36
ISOLATION OF P L A T E L E T COMPONENTS
[5]
2. Combine EDTA, 1.02 g Tris, 2.6 g EGTA, 9.51 g Add to 50 ml water and dissolve by the addition of 10 M NaOH 3. Combine solutions 1 and 2, adjust to pH 7.4, and make up to 250 ml Buffer C: Dilute buffer B 10 x and add leupeptin (50 p.g/ml) and PMSF (2 mM) Comments. The yield of membranes by this technique is approximately 3 mg/unit of platelet concentrate, or about 50% increase over the original procedure. Purity is equal to that of the original procedure based on marker enzymes for plasma membranes. The procedure has also been used satisfactorily in our hands for the preparation of plasma membranes following proteolysis of intact platelets with Serratia marcescens protease.
[5] I s o l a t i o n o f D e n s e G r a n u l e s f r o m H u m a n P l a t e l e t s By MmIAM H. FUKAMI
Introduction Human platelet dense granules are secretory organelles that store most of the calcium in the cell, about 90% of its ADP and lesser amounts of ATP, AMP, and guanine nucleotides, as well as nearly all of the platelet serotonin that is taken up by an active transport mechanism localized on the granule membrane. ~The high content of stored calcium and nucleotides gives the granules a high buoyant density of about 1.21 in sucrose gradients and makes it possible to separate them readily from the numerous other subcellular organelles present in platelets. The ease with which the dense granules in intact platelets can be labeled with exogenous serotonin provides a convenient method for monitoring granule isolation and purification. Labeling and Washing of Cells Platelet-rich plasma is prepared from blood collected into 0.1 vol of an anticoagulant solution such as acid-citrate-dextrose [1.4% (w/v) citric ' G. R u d n i c k , in " P l a t e l e t R e s p o n s e s a n d M e t a b o l i s m " (H. H o l m s e n , ed.), Vol. 2, p. 119. C R C P r e s s , B o c a R a t o n , F l o r i d a , 1987.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
PLATELET DENSE GRANULES
37
acid, 2.5% (w/v) sodium citrate, and 2% (w/v) dextrose, ACD] or 3.8% sodium citrate by centrifugation at 200 g for 15 min at room temperature. The supernatant platelet-rich plasma is carefully removed from the sedimented red cells with a pipette. The platelets can be incubated with [3H]or [~4C]serotonin at this stage if one elects to monitor the isolation of dense granules by isotopic labeling. Incubation for 15 min at 37 ° with 1/xM serotonin, including the desired amount of isotope, is adequate for labeling. Then 0.1 M sodium EDTA, pH 7.4, is added to the platelet-rich plasma to give a final EDTA concentration of 5 mM, and the platelets are pelleted by centrifugation at 1000 g for 15 min. The platelets are washed once with an ice-cold medium consisting of 0.13 M NaC1, 0.02 M HEPES, and 0.001 mM EDTA, pH 6.5, 2 and resuspended in the same medium. (Platelets obtained from 150 ml of whole blood were resuspended in a volume of 15-20 ml.)
Homogenization Some special problems exist for the homogenization of platelets that do not occur with tissues such as liver. As with other free cell suspensions, it is difficult to apply a mechanical shear force with a conventional homogenizer such as a Potter-Elvehjem homogenizer, which works very well on minced tissue. The high content of actin and myosin in the platelet ultrastructure seems to confer resilient properties to the plasma membrane, which makes them more resistant to gentle homogenization. Platelets present an additional difficulty since they are activated to release the contents of their secretory organelles by contact with foreign surfaces and vigorous manipulations. (EDTA protects from activation only under moderately gentle conditions, such as low-speed centrifugation and washing.) Ultrasonication and nitrogen cavitation methods 3 (see also [3] in this volume) are more suitable for application of shear forces necessary for disruption of cell suspensions than mortar-and-pestle techniques. Although ultrasonication has been used successfully by others for the isolation of platelet granules, 4,5 it has not been satisfactory in our hands with respect to reproducibility and yield of intact granules. The method de-
2 Platelet secretion occurs less readily at pH values lower that 7. 3 p. F. Zurendock, M. E. Tischler, T. P. M. Akerboom, R. Van der Meer, J. R. Williamson, and J. M. Tager, this series, Vol. 56, p. 214. 4 F. Rendu, M. Lebret, A. T. Nurden, and J. P. Caen, Br. J. Haematol. 52, 241 (1982). 5 M. Da Prada, J. P. Tranzer, and A. Pletscher, Experientia 28, 1328 (1972).
