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Biochimica et Biophysica Acta, 3 9 2 ( 1 9 7 5 ) 2 4 2 - - 2 5 4 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA 27664

EFFECTS OF THROMBIN ON WASHED, HUMAN PLATELETS: CHANGES IN THE SUBCELLULAR FRACTIONS*

INGER HAGEN

Institute for Thrombosis Research, Rikshospitalet, Oslo I (Norway) (Received December 24th, 1974)

Summary Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 -+ 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control {mol. wt 147 000 + 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37°C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 -+ 3500) had disappeared. The distribution of protein and ~-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (>77%). The granule fractions, D and E of the control contained 3.0% -+ 0.8% and 6.4% + 1.3% of the total a m o u n t of ~-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% + 1.0% of total while fraction D was lacking.

* Part of this work was presented at the 4th Congress of Thrombosis and Haemostasis, Vienna 1973.

243 Introduction Blood platelets undergo "release reaction" [1] when exposed to different stimuli, such as thrombin [2], collagen [3], ADP [4] and adrenaline [5]. During release, adenine nucleotides, Ca 2÷ serotonin, platelet factor 4, glycosaminoglycans, platelet fibrinogen and acid hydrolases are extruded to the extracellular milieu [6,7]. Subcellular fractionation has shown that substances released originate from the granular fractions, whereas those retained are localized mainly in the membrane and soluble fractions [6,8]. Electron microscopy of platelets treated with thrombin has revealed profound morphological alterations compared to control platelets [9]. Holme [10] and Droller and Fox [11] found that parallel to thrombin-induced release of adenine nucleotides and Ca 2÷, there occurred an almost complete loss of granules from the cytoplasm. Partial inhibition of the release reaction correlated with fewer granules discharged. In this study, subcellular fractionation was performed to reveal changes in the fractions after thrombin-induced release reaction. To examine the granule localization of the releasable substances, the a m o u n t of the lysosomal marker enzyme, fi-N-acetylglucosaminidase, was compared for the subcellular fractions of thrombin-treated and control platelets. Sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed to reveal changes in the protein pattern. The initial interaction between thrombin and the platelet membrane has been thought to involve proteolysis [1,12,13]. Because of the subsequent rapid release of intracellular material, it has been difficult to study the different effects of thrombin separately. In these experiments, the thrombin-platelet interaction has been studied by comparison of the protein pattern after sodium dodecylsulfate-polyacrylamide gel electrophoresis of membranes isolated from thrombin-treated and control platelets.

Materials and Methods

Chemicals Thrombin, Topical (bovine origin, Parke, Davis and Co., Detroit), was made up to 100 N.I.H. units/ml with 0.12 M NaC1 containing 0.03 M Tris and 0.003 M EDTA, and stored a t - - 2 0 ° C , fi-Galactosidase, bovine serum albumin, ovalbumin, trypsin and Coomassie brilliant blue R were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A. Acrylamide and N,N'-methylenebisacrylamide were purchased from Eastman Organic Chemicals, Rochester, N.Y., U.S.A., N,N,N'N'-tetramethylethylenediamine was from Fluka AG, Switzerland and fuchsin from E. Merck, Darmstadt. All chemicals were reagent grade. Platelet thrombosthenin was a gift from Dr P.C. French. Biochemical materials Platelets were obtained from 500 ml of human blood collected into Fenwal Double Blood Pack Haemosystem containing 0.15 vol. of acid/citrate/dextrose solution, and processes as described elsewhere [14].

