Cell, Vol. 18, 1015-l

023.

December

1979,

Copyright

0 1979

by MIT

A Study of Protein Phosphorylation in Shape Change and Ca”-Dependent Serotonin Release by Blood Platelets William F. Bennett,* John S. Belville Gary Lynch School of Biological Sciences University of California Irvine, California 92717

and

Summary Upon treatment with agents such as thrombin, collagen or concanavalin A, blood platelets change shape, secrete serotonin and phosphorylate two proteins having molecular weights of approximately 20,000 and 40,000. We have analyzed the relationship of this protein phosphorylation to shape change and release aided by the fact that while shape change occurs independently of extracellular calcium, release of serotonin displays a rather strict calcium requirement. Under limited calcium conditions, where virtually no serotonin release occurs, (Con A)-stimulated phosphorylation is uninhibited. Divalent cations (Mg++, Co++ and Zn++) also inhibit release but not phosphorylation. The microtubule effectors colchicine and D20 show concomitant effects on release and phosphorylation, Indicating a microtubule involvement prior to phosphorylation. Papaverine inhibits release and phosphorylation while not strongly influencing shape change, suggesting that shape change does not require phosphorylation. We therefore conclude that phosphorylation of these proteins takes place after shape change but prior to release, and although it may be required for secretion to occur, the two processes are easily separated. Thus phosphorylation of these proteins is not likely to be an integral component of the release mechanism. Introduction The biochemical mechanisms by which extracellular stimuli elicit the release of transmitters and hormones are very poorly understood. It is apparent that in many cases a secretagogue (for example, depolarization for nerve terminals, or acetylcholine for adrenal chromaffin cells) produces an inward calcium flux which then activates exocytosis of the effector substance, probably from small storage vesicles (see Douglas, 1974 for a review). Although the means by which calcium achieves this effect remain unclear, it has recently become evident that some of the intracellular actions of calcium are mediated by protein phosphorylation (Shulman and Greengard, 1978a, 1978b; Greengard, 19791, and it has been proposed that phosphorylation is a key intermediate step in the release reaction (Lyons, Stanford and Majerus, 1975; Haslam and ’ To whom

all correspondence

should

be addressed.

Lynham, 1977; Sieghart et al., 1978). A major difficulty in evaluating this hypothesis lies in determining which biochemical changes are due to unique contributions of extracellular calcium and which are among the many other cellular changes occurring before, during and after exocytosis. Previous studies showing correlations between protein phosphorylation and secretion by platelets and mast cells (Haslam and Lynham, 1977; Sieghart et al., 1978) have not addressed the issue of whether, or to what extent, release in those systems was dependent on extracellular calcium. Therefore, little information is presently available on the relative stages of the exocytotic process at which Ca++ and protein phosphorylation might function. Our approach to this problem makes use of the blood platelet, a relatively simple anucleate secretory cell that carries out high affinity uptake, granular storage and secretagogue-coupled release of serotonin (5-hydroxytryptamine-5-HT). We have recently found that platelets possess a calcium-sensitive release system, and that the stages leading from application of the secretagogue to the appearance of extracellular serotonin can to some extent be separated (Bennett, Belville and Lynch, 1979). Specifically, concanavalin A applied in the absence of calcium binds to platelets and elicits a normal shape change response but no serotonin secretion, whereas if calcium is included, serotonin release accompanies the shape change (Belville, Bennett and Lynch, 1979). On the basis of these findings, we have suggested that the Con A-activated platelet may be used as a model for study of the cellular events and biochemical processes mediated by extracellular calcium. In this report, we assess 5-HT release, protein phosphorylation and shape change under a number of conditions, and present data indicating that although selective phosphorylation is apparently a necessary antecedent of release, it is not the mechanism by which extracellular calcium activates exocytosis. Results Turbidimetric analysis has shown that Con A elicits the platelet shape change reaction to its full extent independently of extracellular calcium (Skjerten, 1968; Patscheke and Brossmer, 1974; Belville et al., 1979). Scanning electron microscopy reveals that Con A-induced shape change involves rounding of the cells and extrusion of numerous pseudopodia (Figure 1). This effect, which occurs at 25°C within 2 min of lectin application, is obtained equally well in the presence of calcium or the calcium-chelating agent, EGTA. In contrast to the effects of most other secretagogues, such as collagen or thrombin, no aggregation is observed under these conditions (Schmukler and Zieve, 1974; Patscheke and Worrier, 1977; Belville et al.,