38
ISOLATION OF PLATELET COMPONENTS
[5]
scribed here utilizes a French pressure cell6 (and hydraulic press, both from Aminco Corp., Silver Springs, MD) for disruption of platelets that have been pretreated with metabolic inhibitors to block secretion 7 and a protease, nagarse (Enzyme Development Corp., New York, NY), which has been used to digest briefly heart and brain tissue before homogenization. 8-10
Procedure for Homogenization and Fractionation The washed platelets obtained from 150 ml of blood and resuspended in 20 ml of the pH 6.5 medium described above are incubated with 20 /xM rotenone (0.2 ml of a 2 mM stock solution in ethanol), 10 mM 2deoxyglucose (0.4 ml of a 0.5 M stock) and 30 mM gluconic acid 8-1actone (108 mg dissolved in 1 ml of washing medium just before addition) at 37° for 10 min. Then 3 mg of nagarse and 10 mg of ATP ~ (both dissolved in 1 ml of medium) are mixed into the suspension, which is kept at room temperature for 5 min. Then 20 mg of soybean trypsin inhibitor in 1 ml of medium is stirred into the suspension to neutralize the nagarse and the platelets are centrifuged at 1000 g for 15 min at 0-4 °. All subsequent procedures are carried out at 0-4 °. The platelet pellet is resuspended in a cold medium consisting of 0.25 M sucrose, 10 mM HEPES, and 1 mM sodium EDTA, pH 7.4. The suspension is then passed through a precooled French pressure cell 6 at 1000 psi and then centrifuged at 1000 g for 15 min. The supernatant fraction is saved and the pellet is resuspended in 15-20 ml of sucrose medium and subjected to another pass through the pressure cell. After the second homogenate is centrifuged again at 1000 g for 15 min, the combined supernatant fractions are centrifuged at 12,000 g for 20 min to give a pellet (about 5-10 mg protein) that consists of a mixture of platelet organelles, including dense granules, ~ granules, acid hydrolasecontaining vesicles, and mitochondria. Table I shows that the markers for the various organelles are 2 to 3.6 times enriched in this pellet compared to the homogenate. This organelle mixture is suitable for some studies on dense granules and can be used as such after two washes with the sucrose 6 L. Salganicoff and M. H. F u k a m i , Arch. Biochem. Biophys. 153, 726 (1972). The valve modification described facilities regulation of the flow rate but is not essential. 7 M. H. F u k a m i , J. S. Bauer, G. J. Stewart, and L. Salganicoff, J. CellBiol. 77, 389 (1978). 8 D. D. Tyler and J. Gonze, this series, Vol. 10, p. 75. 9 A. L. Smith, this series, Vol. 10, p. 84. 10 R. E. Basford, this series, Vol. 10, p. 98. it O m i s s i o n o f nagarse gives preparations that are difficult to r e s u s p e n d and do not separate well on s u c r o s e density gradient centrifugation. A T P s e e m s to prevent the aggregation that occurs occasionally at this step, probably via its action as a potent inhibitor of ADP, small a m o u n t s of which m a y be released from the cells by nagarse.
[5]
PLATELET DENSE GRANULES
39
TABLE I MARKER ENZYMES IN ORGANELLE MIXTURE
Marker
Relative specific activity ~
Yield (%)
[t4C]Serotonin (dense granules) a-Glycerophosphate oxidase (mitochondria) /3-N-Acetylglucosaminidase (acid hydrolase vesicles) Platelet factor 4 (a granules)
2.82 --- 0.5 3.04 - 1.6
36.4 49.6
1.92 -+ 1.3
36.4
2.96 -+ 0.5
46.7
c, The relative specific activity is given as the ratio of the specific activity of the fraction divided by the specific activity of the homogenate, mean -+ SD, n = 3-4.