244

Release experiments and subcellular fractionation Two 5-ml aliquots of washed platelet suspension were incubated for 5 or 30 min at 37°C with, respectively; (1), 50 pl of suspending medium (control) and (2), 50 pl of 100 N.I.H. units/ml thrombin (thrombin-treated platelets) The cells were separated from the extracellular phase by centrifugation (2000 × g, 30 min, 4 ° C), resuspended in 2.5 ml 0.27 M sucrose and homogenized in the Aminco-French pressure cell (2 X 1 min, 1.361 atm) [15]. The sediment after homogenate centrifugation (2000 × g, 30 min, 4°C) was suspended in 1.0 ml of buffer (0.12 M NaC1, 0.03 M Tris and 0.003 EDTA, pH 7.4) and stored at --20°C. 1.4 ml of the supernatant after homogenate centrifugation was layered on a continuous sucrose gradient (d: 1.020--1.230) and centrifuged in a Spinco preparative ultracentrifuge (SW39 rotor 37 000, 2 h, 2°C) [15]. The sucrose density was monitored with a Zeiss Abbe (model B) refractometer. The gradient was separated into six fractions (A--F) as shown in Fig. 1. Protein determination was done using Miller's modification of Lowry's method [16], and ~-N-acetyl-glucosaminidase as described by Day et al. [8], defining 1 unit as 1 pmol p-nitrophenol liberated per 30 min. Polyacrylam ide gel elec trophoresis The polyacrylamide gel electrophoresis was essentially performed as described by Weber and Osborn [17], except for use of 5% polyacrylamide gels. Before application, the samples were incubated for 30 min at 37°C in a buffer containing 0.1 M sodium phosphate, 2% sodium dodecylsulfate and 1.5% ~-mercaptoethanol (pH 7.5). 10--50 pg of protein was applied to each gel. The electrophoresis was performed at 6.7 mA per gel for 3.5 h, in a 0.1 M sodium phosphate buffer, pH 7.5, containing 0.1% sodium dodecylsulfate. Proteins were stained with Coomassie brilliant blue as described by Fairbanks et al. [181. Molecular weight determinations Platelet myosin (200 000), ~-galactosidase (130 000), albumin (68 000), platelet actin (46 000), ovalbumin (45 000) and trypsin (23 000) were used to prepare the standard curve. Electron microscopy The fractions were fixed in 2.5% glutaraldehyde in 0.1 M Sorensen's buffer, pH 7.2, for 1.5 h, and post-fixed 1 h in 1% OsO4 in Tyrode's solution, pH 7.2. Dehydration was performed in graded ethanols. The samples were embedded in EPON 812, and sent to Dr J.J. Sixma, Utrecht, for examination under the electron microscope. Results

The amount of protein in the centrifuged control homogenate was 66% ± 7% of the protein content of the original platelet suspension. This was taken as a measure of the degree of homogenization. Fig. 1 shows the distribution of the subcellular fractions from control and thrombin-treated platelets after sucrose density gradient centrifugation. Three particulate zones were observed in the

245

Fig. 1. S u c r o s e ~ ' a d i e n t a f t e r u l t r a c e n t r i f u g a t i o n o f p l a t e l e t h o m o g e n a t e . L e f t : f r o m c o n t r o l p l a t e l e t s . R i g h t : f r o m t h r o m b i n - t r e a t e d p l a t e l e t s (1 N.I.H. u n i t s / m l f o r 5 rain). F o r d e f i n i t i o n o f t h e s u b f r a c t i o n s A - - F see M a t e r i a l s a n d M e t h o d s . N o t e t h e a b s e n c e of f r a c t i o n D f r o m t h e t h r o m b i n - t r e a t e d cells.

control sample, whereas one of these (d = 1.1562) was lacking in the thrombintreated sample. Electron microscopy demonstrated that fraction B (see Materials and Methods and Fig. 1) contained membrane vesicles, glycogen particles and a certain a m o u n t of small granules (Fig. 2). Fraction D contained a lot of small granules, vesicles and a mass of homogeneous material (Fig. 3), whereas fraction E contained granules and mitochondria (Fig. 4}. A background material, often of a fibrillar structure, was also observed. The most striking effect of thrombin treatment of the platelets was that no particulate material corresponding to fraction D could be observed after sucrose density gradient centrifugation (Fig. 1), and also that only few non-mitochondrial granules were present in fraction E in this case. (Fig. 5). Sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on each fraction from the sucrose gradient, and on the extracellular phase (supernatant after sedimentation of the platelets}. A major protein with a molecular weight of 145 500 + 1000 appeared in the extracellular phase from

246

F i g . 2. E l e c t r o n m i c r o g r a p h o f f r a c t i o n B i s o l a t e d f r o m c o n t r o l p l a t e l e t s . M e m b r a n e p a r t i c l e s a n d a c e r t a i n a m o u n t o f s m a l l g r a n u l e s are s e e n . M a g n i f i c a t i o n ; 2 . 6 X 3 0 0 0 0 .

