found between the observed fractions and the major apoproteins of alpha, beta, and prebeta serum lipoproteins. The SDS platelet extract, adjusted at 4 mg/ml o f protein, was tested against antisera to h u m a n alpha and beta serum lipoproteins using as controls h u m a n alpha, beta, and prebeta lipoproteins adjusted to the same protein concentration in 0.1 ~°/oo SDS (Fig. 5). Precipitation lines were detected with the purified serum lipoproteins but no precipitation was observed with the platelet extract. A similarly negative result was obtained when fractions separated by G-200 gel filtration from an SDS-platelet extract were studied. DISCUSSION Langdon, in 1974 [9], suggested that the major intrinsic lipophilic proteins of the erythrocyte membrane contained apoproteins similar to those of serum lipc-
346
Fig. 5. Double immunodiffusion study of platelet lipoproteins in a SDS-solubilized platelet extract. In the two represented systems, peripheral wells were filled with beta lipoprotein dissolved in SDS (1), Prebeta lipoprotein dissolved in SDS (2), SDS-phosphate buffer (3) alpha lipoprotein dissolved in SDS (4), and SDS platelet extract (5) and normal human serum dissolved in SDS. The central well of the left system was filled with antisera against human serum beta lipoprotein, and in the right system the central well was filled with antisera against human serum alpha lipoprotein. proteins based on immunological evidence of identity of antigenic determinavts for both serum alpha and beta lipoproteins and those of lipoproteins in erythrocyte membranes. This author also showed that the NH2 terminal aminoacids of serum beta or alpha lipoproteins and the NH2 terminal aminoacids of erythrocyte membrane proteins are similar. This observation encouraged our studies aimed at comparing the antigenicity of serum and platelet lipoproteins. However, no shared determinants were detected with saline extracts and platelet homogenates and negative results were also obtained when membrane proteins solubilized with detergents were submitted to immunochemical analysis. A possible inhibitory effect of detergents on precipitin reactions could be ruled out by the fact that we could obtain precipitation with purified serum lipoproteins dissolved in 0.1 ~i~ SDS solution (w/v). Further, several authors have described the possibility of immunochemical testing of lipoproteins dissolved in the presence of detergent [9, 28-30]. The lack of identity between serum and platelet lipoproteins is also supported by the fact that the majo~ apoprotein bands revealed by SDS-polyacrylamide electrophoresis of serum lipoproteins were absent in platelet extracts. Our results, therefore, suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoprotOns appear to be dissimilar immunochemically and electrophoretically from plasma lipoproteins. If platelet lipoproteins were derived from circulating plasma lipoproteins by absorption or active uptake, it would be expected that some structural and antigenic properties would be shared between the two types of lipoproteins. Since no evidence of similarity was obtained by the different techniques used in this work, this hypothesis seems to be unlikely.
347 The possible role of q u a n t i t a t i v e or qualitative changes of platelet m e m b r a n e lipoproteins appears to be a most challenging field. It is k n o w n that no correlation exists between the severity of t h r o m b o t i c arterial disease and serum lipoprotein levels. A relation between increased platelet adhesiveness and arterial thrombosis has been the object ol much speculation and it is likely that platelet lipoproteins play an i m p o r t a n t role in d e t e r m i n i n g platelet adhesiveness. F u r t h e r work, including attempts to develop