isolation m e d i u m if one is certain that the presence of the other organelles does not interfere with the activity to be monitored. The dense granules in this organelle mixture are considerably more stable to leakage 12 than the highly enriched preparation described below. Subfractionation on a Sucrose Density Gradient The sucrose stepwise gradient used for subfractionation of the granule mixture consists of seven steps from 0.8 to 2.0 M sucrose in 0.2 M increments. The sucrose steps are buffered with 5 m M H E P E S , p H 7.4, and contain 0.1 m M sodium E D T A , p H 7.4. Starting with the 2 M step, 1.5 ml of each step is layered into a tube(s) suitable for use in a B e c k m a n (Palo Alto, CA) SW 40 rotor (or c o m p a r a b l e swing-out ultracentrifuge rotor). The gradients are incubated for 1 hr at 37 ° to soften the interfaces and then cooled on ice to 0 °. A b o u t 1.5-2.0 ml of the organelle mixture (protein concentration not m o r e than 10 mg/ml) is then layered onto each gradient tube and centrifuged at 100,000 g and 4 ° for 60 min. At least three bands of organelles and a pellet should be observed. 7 The dense granules are to be found in the pellet, which we have usually resuspended in the homogenization m e d i u m and used only for analysis. H o w e v e r , in fact, pig platelet dense granules p r e p a r e d by the same method and used for nuclear magnetic r e s o n a n c e (NMR) studies at 31 ° were found to leak and slowly lose their stored contents, 12 and it is reasonable to assume that the h u m a n platelet preparation does the same. A modification of the sucrose gradient that resulted in a m o r e stable preparation of pig platelet dense granules 12 and that is p r o b a b l y applicable to the h u m a n preparation is described here for those uses that require incubations at temperatures a b o v e 0 - 4 ° . Bovine 12K. Ugurbil, M. H. Fukami, and H. Holmsen, Biochemistry 23, 416 (1980).
40
ISOLATION OF PLATELET COMPONENTS
[5]
TABLE II MARKER ENZYMES IN SUCROSE GRADIENT-ENRICHED DENSE GRANULES
Marker
Relative specific activitya
Yield (%)
[14C]Serotonin (dense granules) a-Glycerophosphateoxidase (mitochondria) fl-N-Acetylglucosaminidase Platelet factor 4 (a granules)
52.3 -- 6.9 ND b
19.9
3.09 -+ 1.5 3.66 -+ 0.5
4.0 2.1
a The relative specific activity is given as the ratio of the specific activity of the fraction divided by the specific activity o f the homogenate, mean -+ SD, n = 3-4. b ND, None detected.
serum albumin (0.6%) was added to all of the gradient steps that were made up with DzO (the D20 made it possible to use 1.8 M as the final gradient step). The final resuspension medium was 0.3 M KCI in D20, 0.4% bovine serum albumin, and 30 mM phosphate buffer, pH 7.4. Some details of these modifications that were required specifically for proton NMR such as use of DzO, and a KCI resuspension medium with phosphate buffer, can be changed. The essence of the modification was to use bovine serum albumin and a somewhat hyperosmotic (compared to 0.25 M sucrose) resuspension medium. A metrizamide gradient for human platelet dense granules 4 and a Percoll gradient for pig platelet dense granules ~3 have also been described. Yield and Purity The pellet consists of about 30-50/zg of protein and contains about 20% of the total serotonin originally present in 150 ml of whole blood starting material. Table II shows that the dense granule marker serotonin is 50-fold enriched compared to the homogenate. The mitochondrial marker was not measurable in this pellet, but the markers for acid hydrolase vesicles and ct granules were some threefold enriched, although only 4 and 2% of the starting material was present. This degree of contamination is negligible for most purposes, but the preparation is not suitable for study of unique dense granule proteins except by functional tests. The reason for this limitation is that the other organelles are not only larger and more numerous than dense granules in the platelet, but they also contain primarily proteins. The dense granules, on the other hand, contain other low molecular weight compounds (see below) and very little protein. 13 S. E. Carty, R. G. J o h n s o n , and A. Scarpa, J. Biol. Chem. 256, 11244 (1981).
[5]
PLATELET DENSE GRANULES
41
T A B L E III Low-MOLECULAR WEIGHT CONSTITUENTS OF HUMAN PLATELET DENSE GRANULESa
Substance PPI Pi ATP ADP Ca 2÷ Mg 2+
Serotonin
Whole platelets 6.3 16.7 23.8 14.5 125
--- 0.05 --- 2.5 --- 3.5 -+ 2.4 -+ 27 -1.5-2.5
Secreted amounts 6.0 4.6 9.7 14.1 88
-+ 0.52 + 1.6 -+ 1.3 + 1.8 + 23 -1.5-2.0
Dense granules 236 -+ 53 248 -+ 65 440 _+ 96 633 -+ 142 2634 -+ 471 98 + 33 90-100
Isolated by the method described in this article (nmol/mg of protein). Values are given as mean -+ SD, n = 4-6.