vesicles, glycogen

the thrombin-treated sample and in fraction E (147 000 -+ 1000) from the control (Fig. 6). This band could neither be observed in the extracellular phase from the control, nor in fraction E from the thrombin-treated platelets (fraction D is lacking after thrombin treatment), and could thus represent protein(s) extruded from the granules during the release reaction. The specific release of intracellular platelet constituents by thrombin (release reaction) is completed within the first 2 min under the conditions used. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of fraction B (membranes) show that no reproducible difference can be observed between the control and the thrombin-treated sample. The first band migrated with the same mobility as isolated platelet myosin (mol. wt 200 000) and the intensively stained, fast-moving band as platelet actin (mol. wt 46 000) (Fig. 7). Thrombin treatment of the platelet suspension was also performed at 0°C to inhibit the

247

Fig. 3. E l e c t r o n m i c r o g r a p h of f r a c t i o n D isolated f r o m c o n t r o l platelets. The p i c t u r e s h o w s a l o t of s m a l l granules, vesicles a n d a m a s s o~ h o m o g e n e o u s m a t e r i a l . M a g n i f i c a t i o n : 2.6 X 6 0 0 0 .

release reaction [19], thus avoiding any effects of extruded substances on the platelet membrane. Membranes from these platelets showed protein patterns identical to membranes obtained from platelets treated with thrombin at 37 ° C. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of platelet membranes isolated from a suspension preincubated with thrombin for 30 rain is shown in Fig. 8. A band of molecular weight 183 000 -+ 3500 showed strongly decreased intensity, or had disappeared completely from the thrombintreated sample during this prolonged incubation. fl-N-Acetylglucosaminidase and total protein were measured in the centrifuged homogenate, the sediment (unhomogenized platelets) and the extraceltular phase. In the extracellular phase from the thrombin-treated sample, the amount of fl-Noacetylglucosaminidase and protein were 18 and 7 times higher than the corresponding fraction from the control platelets (Table 1). A representative experiment of the subcellular distribution of total protein and fl-N-acetylglucosaminidase is shown in Table II. The major part of the protein and of the enzyme activity were recovered in the soluble fraction (A}.

248

F i g . 4. E l e c t r o n m i e r o g r a p h o f f r a c t i o n E i s o l a t e d f r o m c o n t r o l p l a t e l e t s . T h e p i c t u r e s h o w s g r a n u l e s ( m o r e t h a n in f r a c t i o n D ) a n d m i t o c h o n d r i a . Larger vesicles and a background material of a fibrillar structure were also seen. Magnification; 2.6 X 20 000.

The fl-N-acetylglucosaminidase f o u n d in the granule fractions D and E o f the c o n t r o l c o n s t i t u t e d a p p r o x . 10% o f total applied on the gradient (D : 3.0% ± 0.8%, E : 6.4% + 1.3%), whereas in the t h r o m b i n - t r e a t e d sample o n l y a b o u t 3% (E : 3.3% -+ 1.0%) o f t o t a l was f o u n d in f r a c t i o n E, and the granule f r a c t i o n D was lacking. The c o r r e s p o n d i n g values f o r t o t a l p r o t e i n were 1.6% -+ 0.6% and 4.2% + 2.7% for the c o n t r o l , and 2.3% ± 1.4% for the t h r o m b i n - t r e a t e d sample. Discussion Pressure h o m o g e n i z a t i o n o f platelets was first described b y Salganicoff and F u k a m i [20] and F r e n c h and H o l m e [15] using pig and h u m a n platelets,

249

Fig. 5. E l e c t r o n m i e r o g r a p h of f r a c t i o n E isolated f r o m t h r o m b i n - t r e a t e d platelets (1 N . I . H . u n i t s ] m l f o r 5 rain). This f r a c t i o n c o n t a i n e d granules, m o s t l y m i t o c h o n d r i a , a c e r t a i n a m o u n t of vesicles ( p r o b a b l y less t h a n in f r a c t i o n E f r o m c o n t r o l p l a t e l e t s ) a n d fibrillar m a t e r i a l . M a g n i f i c a t i o n ; 3.1 X 21 000.

respectively. By this method well preserved membrane vesicles, mitochondria and storage granules were obtained. The efficiency of the homogenization has been given as 95 [15] and 78% [20] as compared to 66% in the present experiments. Electron microscopy of the particulate fractions obtained in this study revealed good separation of membrane vesicles from granules, and of small granules from mitochondria and larger granules. Fractions isolated from thrombin-treated cells showed only a few non-mitochondrial granules, which would fit the view that substances extruded during the release reaction are located in such granules [6,8]. This finding is also consistent with the loss of granules observed in the electron microscope [10,11 ]. Various enzymes extruded during the release reaction are reported to have their highest specific activity in the granule fraction [21]. This was also indicated in our experiments using fl-Nacetylglucosaminidase as marker. However, the difference in specific activity