Cell 1016

Figure

1. Scanning

Electron

Microscopy

of Resting

and Con A-Treated

Platelets

Platelets prepared as described in Experimental Procedures were split into two aliquots in medium B (no Ca”). One portion was incubated 50 pg/ml Con A for 2 min prior to fixation with glutaraldehyde; the remaining cells were left untreated. Both samples were fixed, prepared examined as described in Experimental Procedures. (A) resting; (B) Con A-treated.

1979; Bennett, et al., 1979). It is clear from these earlier studies that in the absence of calcium, Con Atreated platelets can undergo radical morphological changes while releasing very little serotonin, and that addition of calcium allows release of 5-HT with no need of further shape change. To examine the role of protein phosphorylation in shape change and/or release, we have assayed protein phosphorylation in platelets by prelabeling with 32P-orthophosphate and 3H-5-HT and treating the cells with increasing concentrations of Con A + Ca++. Whole platelet proteins were then separated by SDS-PAGE, and the proteinbound phosphate was assessed by autoradiography. Three things are apparent from these autoradiographic data (Figure 2). First, Con A induces a concentration-dependent phosphorylation of the -40,000 and -20,000 molecular weight proteins, these effects being detectable at lectin concentrations as low as 10 pg/ml; second, other cellular phosphoproteins are changed relatively little under these conditions; and third, especially at lower Con A concentrations, Ca++ has significant influence on phosphorylation. Figure 3 is a graphical representation of the same type of experiment, showing lectin-induced changes in specific phosphorylation of the 40,000

with and

molecular weight proteina and concomitantly measured 5-HT release. It is obvious from the figure that even though Ca++ is required for release, phosphorylation of the protein to near its maximum level can occur in the absence of added Ca++. Again, there is a marked effect of Ca++ on phosphorylation at low Con A levels, but further analysis (shown below) indicates that this is probably a divalent cation effect, separate from effects on release. Figure 4 shows the time course of 40K phosphorylation and 5-HT release in response to Con A treatment 1 Ca. Release, which is obtained only in the linearly for 5-10 min presence of Ca++, proceeds under these conditions. Phosphorylation of the protein takes a similar time course, but also occurs in the absence of added Ca++. The Con A concentration used in this experiment (100 pg/ml) is optimal for both phosphorylation (5-7 fold increase over baseline) and release (7-l 0 fold), as was shown in Figure 3. At that a The figures in this paper show only the data for the 40,000 molecular weight protein. This was done for simplicity and because this protein shows a more robust effect than the 20,000 molecular weight band. Under all conditions reported here, however, the 20.000 and 40,000 molecular weight bands co-vary.

Blood Platelet 1017

Protein

Phosphorylation

ConA Ca

1

5

+ -I+

-I+

10

50

loo

500

I

c

A. 40K

PHOSPHORYLATION

1

14-

-I+-/+

12-

96-

2

IO-

& I-

8-

6 *.