specific antisera to plateleo lipoproteins that could provide an easy method for quantification, will be pursued with the objective of testing some of these assumptions. ACKNOWLEDGEMENTS The authors wish to t h a n k Dr. H a l d o r T. Jonsson, Associate Professor of Biochemistry, Medical University of South Carolina, for discussions and c o m m e n t s on this work and manuscript, a n d Dr. R. Nair, Assistant Professor of Medicine a n d Biochemistry of the same Universi.ty for his help in the ultracentrifugation and gel c h r o m a t o g r a p h y studies. They also wish to t h a n k Miss H. Schraibman for her valuable technical assistance. REFERENCES 1 Lacave, C. and Panos, C. (1973) Biochim. Biophys. Acta 307, 118-132 2 Srinivasan, S. R., Dolan, P., Radhakrishnamurthy, B. and Berenson, G. S. (1972) Preparative Biochemistry 2, 83-91 3 Marcus, A. J., Ullman, H. L. and Sailer, L. B. (1969) J. Lipid Res. 10, 108-114 4 Glossmann, H. and Neville, Jr., D., (1971) J. Biol. Chem. 246, 6339-6346 5 Nordoy, A., Bjorge, J. M. and Strom, E. (1973) Acta Med. Scand. 193, 59-64 6 Srinivasan, S. R., Dolan, P., Radhakrishnamurthy, B., Pargaonkar, P. S. and Berenson, G. S. (1975) Biochim. Biophys. Acta 388, 58-70 7 Marcus, A. J., Zucker-Franklin, D., Sailer, L. B. and Ullman, H. L. (1966) J. Clin. Invest. 45, 14-28 8 Ames, G. F. (1968) J. Bacteriol. 95, 833-843 9 Langdon, R. G. (1974) Biochim. Biophys. Acta, 342, 213-228 10 Marcus, A. J. (1969) N. Engl. J. Med. 280, 1330-1335 11 Marcus, A. J. (1969) N. Engl. J. Med. 280, 1213-1220 12 Sarji, K. E., Stratton, R. D., Wagner, R. H. and Brinkhous, K. M. (1974) Proc. Natl. Acad. Sci. U.S. 71~ 2937-2941 13 Westphal, O., Luderitz, O. and Bister, F. (1952) Z. Naturforsch 7b, 148-155 14 Okuda, S. and Weinbaum, G. (1968) Biochemistry 7, 2819-2825 15 Lopes-Virella, M. F., Virella, G., Parreira, F. and Sequerra-Amran, S. (1973) Rev. Clin. Espafiola 133, 195-204 16 Naito, H. K., Wada, M., Ehrhart, L. A. and Lewis, L. A. (1973) Clin. Chem. 19, 228-234 17 Frings, C. S., Foster, L. B. and Cohen, P. S. (1971) Clin. Chem. 17, 111-114 18 Masket, B. H., Levy, R. I. and Fredrickson, D. S. (1973) J. Lab. Clin. Med. 81,794-802 19 Outcherlony, O. (1968) Handbook of Immunodiffusion and Immunoelectrophoresis, pp. 21-47, Ann Arbor Sciences Publishers, Inc., Ann Arbor, Michigan 20 Summers, D. T., Maizel, J. F. and Darnell, J. E. (1965) Proc. Natl. Acad. Sci. U.S. 54, 505-513 21 Parkhouse, R. M. E. and Askonas, B. A. (1969) Biochem. J. 115, 163-169 22 Virella, G. and Parkhouse, R. M. E. (1973) Prot. Biol. Fluids 20, 4348 23 Burnstein, M., Scholnick, H. R. and Morfin, R. (1970) J. Lipid Res. 11, 583-595 24 Burnstein, M. (1963) Nouv. Rev. Franc. Hemat. 3, 139-148 25 Lowry,~O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
348 26 Block, W. D., Jarretl, Jr., K. J. and Levine, J. B. (1966) Clin. C h e m . 12. 681 689 27 G r a b a r , P. a n d Williams, C. A. (1953) Biochim. Biophys. Acta 10, 1 9 3 194 28 Fredrickson, D. S., G o t t o , A. M. a n d Levy~ R. 1. (1972) i~a The Metabolic Basis of Inherited Disease (Stanbury, J. B., W y n g a a r d e n , J. B. and Fredrickso~a, D. S., eds.) 3rd edn., pp. 493 496, McGraw-Hill, New York 29 Shore, B. (1957) Arch. Biochem. Biophys. 71, 1 10 30 Shore, V. G. and Shore, B. (1973) Biochemistry 12, 502 507
¢~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 37412 PLATELET LIPOPROTEINS A COMPARATIVE STUDY WITH SERUM LIPOPROTEINS*
MARIA FERNANDA LOPES-VIRELLA a, K. SARJP, G. VIRELLA b and J. A. COLWELL a ~ Endocrinology, Metabolism and Nutrition Division, Department o f Medicine, Medical University o f South Carolina and Veterans Administration Hospital, ~ Department o f Basic and Clinical hnmunology and Microbiology, Medical University o f South Carolina, Charleston, S.C. (U.S.A.)