Therefore, the relative amounts of protein contributed by the various organelles are disproportionate to their concentration in the preparation. Properties of Human Platelet Dense Granules The concentrations of low molecular weight compounds in dense granules are given in Table III. The amounts contained in whole platelets and that released by thrombin are also shown in order to indicate which of the constituents are contained primarily in the dense granules. Most of the platelet content of PPi,14 ADP, serotonin, and much of the calcium is secreted and therefore is stored in secondary granules. Significant amounts of ATP and Pi that are required for metabolic activity are present in sites other than in the dense granules and are not secreted. Nuclear magnetic resonance studies on intact human platelets suggest that the nucleotides and calcium in the dense granules form high molecular weight aggregates that are more or less insoluble, which reduce the hyperosmolarity of the granule interior and which bind serotonin. ~5a6 Similar studies on pig platelets and isolated pig platelet dense granules in which magnesium is the predominant divalent cation have confirmed the existence of these nucleotide-divalent cation-amine interactions. ~2'~7'~8 14 M. H. Fukami, C. A. Dangelmaier, J. S. Bauer, and H. Holmsen, Biochem. J. 192, 99 (1980). 15 j. L. Costa, C, M. Dobson, K. L. Kirk, F. M. Poulsen, C. R. Valeri, and J. J. Vecchione, FEBS Lett. 99, 141 (1979). 16 K. Ugurbil, H. Holmsen, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 76, 2227 (1979). ~7j, L. Costa, C.M. Dobson, K. L. Kirk, F.M. Poulsen, C. R. Valeri, and J. L. Vecchione, Philos. Trans. R. Soc. London, Ser. B. 289, 413 (1980). I8 M. H. Fukami, H. Holmsen, and K. Ugurbil, Biochem. Pharmacol. 33, 3869 (1984).
42
ISOLATION OF PLATELET COMPONENTS
[6]
Other mechanisms shown to be involved in serotonin uptake and storage in platelet dense granules from species other than human, such as an intragranular pH of 5.5,19 an ATP-driven proton pump, 2° and a reserpinesensitive amine transport system on the granule membrane, 2° most probably also exist in human platelet granules, The similarities in serotonin uptake, storage, and secretion by intact platelets of the various species indicate that the granular mechanisms are also similar. Finally, it should be mentioned that there is a heterogeneous group of inherited bleeding disorders, referred to as storage pool deficiency, both in humans and various animal species in which the platelet dense granule constituents are considerably diminished or absent, as shown both by secretion studies and by electron microscopy. 2~ 19 R. G. Johnson, A. Scarpa, and L. Salganicoff, J. Biol. Chem. 253, 7061 (1978). 20 G. Rudnick, H. Fishkes, P. J. Nelson, and S. Schuldiner, J. Biol. Chem. 2S5, 3638 (1980). :1 K. M. Meyers and M. M6nard, in "The Platelet Amine Storage Granule" (K. M. Meyers and C. D. Barnes, eds.), pp. 149-85. CRC Press, Boca Raton, Florida, 1992.
[6] S t u d y i n g t h e P l a t e l e t C y t o s k e l e t o n in T r i t o n X-100 Lysates
By JOAN E. B. Fox, CLIFFORD C. REYNOLDS, and JANET K. BOYLES The platelet cytoskeleton is composed primarily of actin filaments. In unstimulated platelets, 40 to 60% of the actin is polymerized into filaments.~ The majority of these filaments are present in networks throughout the body of the platelet. These are referred to as the cytoplasmic actin filaments. However, a small pool of the filaments appears to be present as part of a two-dimensional latticework that coats the inner surface of the lipid bilayer in much the same way as the red blood cell skeleton coats the membrane of the erythrocyte. This lattice-like structure is referred to as the platelet membrane skeleton. The filaments of the platelet membrane skeleton appear to be cross-linked by actin-binding protein (otherwise known as filamin), spectrin, and other unidentified proteins. 2,3 During platelet activation there is a rapid burst in actin polymerization, and about i j. E. B. Fox, J. K. Boyles, C. C. Reynolds, and D. R. Phillips, J. CellBiol. 98, 1985 (1984). -' J. E. B. Fox, J. K. Boyles, M. C. Berndt, P. K. Steffen, and L. K. Anderson, J. CellBiol. 106, 1525 (1988). 3 j. H. Hartwig and M. DeSisto, J. Cell Biol. 112, 407 (1991).
METHODS 1N ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, lnc, All rights of reproduction in any form reserved.