64-

40-

I Figure 2. Con Platelets

A-Dependent

Protein

Phosphorylation

by

8. 3H-5HT

RELEASE

Isolated

Cells were labeled as described in Experimental Procedures, then treated with Con A at the indicated concentrations (from l-500 Fg/ ml) in either the presence or absence of 1 .O mM CaCI,. Well “C” is an untreated control. Incubations were carried out for 10 min at 25°C. Molecular weights were determined using the following proteins as standards: cytochrome C, 13,500; glyceraldehyde-3-phosphate dehydrogenase. 37,000; creatine phosphokinase, 41.000; platelet actin, 43,000; bovine serum albumin, 67.000; phosphorylase 6, 96.000. As indicated by the figure, the phosphoproteins of interest have molecular weights close to 40,000 and 20,000 in this gel system, and to avoid confusion will be referred to as having those molecular weights.

lectin concentration there is very little further effect of Ca++ on phosphorylation. The above results indicate that phosphorylation of these proteins, like the shape change, proceeds independently of the release process. This effectively rules out the possibility that phosphorylation is either a consequence of the release reaction or an integral part of the secretion mechanism. It might then be either a parallel (unrelated) process or a required antecedent component of release. To investigate this further, we have tested the effects of colchicine and deuterium, microtubule effecters which influence secretion in other cell types (see Gillespie, 1975), for their effects on the platelet system. Colchicine induces a concentration-dependent block of release (Figure 5) and, as shown in the upper panel of the figure, it inhibits phosphorylation of the 40K material across the same range. On the other hand, deuterium facilitates release of serotonin (Figure 6) and likewise facilitates the effects of Con A on phosphorylation of the 40K protein. Thus it appears that facilitation and depression of phosphorylation produce comparable effects on release, which suggests that phosphorylation might be a required antecedent to the calciumdependent secretion step. [We should note that the

Figure 3. Dependence centrations

of Specific

Phosphorylation

on Con A Con-

Labeled platelets were incubated with the indicated concentration of Con A f 1 .O mM Ca++. After 10 min at 25°C. protein phosphorylation and ‘t-t-serotontn release were assessed as described in Experimental Procedures.

effect of D20 in these experiments appears to be due to deuterium ion, and not to the increased mass of water, since H2”0 has no facilitatory effect on either release or phosphorylation (data not shown).] Colchitine and D20 are generally believed to influence microtubules by disruption (Borisy and Taylor, 1967; White, 1968) and stabilization (Inoue and Sato, 1987), respectively. These data therefore suggest a relationship between microtubule assembly and phosphorylation of the two polypeptides. In this regard, it has been reported that colchicine inhibits shape change (Patscheke and Brossmer, 1974; see also White and Gerrard, 1979) and in addition to confirming this observation, we have also found that the presence of D20 at a concentration of 30% approximately doubles the rate of shape change (data not shown). Taken together, these data imply that a microtubule assembly step which occurs prior to phosphorylation and correlated with the shape change influences the rate (or extent) of phosphorylation of the 20 and 40K bands. The notion that specific phosphorylation occurs antecedent to any effect of extracellular calcium is fur-

Cell 1018

A. 40K PHOSPHORYLATKIN

24-

25

y-yql

20g

16-

e LL

‘2-

c

A. 40K PHOSPHORYLATlON

t-1 Con A

s

B-

b-

4-

o- (

I

I

I B. ‘H-5HT

I RELEASE

,r

B. ‘H-5HT

5-

RELEASE

30‘: e x

20-

B IO-6 0

I

2

-5

-3

log [CGhicinr]

1

3

4

5

..

lo

MINUTES AT 25’ Figure 4. Time Course of 40K Protein Release in Response to Con A

Phosphorylation

and ‘H-5-HT

Labeled platelets were treated at 25’C with 100 pg/ml Con A + 1 mM Ca++ (MI. 100 Fg/ml Con A without added Ca++ (A-A) or were left untreated, for the indicated times @SD). Because of the physical manipulations involved, the earliest time point for the release assay was 1 min. the zero time point for release being determined by the untreated samples. No further increases or decreases were observed for either the 20K or 40K protein, even at 30 min after Con A addition, although slight decreases occurred for both proteins after 1 hr.