(Received December 23rd, 1975)
SUMMARY The nature of human platelet lipoproteins was studied in two series of experiments. In the first series, whole platelets were utilized for extraction of lipoproteins by three different methods : chloroform/methanol/phenol; saline; or sucrose-gradient ultracentrifugation of platelet homogenates. By polyacrylamide gel electrophoresis we were able to demonstrate the existence of lipoprotein in the extracts obtained by the last two methods. These lipoproteins were found not to share antigenic determinants with alpha and beta serum lipoproteins. The second series of experiments utilized platelets solubilized either in sodium deoxycholate or sodium dodecyl sulfate. The solubilized product was characterized by double immunodiffusion and polyacrylamide gel electrophoresis. The nonidentity between plasma and platelet lipoproteins previously demonstrated in the first series of experiments was confirmed. This nonidentity was also supported by a comparison between the apoproteins of purified serum lipoproteins and platelet proteins released after solubilization with sodium dodecyl sulfate. No identical protein fractions were found. Our results suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoproteins are structurally different from plasma lipoproteins. _
_
_
INTRODUCTION In recent years, several reports have been published concerning the lipid and protein composition of cell membranes and tissues [1-9]. Most authors have been concerned with the study of the lipid composition of cell membranes, while lipoproteins have usually not been characterized. This is particularly true in the case of platelets. Recently, Langdon [9] published an interesting report suggesting that the apoproteins of the serum lipoproteins are immunologically and biochemically similar Abbreviations: LDL, low density lipoprotein (d ~ 1.006-1.063 g/ml); VLDL, very low density lipoprotein (d < 1.006 g/ml); HDL, high density lipoprotein (d ~ 1.063-1.21 g/ml); SDS, sodium dodecyl sulfate. * Publication No. 34 of the Department of Basic and Clinical Immunology and Microbiology.
340 to thoe sof the erythrocyte membrane. In platelets, however, the relation between plasma lipoproteins and platelet lipoproteins is not known [5]. In view of these observations and because of the importance of platelet lipoproteins in platelet function and coagulation [10, I1], we have undertaken a study to compare serum and platelet lipoproteins of normal individuals. MATERIALS AND METHODS Platelets. Human platelets were obtained from freshly drawn blood of healthy donors whose serum lipoproteins had been characterized as normal. The platelet suspensions were prepared according to the method described by Sarji et al. [12], exhaustively washed to avoid any contamination with plasma, and finally resuspended in sodium phosphate buffer 0.5 mM, pH 8.0. The final suspension contained 1 000 000 platelets/mm 3. Platelet lipoprotein extraction. I_ipoproteins were extracted from platelets by three methods. The first was based on a modification by Lacave [I] of the methods described by Ames [8], Westphal [13] and Okuda [14]. Free lipids of whole platelets were removed according to Ames [8]. The remaining residue was then extracted with phenol [13] and phenol was finally removed by dialysis against glycerol [14]. The fraction obtained in the phenolic phase was dialyzed against distilled water for three days and then was tested by conventional lipoprotein polyacrylamide gel electrophoresis [15-18]. Secondly, we used a modification of the method described by Srinivasan for the extraction of lipoproteins from fatty streaks of human aorta [2, 6]. This consisted of extracting water-soluble material from a platelet suspension (109 platelets) with 4 volumes of 0.15 M sodium chloride by gentle shaking at 4 °C for 24 h, followed by low speed centrifugation (2000 rev./min) for 30 min. Finally, we used the method described by Marcus [7], consisting of homogenizing a platelet suspension with a Teflon pestle rotated at 1700 rev./min for 5 min. After washing the tube and plunger with sodium phosphate buffer and mixing homogenate and washes, the product was centrifuged for 30 min at 2000 ~ g. The supernatant obtained after centrifugation was layered on 30 to 60~o sucrose gradients, which were prepared at 4 °C immediately before use. Ultracentrifugation was carried out in a Beckman L5-75 ultracentrifuge with SW 50.1 swinging bucket rotor at 130 000 g at 4 °C for 