250

1 Fig. 6. S o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r c s i s of 1, c x t r a c e U u l a r p h a s e ( s u D e r n a t a n t ) f r o m c o n t r o l p l a t e l e t s ; 2, e x t r a c e l l u l a r p h a s e f r o m t h r o m b i n - t r e a t c d p l a t e l e t s ; 3, f r a c t i o n D f r o m c o n t r o l platelets; 4, f r a c t i o n E f r o m c o n t r o l p l a t e l e t s : 5, f r a c t i o n E f r o m t h r o m b i n - t r e a t e d platelets. 10 50 pg of p r o t e i n was a p p l i e d to e a c h gel. F r a c t i o n s D and E, see Fig. 1.

TABLE I D I S T R I B U T I O N OF ~ - N - A C E T Y L G L U C O S A M I N I D A S E AND P R O T E I N A F T E R P L A T E L E T HOMOGENIZATION Platelets o b t a i n e d f r o m 1 u n i t of b l o o d . Sample

Supernatant after homogenate centrifugation

~-N-Acetylglucosaminidase

P r o t e i n (rag)

A b s e n c e of t h r o m b i n

P r e s e n c e of t h r o m b i n

Total activity (units)

Spec. act. (units/mg protein)

Total activity (units)

A b s e n c e of thrombin

Presence of t h r o m bin

Spec. act. (units/mg protein)

15.18

0.36

8.76

0.41

41.80

21.16

Sediment after homogenate centrifugation

2.92

0.42

4.61

0.28

6.88

16.19

Extracellular phase

0.25

0.25

0.67

0.97

6.84

49.65

44.19

Sum

18.25

4.59 17.96

251

Fig. 7. S o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . L e f t ; m e m b r a n e s i s o l a t e d f r o m c o n t r o l p l a t e l e t s . Middle; m e m b r a n e s i s o l a t e d f r o m t h r o m b i n - t r e a t e d p l a t e l e t s (1 N.I.H. u n i t / m l f o r 5 m i n ) . R i g h t ; Platelet actomyosin.

between the membrane and the granule fraction was t o o low to draw any final conclusion. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed one major protein band in the granule fractions (D and E} from the control and in the extracellular phase from the thrombin-treated sample. This band could neither be observed in the extracellular phase from the control nor in fraction E from the thrombin-treated cells, and could thus represent protein(s) extruded from the granules during the release reaction. This protein(s) is probably identical to the "thrombin-sensitive protein" reported by Baenziger et al. [13,22] and which was firstly believed to represent the target protein for thrombin in the platelet membrane. Enzymatic analyses have shown that various enzymes are released to the extracellular phase during the release reaction [6,2]. Possibly, most of these are present in too low amounts to be seen on sodium dodecylsulfate-polyacrylamide gels. The unhomogenized platelets from the thrombin-treated cells showed a higher content of/~-N-acetylglucosaminidase and protein than the corresponding control platelets. This could mean that the thrombin-treated platelets are

252

183000

,

ir

Fig. 8. S o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . L e f t : m e m b r a n e s i s o l a t e d f r o m c o n t r o l platelets. Right: m e m b r a n e s isolated f r o m t h r o m b i n - t r e a t e d p l a t e l e t s (1 N.I.H. u n i t / m l f o r 30 rain).

more resistant to homogenization than the control cells. The high total activity of ~-N-acetylglucosaminidase in the soluble fraction (A) is probably due to disruption of some granules during the homogenization procedure. This is also consistent with the earlier observations of Day et al. [8] and Fukami (personal communication). Due to the release reaction, the a m o u n t of ~-N-acetylglucosaminidase and protein were higher in the subcellular fractions from the control platelets than the corresponding values from the thrombin-treated cells, and the most pronounced reduction was observed in the granule fraction. Together with the observation that fraction D is lacking in the thrombin-treated sample, we find it highly probable that the extruded substances were located in the granules. Similar release experiments have been performed on other secretory cells. Woodin [23] reported that during the release of certain enzymes from polymorphonuclear leukocytes, there was a decrease in the corresponding enzyme activities in the granule fraction of the cells. Upon stimulation of the adrenal medulla an increased o u t p u t of catecholamines and protein occurred [24,25]. The secretion caused a reduction in the protein c o n t e n t of chromaffin granules which correspond to the a m o u n t of amines released. Lagernoff [26] found that