ISOLATION OF P L A T E L E T COMPONENTS
[5]
2. Combine EDTA, 1.02 g Tris, 2.6 g EGTA, 9.51 g Add to 50 ml water and dissolve by the addition of 10 M NaOH 3. Combine solutions 1 and 2, adjust to pH 7.4, and make up to 250 ml Buffer C: Dilute buffer B 10 x and add leupeptin (50 p.g/ml) and PMSF (2 mM) Comments. The yield of membranes by this technique is approximately 3 mg/unit of platelet concentrate, or about 50% increase over the original procedure. Purity is equal to that of the original procedure based on marker enzymes for plasma membranes. The procedure has also been used satisfactorily in our hands for the preparation of plasma membranes following proteolysis of intact platelets with Serratia marcescens protease.
[5] I s o l a t i o n o f D e n s e G r a n u l e s f r o m H u m a n P l a t e l e t s By MmIAM H. FUKAMI
Introduction Human platelet dense granules are secretory organelles that store most of the calcium in the cell, about 90% of its ADP and lesser amounts of ATP, AMP, and guanine nucleotides, as well as nearly all of the platelet serotonin that is taken up by an active transport mechanism localized on the granule membrane. ~The high content of stored calcium and nucleotides gives the granules a high buoyant density of about 1.21 in sucrose gradients and makes it possible to separate them readily from the numerous other subcellular organelles present in platelets. The ease with which the dense granules in intact platelets can be labeled with exogenous serotonin provides a convenient method for monitoring granule isolation and purification. Labeling and Washing of Cells Platelet-rich plasma is prepared from blood collected into 0.1 vol of an anticoagulant solution such as acid-citrate-dextrose [1.4% (w/v) citric ' G. R u d n i c k , in " P l a t e l e t R e s p o n s e s a n d M e t a b o l i s m " (H. H o l m s e n , ed.), Vol. 2, p. 119. C R C P r e s s , B o c a R a t o n , F l o r i d a , 1987.
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
PLATELET DENSE GRANULES
37
acid, 2.5% (w/v) sodium citrate, and 2% (w/v) dextrose, ACD] or 3.8% sodium citrate by centrifugation at 200 g for 15 min at room temperature. The supernatant platelet-rich plasma is carefully removed from the sedimented red cells with a pipette. The platelets can be incubated with [3H]or [~4C]serotonin at this stage if one elects to monitor the isolation of dense granules by isotopic labeling. Incubation for 15 min at 37 ° with 1/xM serotonin, including the desired amount of isotope, is adequate for labeling. Then 0.1 M sodium EDTA, pH 7.4, is added to the platelet-rich plasma to give a final EDTA concentration of 5 mM, and the platelets are pelleted by centrifugation at 1000 g for 15 min. The platelets are washed once with an ice-cold medium consisting of 0.13 M NaC1, 0.02 M HEPES, and 0.001 mM EDTA, pH 6.5, 2 and resuspended in the same medium. (Platelets obtained from 150 ml of whole blood were resuspended in a volume of 15-20 ml.)
Homogenization Some special problems exist for the homogenization of platelets that do not occur with tissues such as liver. As with other free cell suspensions, it is difficult to apply a mechanical shear force with a conventional homogenizer such as a Potter-Elvehjem homogenizer, which works very well on minced tissue. The high content of actin and myosin in the platelet ultrastructure seems to confer resilient properties to the plasma membrane, which makes them more resistant to gentle homogenization. Platelets present an additional difficulty since they are activated to release the contents of their secretory organelles by contact with foreign surfaces and vigorous manipulations. (EDTA protects from activation only under moderately gentle conditions, such as low-speed centrifugation and washing.) Ultrasonication and nitrogen cavitation methods 3 (see also [3] in this volume) are more suitable for application of shear forces necessary for disruption of cell suspensions than mortar-and-pestle techniques. Although ultrasonication has been used successfully by others for the isolation of platelet granules, 4,5 it has not been satisfactory in our hands with respect to reproducibility and yield of intact granules. The method de-
2 Platelet secretion occurs less readily at pH values lower that 7. 3 p. F. Zurendock, M. E. Tischler, T. P. M. Akerboom, R. Van der Meer, J. R. Williamson, and J. M. Tager, this series, Vol. 56, p. 214. 4 F. Rendu, M. Lebret, A. T. Nurden, and J. P. Caen, Br. J. Haematol. 52, 241 (1982). 5 M. Da Prada, J. P. Tranzer, and A. Pletscher, Experientia 28, 1328 (1972).