2 h. After ultracentrifugation, 0.5 ml samples were collected. Platelet solubilization. In the second series of experiments, whole platelets were solubilized using sodium deoxycholate and sodium dodecyl sulfate. Solubilization with sodium deoxycholate was accomplished by mixing equal quantities of platelet suspension containing 1.106 cells/mm 3 and 0.1 M sodium deoxycholate solution. The solubilization with sodium dodecyl sulfate (SDS) was carried out as described by Langdon [9] for erythrocyte membrane. To 1 ml o f a platelet suspension (109 platelets) prepared as described above, 0.1 ml of 20 ~ SDS solution was added. The solution obtained was heated in a steam bath for 1.5 rain. When the resulting extract was to be tested immunochemically or by SDS-polyacrylamide electrophoresis, it was dialyzed for 48 h against 0.1 M sodium phosphate buffer pH 7.2, containing 0.1 ~,/, (w/v) of SDS. When the extract was to be submitted to gel chromatography, after it had
341 cooled to room temperature, we added 50 #1 of N-ethyl morpholine and 100 #1 of dimethylformamide containing 2 mg of dansyl chloride. This was used as a fluorescent protein marker. The solution was allowed to stand 30 minutes at room temperature and was then layered on the top o f a 1 x 30 cm Sephadex G-200 column equilibrated and eluted with 0.1 ~o SDS in pH 7.2, 0.1 M sodium phosphate buffer. 0.5 ml fractions were collected and the fluorescence of the fractions was detected by illuminating the test tubes with a 366 nm light source. All the fluorescent fractions were tested by double diffusion in agar gel [19] against antisera to human alpha and beta serum lipoproteins and studied by SDS-polyacrylamide gel electrophoresis [20-22]. Isolation of serum lipoproteins. Pooled fresh sera from fasting healthy donors was used. Platelet donors were represented in this pool. The isolation of serum lipoproteins was done according to the methods described by Burnstein [23-24]. Low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were isolated by precipitation with heparin and MgCI2 in presence of sucrose, followed by ultracentrifugation for 24 h at 100 000 ;~, g. High density lipoprotein (HDL) was isolated by precipitation with sodium phosphotungstate and MgClz. The purity of isolated serum lipoproteins was verified by conventional lipoprotein polyacrylamide electrophoresis, immunodiffusion and immunoelectrophoresis. Some albumin was found as a contaminant of beta lipoprotein. Quantitative studies on platelet extracts and homogenates and purified lipoprotein fractions. Total protein concentration was determined by the method of Lowry et al. [25]. Total cholesterol was determined by a two-step method involving an isopropanol extract (1 :I0) of the preparations to study and a colorimetric determination of cholesterol based on the reaction of concentrated sulfuric acid and ferric chloride in acetic acid with steroids having the 5-erie 3 beta-01 grouping [26]. Electrophoretic studies. The l ipoproteins of serum, platelet extracts and platelet homogenates were separated by conventional polyacrylamide gel electrophoresis as described for the separation of lipoproteins [15-18]. The protein content of the samples applied in each gel varied from 300-700 /~g of total protein. The total cholesterol content in the samples varied from 7 to 12/~g. SDS-polyacrylamide electrophoresis in 5~o (monomer w/v) gel [20-22] was used to compare the mobility of the apolipoproteins of alpha, beta and pre-beta lipoproteins, proteins contained in a SDS solubilized platelet suspension, whole serum proteins and several isolated proteins used as controls. The protein concentration of the platelet suspension, purified lipoproteins, normal serum and each control protein was adjusted to approximately 4 mg/ml prior to incubation with 0.1 ml of 20 ~o SDS and subsequent dialysis against 0.1 M phosphate buffer, pH 7.2, containing 0.1 ~ SDS (w/v). Approximately 200/~g of protein were applied to each gel. hnmunochemical studies. The purity of isolated serum lipoproteins was tested immunoelectrophoretically [27] using goat anti-human beta lipoprotein anti-~erum (Lot No. 220 s2, 9.1 mg of antibody protein per ml), obtained from Miles Laboratories, and rabbit anti-human alpha lipoprotein antiserum (Lot No. 2348 T, 0.1 units of antibody per ml), obtained from Behring Diagnostics. Antigenic analysis of serum and platelet lipoprotein extracts was carried out by double-immunodiffusion using the same antisera.