253 T A B L E II S U B C E L L U L A R - D I S T R I B U T I O N OF P L A T E L E T ~ - N - A C E T Y L G L U C O S A M I N I D A S E A N D P R O T E I N V o l u m e o f c e n t r i f u g e d h o m o g e n a t e a p p l i e d on t h e sucrose g r a d i e n t : 1.4 m l . Sample

Applied on t h e gradient Fraction A Fraction B Fraction C Fraction D Fraction E Fraction F

f l - N - A c e t y l g l u c o s a m i n i d a s e (units)

Protein (mg)

Absence of t h r o m b i n

Presence of t h r o m b i n

Total activity (units)

Spec. act. (units/rag protein)

Total activity (units)

Spec. act. (units/rag protein)

A b s e n c e of thrombin

P r e s e n c e of thrombin

8.50

0.36 0.33

6.12 5.57 ( 9 1 . 2 % )

0.41 0.47

23.53 17.23 (83.9%)

14.80 11.93 (88.1%)

0.37 (5.0%)

0.62

0.12 (2.0)

0.33

0 . 6 0 (2.9%)

0.39 (2.7%)

0 . 5 0 (6.7%)

0.56

0 . 2 0 (3.3%)

0.27

0 . 9 0 (4.4%)

0.73 (5.4%)

0 . 2 4 (3.2%)

0.63

--

--

0 . 3 8 (1.9%)

0.53 (7.2%)

0.43

0 . 1 4 (2.2%)

0.40

1.23 (6.0%)

0.06 (0.8%)

0.31

0 . 0 8 (1.3%)

0.44

5.73 (77%)

Sum 7.43 ( 1 0 0 % ) R e c o v e r y 87.0%

6.11 ( 1 0 0 % ) 100%

-0.34 (2.5%)

0.19 (0.9%)

0 . 1 8 (1.3%)

20.53 (100%) 87.5%

13.54 (100%) 91.5%

the release of ~-N--acetylglucosaminidase from polymyxin B-stimulated mast cells, closely parallelled the released of histamine, which is known to be located in the granules. No reproducible changes in the protein pattern could be observed after sodium dodecylsulfate-polyacrylamide gel electrophoresis of membranes isolated from platelets incubated 5 min with thrombin. Phillips [27,28] and Steiner [29], using an isotope labelling technique observed a membrane protein which showed reduced radioactivity after thrombin treatment. However, no changes could be seen on the sodium dodecylsulfate-polyacrylamide gels, probably because this technique is not sensitive enough to reveal small changes in the protein The disappearance of a protein band (mol. wt 183 000) after prolonged incubation with thrombin (30 min) was probably a result of a slow proteolysis. Since this effect could not be seen after 5 min incubation, it is unlikely that it has any connection with the initial thrombin-platelet interaction leading to aggregation and release reaction. The commercial thrombin preparation used in these experiments was not further purified, and the effect observed on the platelet membrane after 30 min incubation might also be due to contaminating enzymes. Cohen et al. [30] purified thrombosthenin M (Platelet myosin) from equine blood platelets and showed by acrylamide gel electrophoresis that a protein band corresponding to thrombosthenin M disappeared during incuba-

254 tion of washed platelets for 1 h with 10 N.I.H. units/ml of thrombin. In view of the poor separation obtained in their electrophoresis system, we consider it possible that this protein could be the same as the one lacking after 30 min incubation with thrombin in our experiments. Although this moved slightly faster than platelet myosin, the two proteins moved as one broad band when high amounts of protein were applied to the gel. Ganguly [31,32] has reported on several thrombin-sensitive proteins in platelet lysates. Due to differences in the separation techniques we find it difficult to correlate any of those to the one we observed.

Acknowledgements I wish to gratefully acknowledge the provision of the electron micrographs by Drs Jan J. Sixma, J. Wester and J. van der Veen of the University Hospital, Utrecht. The helpful advice of Drs P.C. French, H. Holmsen and N.O. Solum, and the skilfut technical assistance of May Britt Nordkild are also appreciated. This work was supported by Nasjonalforeningen, Det Norske Rfid for Hjerte- og Karsykdommer, Anders Jahres fond til Videnskapens Fremme, Dr med. Carl Sembs Fond og Direkt0r Godtfred Lie og hustru Marie Lies Fond.

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