38
ISOLATION OF PLATELET COMPONENTS
[5]
scribed here utilizes a French pressure cell6 (and hydraulic press, both from Aminco Corp., Silver Springs, MD) for disruption of platelets that have been pretreated with metabolic inhibitors to block secretion 7 and a protease, nagarse (Enzyme Development Corp., New York, NY), which has been used to digest briefly heart and brain tissue before homogenization. 8-10
Procedure for Homogenization and Fractionation The washed platelets obtained from 150 ml of blood and resuspended in 20 ml of the pH 6.5 medium described above are incubated with 20 /xM rotenone (0.2 ml of a 2 mM stock solution in ethanol), 10 mM 2deoxyglucose (0.4 ml of a 0.5 M stock) and 30 mM gluconic acid 8-1actone (108 mg dissolved in 1 ml of washing medium just before addition) at 37° for 10 min. Then 3 mg of nagarse and 10 mg of ATP ~ (both dissolved in 1 ml of medium) are mixed into the suspension, which is kept at room temperature for 5 min. Then 20 mg of soybean trypsin inhibitor in 1 ml of medium is stirred into the suspension to neutralize the nagarse and the platelets are centrifuged at 1000 g for 15 min at 0-4 °. All subsequent procedures are carried out at 0-4 °. The platelet pellet is resuspended in a cold medium consisting of 0.25 M sucrose, 10 mM HEPES, and 1 mM sodium EDTA, pH 7.4. The suspension is then passed through a precooled French pressure cell 6 at 1000 psi and then centrifuged at 1000 g for 15 min. The supernatant fraction is saved and the pellet is resuspended in 15-20 ml of sucrose medium and subjected to another pass through the pressure cell. After the second homogenate is centrifuged again at 1000 g for 15 min, the combined supernatant fractions are centrifuged at 12,000 g for 20 min to give a pellet (about 5-10 mg protein) that consists of a mixture of platelet organelles, including dense granules, ~ granules, acid hydrolasecontaining vesicles, and mitochondria. Table I shows that the markers for the various organelles are 2 to 3.6 times enriched in this pellet compared to the homogenate. This organelle mixture is suitable for some studies on dense granules and can be used as such after two washes with the sucrose 6 L. Salganicoff and M. H. F u k a m i , Arch. Biochem. Biophys. 153, 726 (1972). The valve modification described facilities regulation of the flow rate but is not essential. 7 M. H. F u k a m i , J. S. Bauer, G. J. Stewart, and L. Salganicoff, J. CellBiol. 77, 389 (1978). 8 D. D. Tyler and J. Gonze, this series, Vol. 10, p. 75. 9 A. L. Smith, this series, Vol. 10, p. 84. 10 R. E. Basford, this series, Vol. 10, p. 98. it O m i s s i o n o f nagarse gives preparations that are difficult to r e s u s p e n d and do not separate well on s u c r o s e density gradient centrifugation. A T P s e e m s to prevent the aggregation that occurs occasionally at this step, probably via its action as a potent inhibitor of ADP, small a m o u n t s of which m a y be released from the cells by nagarse.
[5]
PLATELET DENSE GRANULES
39
TABLE I MARKER ENZYMES IN ORGANELLE MIXTURE
Marker
Relative specific activity ~
Yield (%)
[t4C]Serotonin (dense granules) a-Glycerophosphate oxidase (mitochondria) /3-N-Acetylglucosaminidase (acid hydrolase vesicles) Platelet factor 4 (a granules)
2.82 --- 0.5 3.04 - 1.6
36.4 49.6
1.92 -+ 1.3
36.4
2.96 -+ 0.5
46.7
c, The relative specific activity is given as the ratio of the specific activity of the fraction divided by the specific activity of the homogenate, mean -+ SD, n = 3-4.