342 In order to ensure that a negative result would not be a consequence of in,correct antigen-antibody proportions, samples to be compared were either adjusted to similar protein concentrations or the tests were repeated under different antigenantibody proportions, obtained by keeping constant the values of antigen and varying the volume of antibody from 5 #1 to 50 #1. Diffusion was prolonged to 48 hours at room temperature and the agar platelets were washed, dried and stained with amidoblack 10B to ensure maximal sensitivity. RESULTS
When platelets were extracted by the method of Lacave [1] and tested by conventional lipoprotein polyacrylamide electrophoresis, no lipid-stained fractions were obtained. However, a lipoprotein fraction remaining on the top of the separation gel was observed when water soluble material obtained by extraction with 0.15 M sodium chloride as described by Srinivasan was studied. When platelet homogenates were prepared according to the method of Marcus [7] and submitted to ultracentrifugation, polyacrylamide electrophoresis of the fractions obtained revealed a sudanophilic band at the top of the separating gel sharper than the one obtained from saline extracts. This band was particularly obvious in the upper fraction of sucrose gradients after preparative ultracentrifugation (Fig. 1). /-
t~ - " ~ "
C
1
"
•
11
Fig. 1. Conventional lipoprotein electrophoresis in polyacrylamide gel of fractions obtained from platelet homogenates through sucrose-gradient ultracentrifugation. Gel C was loaded with 30/d of normal human serum. Gels 1 to 11 were loaded with 100/~1 of sucrose gradient fractions 1 to 11, starting from the bottom (fraction 1).
343
A
B
Fig. 2. Conventional lipoprotein electrophoresis of two platelet extracts obtained respectively with sodium deoxycholate (Gel A) and sodium dodecyl sulfate (gel B). Note that the top fraction seems to be constituted by two sub-fractions, more obviously separated in the sodium dodecyl sulfate extract. Exposure o f saline extracted platelet material and platelet homogenates to sodium dodecyl sulfate and sodium deoxycholate resulted in the separation of the sudanophilic material into two fractions, one migrating deep into the gel and the other remaining at the top of the separating gel as in the original untreated extracts (Fig. 2). The platelet extracts obtained according to the different extraction methods
344
Fig. 3. Double immunodiffusion study of the antigenicity of platelet lipoproteins. Wells 1, 2, and 3 contain three normal sera, wells 4 to 14 contain sucrose gradient fractions, 1 to 11, from bottom. Well 15 and 18 contain a saline platelet extract. Well 16 contains a platelet homogenate. Well 17 contains a methanol/chloroform/phenol platelet extract. The upper three central wells were filled with antisera against human serum beta lipoprotein. The lower central wells were filled with antisera against human serum alpha lipoprotein. d e s c r i b e d were tested by d o u b l e diffusion in a g a r against a n t i s e r a to h u m a n a l p h a a n d beta s e r u m l i p o p r o t e i n s . N o p r e c i p i t a t i o n lines were d e t e c t e d (Fig. 3). E l e c t r o p h o r e t i c s e p a r a t i o n s o f e x t r a c t s o b t a i n e d after p l a t e l e t s o l u b i l i z a t i o n w i t h s o d i u m d e o x y c h o l a t e were n o t v e r y s a t i s f a c t o r y and t h e i r i n t e r p r e t a t i o n difficult. H o w e v e r , e l e c t r o p h o r e t i c s e p a r a t i o n s f r o m e x t r a c t s o b t a i n e d after s o l u b i l i z a t i o n with S D S w e r e r e p r o d u c i b l e and t h e i r i n t e r p r e t a t i o n easier. W e f o u n d n o c o r r e l a t i o n b e t w e e n the p r o t e i n f r a c t i o n s s e p a r a t e d f r o m platelets extracts a n d the a p o p r o t e i n s s e p a r a t e d f r o m i s o l a t e d s e r u m l i p o p r o t e i n s (Fig. 4).