isolation m e d i u m if one is certain that the presence of the other organelles does not interfere with the activity to be monitored. The dense granules in this organelle mixture are considerably more stable to leakage 12 than the highly enriched preparation described below. Subfractionation on a Sucrose Density Gradient The sucrose stepwise gradient used for subfractionation of the granule mixture consists of seven steps from 0.8 to 2.0 M sucrose in 0.2 M increments. The sucrose steps are buffered with 5 m M H E P E S , p H 7.4, and contain 0.1 m M sodium E D T A , p H 7.4. Starting with the 2 M step, 1.5 ml of each step is layered into a tube(s) suitable for use in a B e c k m a n (Palo Alto, CA) SW 40 rotor (or c o m p a r a b l e swing-out ultracentrifuge rotor). The gradients are incubated for 1 hr at 37 ° to soften the interfaces and then cooled on ice to 0 °. A b o u t 1.5-2.0 ml of the organelle mixture (protein concentration not m o r e than 10 mg/ml) is then layered onto each gradient tube and centrifuged at 100,000 g and 4 ° for 60 min. At least three bands of organelles and a pellet should be observed. 7 The dense granules are to be found in the pellet, which we have usually resuspended in the homogenization m e d i u m and used only for analysis. H o w e v e r , in fact, pig platelet dense granules p r e p a r e d by the same method and used for nuclear magnetic r e s o n a n c e (NMR) studies at 31 ° were found to leak and slowly lose their stored contents, 12 and it is reasonable to assume that the h u m a n platelet preparation does the same. A modification of the sucrose gradient that resulted in a m o r e stable preparation of pig platelet dense granules 12 and that is p r o b a b l y applicable to the h u m a n preparation is described here for those uses that require incubations at temperatures a b o v e 0 - 4 ° . Bovine 12K. Ugurbil, M. H. Fukami, and H. Holmsen, Biochemistry 23, 416 (1980).
40
ISOLATION OF PLATELET COMPONENTS
[5]
TABLE II MARKER ENZYMES IN SUCROSE GRADIENT-ENRICHED DENSE GRANULES
Marker
Relative specific activitya
Yield (%)
[14C]Serotonin (dense granules) a-Glycerophosphateoxidase (mitochondria) fl-N-Acetylglucosaminidase Platelet factor 4 (a granules)
52.3 -- 6.9 ND b
19.9
3.09 -+ 1.5 3.66 -+ 0.5
4.0 2.1
a The relative specific activity is given as the ratio of the specific activity of the fraction divided by the specific activity o f the homogenate, mean -+ SD, n = 3-4. b ND, None detected.
serum albumin (0.6%) was added to all of the gradient steps that were made up with DzO (the D20 made it possible to use 1.8 M as the final gradient step). The final resuspension medium was 0.3 M KCI in D20, 0.4% bovine serum albumin, and 30 mM phosphate buffer, pH 7.4. Some details of these modifications that were required specifically for proton NMR such as use of DzO, and a KCI resuspension medium with phosphate buffer, can be changed. The essence of the modification was to use bovine serum albumin and a somewhat hyperosmotic (compared to 0.25 M sucrose) resuspension medium. A metrizamide gradient for human platelet dense granules 4 and a Percoll gradient for pig platelet dense granules ~3 have also been described. Yield and Purity The pellet consists of about 30-50/zg of protein and contains about 20% of the total serotonin originally present in 150 ml of whole blood starting material. Table II shows that the dense granule marker serotonin is 50-fold enriched compared to the homogenate. The mitochondrial marker was not measurable in this pellet, but the markers for acid hydrolase vesicles and ct granules were some threefold enriched, although only 4 and 2% of the starting material was present. This degree of contamination is negligible for most purposes, but the preparation is not suitable for study of unique dense granule proteins except by functional tests. The reason for this limitation is that the other organelles are not only larger and more numerous than dense granules in the platelet, but they also contain primarily proteins. The dense granules, on the other hand, contain other low molecular weight compounds (see below) and very little protein. 13 S. E. Carty, R. G. J o h n s o n , and A. Scarpa, J. Biol. Chem. 256, 11244 (1981).
[5]
PLATELET DENSE GRANULES
41
T A B L E III Low-MOLECULAR WEIGHT CONSTITUENTS OF HUMAN PLATELET DENSE GRANULESa
Substance PPI Pi ATP ADP Ca 2÷ Mg 2+
Serotonin
Whole platelets 6.3 16.7 23.8 14.5 125
--- 0.05 --- 2.5 --- 3.5 -+ 2.4 -+ 27 -1.5-2.5
Secreted amounts 6.0 4.6 9.7 14.1 88
-+ 0.52 + 1.6 -+ 1.3 + 1.8 + 23 -1.5-2.0
Dense granules 236 -+ 53 248 -+ 65 440 _+ 96 633 -+ 142 2634 -+ 471 98 + 33 90-100
Isolated by the method described in this article (nmol/mg of protein). Values are given as mean -+ SD, n = 4-6.