345
M A(d) G
TR ALB OVA CHY LYS
i
2
3
4
5
6
Fig. 4. Sodium dodecyl sulfate polyacrylamide electrophoresis of different lipoprotein preparations dissolved with SDS. Purified alpha lipoprotein was separated in gel 1, purified beta lipoprotein in gel 2, and purified prebeta lipoprotein in gel 3. An SDS solubilized platelet extract was separated gel 4. Gels 5 and 6 were used to separate controls, respectively ,normal serum (gel 5) and a mixture of purified proteins (gel 6). lgM (M); lgA dimeric (A(d)) ; lgG (G) ; transferrin (TR); human albumin (ALB) ; ovalbumin (OVA); alpha-chymotrypsinogen (CHY); and eggwhite lysozyme (LYS). The fluorescent fractions obtained after gel c h r o m a t o g r a p h y in Sephadex G-200 o f a SDS platelet extract treated with dansyl chloride were also studied by SDS polyacrylamide electrophoresis and again no correspondence was found between the observed fractions and the major apoproteins of alpha, beta, and prebeta serum lipoproteins. The SDS platelet extract, adjusted at 4 mg/ml o f protein, was tested against antisera to h u m a n alpha and beta serum lipoproteins using as controls h u m a n alpha, beta, and prebeta lipoproteins adjusted to the same protein concentration in 0.1 ~°/oo SDS (Fig. 5). Precipitation lines were detected with the purified serum lipoproteins but no precipitation was observed with the platelet extract. A similarly negative result was obtained when fractions separated by G-200 gel filtration from an SDS-platelet extract were studied. DISCUSSION Langdon, in 1974 [9], suggested that the major intrinsic lipophilic proteins of the erythrocyte membrane contained apoproteins similar to those of serum lipc-
346
Fig. 5. Double immunodiffusion study of platelet lipoproteins in a SDS-solubilized platelet extract. In the two represented systems, peripheral wells were filled with beta lipoprotein dissolved in SDS (1), Prebeta lipoprotein dissolved in SDS (2), SDS-phosphate buffer (3) alpha lipoprotein dissolved in SDS (4), and SDS platelet extract (5) and normal human serum dissolved in SDS. The central well of the left system was filled with antisera against human serum beta lipoprotein, and in the right system the central well was filled with antisera against human serum alpha lipoprotein. proteins based on immunological evidence of identity of antigenic determinavts for both serum alpha and beta lipoproteins and those of lipoproteins in erythrocyte membranes. This author also showed that the NH2 terminal aminoacids of serum beta or alpha lipoproteins and the NH2 terminal aminoacids of erythrocyte membrane proteins are similar. This observation encouraged our studies aimed at comparing the antigenicity of serum and platelet lipoproteins. However, no shared determinants were detected with saline extracts and platelet homogenates and negative results were also obtained when membrane proteins solubilized with detergents were submitted to immunochemical analysis. A possible inhibitory effect of detergents on precipitin reactions could be ruled out by the fact that we could obtain precipitation with purified serum lipoproteins dissolved in 0.1 ~i~ SDS solution (w/v). Further, several authors have described the possibility of immunochemical testing of lipoproteins dissolved in the presence of detergent [9, 28-30]. The lack of identity between serum and platelet lipoproteins is also supported by the fact that the majo~ apoprotein bands revealed by SDS-polyacrylamide electrophoresis of serum lipoproteins were absent in platelet extracts. Our results, therefore, suggest that, unlike erythrocyte membrane lipoproteins, the platelet lipoprotOns appear to be dissimilar immunochemically and electrophoretically from plasma lipoproteins. If platelet lipoproteins were derived from circulating plasma lipoproteins by absorption or active uptake, it would be expected that some structural and antigenic properties would be shared between the two types of lipoproteins. Since no evidence of similarity was obtained by the different techniques used in this work, this hypothesis seems to be unlikely.
347 The possible role of q u a n t i t a t i v e or qualitative changes of platelet m e m b r a n e lipoproteins appears to be a most challenging field. It is k n o w n that no correlation exists between the severity of t h r o m b o t i c arterial disease and serum lipoprotein levels. A relation between increased platelet adhesiveness and arterial thrombosis has been the object ol much speculation and it is likely that platelet lipoproteins play an i m p o r t a n t role in d e t e r m i n i n g platelet adhesiveness. F u r t h e r work, including attempts to develop specific antisera to plateleo lipoproteins that could provide an easy method for quantification, will be pursued with the objective of testing some of these assumptions. ACKNOWLEDGEMENTS The authors wish to t h a n k Dr. H a l d o r T. Jonsson, Associate Professor of Biochemistry, Medical University of South Carolina, for discussions and c o m m e n t s on this work and manuscript, a n d Dr. R. Nair, Assistant Professor of Medicine a n d Biochemistry of the same Universi.ty for his help in the ultracentrifugation and gel c h r o m a t o g r a p h y studies. They also wish to t h a n k Miss H. Schraibman for her valuable technical assistance. REFERENCES 1 Lacave, C. and Panos, C. (1973) Biochim. Biophys. Acta 307, 118-132 2 Srinivasan, S. R., Dolan, P., Radhakrishnamurthy, B. and Berenson, G. S. (1972) Preparative Biochemistry 2, 83-91 3 Marcus, A. J., Ullman, H. L. and Sailer, L. B. (1969) J. Lipid Res. 10, 108-114 4 Glossmann, H. and Neville, Jr., D., (1971) J. Biol. Chem. 246, 6339-6346 5 Nordoy, A., Bjorge, J. M. and Strom, E. (1973) Acta Med. Scand. 193, 59-64 6 Srinivasan, S. R., Dolan, P., Radhakrishnamurthy, B., Pargaonkar, P. S. and Berenson, G. S. (1975) Biochim. Biophys. Acta 388, 58-70 7 Marcus, A. J., Zucker-Franklin, D., Sailer, L. B. and Ullman, H. L. (1966) J. Clin. Invest. 45, 14-28 8 Ames, G. F. (1968) J. Bacteriol. 95, 833-843 9 Langdon, R. G. (1974) Biochim. Biophys. Acta, 342, 213-228 10 Marcus, A. J. (1969) N. Engl. J. Med. 280, 1330-1335 11 Marcus, A. J. (1969) N. Engl. J. Med. 280, 1213-1220 12 Sarji, K. E., Stratton, R. D., Wagner, R. H. and Brinkhous, K. M. (1974) Proc. Natl. Acad. Sci. U.S. 71~ 2937-2941 13 Westphal, O., Luderitz, O. and Bister, F. (1952) Z. Naturforsch 7b, 148-155 14 Okuda, S. and Weinbaum, G. (1968) Biochemistry 7, 2819-2825 15 Lopes-Virella, M. F., Virella, G., Parreira, F. and Sequerra-Amran, S. (1973) Rev. Clin. Espafiola 133, 195-204 16 Naito, H. K., Wada, M., Ehrhart, L. A. and Lewis, L. A. (1973) Clin. Chem. 19, 228-234 17 Frings, C. S., Foster, L. B. and Cohen, P. S. (1971) Clin. Chem. 17, 111-114 18 Masket, B. H., Levy, R. I. and Fredrickson, D. S. (1973) J. Lab. Clin. Med. 81,794-802 19 Outcherlony, O. (1968) Handbook of Immunodiffusion and Immunoelectrophoresis, pp. 21-47, Ann Arbor Sciences Publishers, Inc., Ann Arbor, Michigan 20 Summers, D. T., Maizel, J. F. and Darnell, J. E. (1965) Proc. Natl. Acad. Sci. U.S. 54, 505-513 21 Parkhouse, R. M. E. and Askonas, B. A. (1969) Biochem. J. 115, 163-169 22 Virella, G. and Parkhouse, R. M. E. (1973) Prot. Biol. Fluids 20, 4348 23 Burnstein, M., Scholnick, H. R. and Morfin, R. (1970) J. Lipid Res. 11, 583-595 24 Burnstein, M. (1963) Nouv. Rev. Franc. Hemat. 3, 139-148 25 Lowry,~O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
348 26 Block, W. D., Jarretl, Jr., K. J. and Levine, J. B. (1966) Clin. C h e m . 12. 681 689 27 G r a b a r , P. a n d Williams, C. A. (1953) Biochim. Biophys. Acta 10, 1 9 3 194 28 Fredrickson, D. S., G o t t o , A. M. a n d Levy~ R. 1. (1972) i~a The Metabolic Basis of Inherited Disease (Stanbury, J. B., W y n g a a r d e n , J. B. and Fredrickso~a, D. S., eds.) 3rd edn., pp. 493 496, McGraw-Hill, New York 29 Shore, B. (1957) Arch. Biochem. Biophys. 71, 1 10 30 Shore, V. G. and Shore, B. (1973) Biochemistry 12, 502 507