Therefore, the relative amounts of protein contributed by the various organelles are disproportionate to their concentration in the preparation. Properties of Human Platelet Dense Granules The concentrations of low molecular weight compounds in dense granules are given in Table III. The amounts contained in whole platelets and that released by thrombin are also shown in order to indicate which of the constituents are contained primarily in the dense granules. Most of the platelet content of PPi,14 ADP, serotonin, and much of the calcium is secreted and therefore is stored in secondary granules. Significant amounts of ATP and Pi that are required for metabolic activity are present in sites other than in the dense granules and are not secreted. Nuclear magnetic resonance studies on intact human platelets suggest that the nucleotides and calcium in the dense granules form high molecular weight aggregates that are more or less insoluble, which reduce the hyperosmolarity of the granule interior and which bind serotonin. ~5a6 Similar studies on pig platelets and isolated pig platelet dense granules in which magnesium is the predominant divalent cation have confirmed the existence of these nucleotide-divalent cation-amine interactions. ~2'~7'~8 14 M. H. Fukami, C. A. Dangelmaier, J. S. Bauer, and H. Holmsen, Biochem. J. 192, 99 (1980). 15 j. L. Costa, C, M. Dobson, K. L. Kirk, F. M. Poulsen, C. R. Valeri, and J. J. Vecchione, FEBS Lett. 99, 141 (1979). 16 K. Ugurbil, H. Holmsen, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 76, 2227 (1979). ~7j, L. Costa, C.M. Dobson, K. L. Kirk, F.M. Poulsen, C. R. Valeri, and J. L. Vecchione, Philos. Trans. R. Soc. London, Ser. B. 289, 413 (1980). I8 M. H. Fukami, H. Holmsen, and K. Ugurbil, Biochem. Pharmacol. 33, 3869 (1984).
42
ISOLATION OF PLATELET COMPONENTS
[6]
Other mechanisms shown to be involved in serotonin uptake and storage in platelet dense granules from species other than human, such as an intragranular pH of 5.5,19 an ATP-driven proton pump, 2° and a reserpinesensitive amine transport system on the granule membrane, 2° most probably also exist in human platelet granules, The similarities in serotonin uptake, storage, and secretion by intact platelets of the various species indicate that the granular mechanisms are also similar. Finally, it should be mentioned that there is a heterogeneous group of inherited bleeding disorders, referred to as storage pool deficiency, both in humans and various animal species in which the platelet dense granule constituents are considerably diminished or absent, as shown both by secretion studies and by electron microscopy. 2~ 19 R. G. Johnson, A. Scarpa, and L. Salganicoff, J. Biol. Chem. 253, 7061 (1978). 20 G. Rudnick, H. Fishkes, P. J. Nelson, and S. Schuldiner, J. Biol. Chem. 2S5, 3638 (1980). :1 K. M. Meyers and M. M6nard, in "The Platelet Amine Storage Granule" (K. M. Meyers and C. D. Barnes, eds.), pp. 149-85. CRC Press, Boca Raton, Florida, 1992.
[6] S t u d y i n g t h e P l a t e l e t C y t o s k e l e t o n in T r i t o n X-100 Lysates
By JOAN E. B. Fox, CLIFFORD C. REYNOLDS, and JANET K. BOYLES The platelet cytoskeleton is composed primarily of actin filaments. In unstimulated platelets, 40 to 60% of the actin is polymerized into filaments.~ The majority of these filaments are present in networks throughout the body of the platelet. These are referred to as the cytoplasmic actin filaments. However, a small pool of the filaments appears to be present as part of a two-dimensional latticework that coats the inner surface of the lipid bilayer in much the same way as the red blood cell skeleton coats the membrane of the erythrocyte. This lattice-like structure is referred to as the platelet membrane skeleton. The filaments of the platelet membrane skeleton appear to be cross-linked by actin-binding protein (otherwise known as filamin), spectrin, and other unidentified proteins. 2,3 During platelet activation there is a rapid burst in actin polymerization, and about i j. E. B. Fox, J. K. Boyles, C. C. Reynolds, and D. R. Phillips, J. CellBiol. 98, 1985 (1984). -' J. E. B. Fox, J. K. Boyles, M. C. Berndt, P. K. Steffen, and L. K. Anderson, J. CellBiol. 106, 1525 (1988). 3 j. H. Hartwig and M. DeSisto, J. Cell Biol. 112, 407 (1991).
METHODS 1N ENZYMOLOGY, VOL. 215
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