ther supported by the results of experiments with divalent cations such as magnesium. This ion is well characterized in its ability to prevent access of Ca++ to its active sites in secretion at the neuromuscular junction (Hubbard, Jones and Landau, 1968) and in the brain (Dunwiddie and Lynch, 1979). As we have shown previously (Bennett et al., 1979) and as illustrated in the bottom panel of Figure 7, magnesium exhibits concentration-dependent inhibition of release in the range between 0.5 and 50 mM. Nevertheless, phosphorylation of the 40K protein remains at a constant level over this entire range of Mg++ concentrations (Figure 7A). Like Mg++, other divalent cations (for example, cobalt and zinc) are inhibitors of Ca++-dependent release by platelets (Bennett et al., 1979). We have found that under conditions of virtually complete re-

Figure 5. Effect and 40K Protein

of Colchicine Phosphorylation

Preincubation

-2

,M on ‘H-5-HT

Release

Labeled cells were incubated with the indicated concentrations of colchicine for 5 min at 25’C prior to addition of Con A. 10 min incubations were then carried out either with or without 50pg/ml Con A. All samples contained 0.5 mM Ca++. Suboptimal stimulus levels were used to allow maximal sensitivity to either inhibition or facilitation by the drug.

lease inhibition by zinc ions (Figure 8), the Con Ainduced phosphorylation remains constant. In the absence of Con A, however, zinc ions alone can effectively induce specific phosphotylation of the 40K protein, so that when the zinc-induced phosphotylation reaches the level produced by Con A treatment, a significant amount of serotonin is released (-50% of the Con A-induced release at 0.2 mM Zn’“). This release is not due to Zn++-induced cell lysis, since 32P-labeled metabolic nucleotides are not released under these conditions (data not shown). In addition to zinc, cobalt and calcium also produce concentration-dependent phosphorylation in the absence of Con A, and in both cases, ionic levels that produce high levels of phosphorylation (>l mM) also produce release (data not shown). Three pertinent aspects of these divalent cation data deserve reiteration at this point: first, platelet Ca’+-dependent release is inhibited by divalent cations, while internal protein phosphorylation is not concomitantly inhibited; second, the effect of Ca++ on protein phosphorylation (shown

Blood Platelet 1019

Protein

Phosphorylation

I

A. 40K

I

PHOSPHORYLATlON

A. 40K

PHOSPHORYLATION

,(-Icf

;: 6I- 20 1

k s

10

l NT.

I

I B. 3H-5HT

I

0

IO

20

I % D$

Figure 6. Effect of D20 Concentration Protein Phosphorylation Labeled indicated samples

on ‘H-5-HT

I

\(+I Cay

7 OHCON

A

I

RELEASE

I

40 Release

I

50 and 40K

platelets were incubated for 5 min in media containing the percentages of Cl20 prior to addition of 50 @g/ml Con A. All contained 0.5 mM Cat+.

in Figures 2 and 3) is not unique to Ca++, but can be produced by other divalent cations that do not support Con A-induced release; and third, when the level of phosphorylation reaches what appears to be a “threshold” level (possibly regardless of the stimulus used to produce the phosphorylation), Ca++-dependent release can be obtained. This suggests the intriguing possibility that phosphorylation of the 40,000 molecular weight protein (or the 20,000 molecular weight protein, or both) might be not only a necessary, but a sufficient condition for release in the presence of Ca++. Since protein phosphorylation is often associated with cyclic nucleotides, and since elevated CAMP levels are known to inhibit activation of platelet secretion (see Haslam et al., 19781, we have investigated the possibility that phosphodiesterase inhibitors might be used to further isolate and characterize various stages of the release process. Figure 9 shows the results obtained by incubating labeled platelets with papaverine before stimulation with Con A. Papaverine inhibits release almost completely with a half-maximal effect, (ICS~) at -2 PM. The effect of papaverine on phosphorylation is also inhibitory, although interestingly, the I&, is 10 fold higher, at approximately 20

Figure 7. Effect of Mg++ 40K Protein Phosphorylation

Concentration

on 3H-5-HT

Release

and

Cells labeled with 32P and 3H-serotonin were treated with 100 pg/ml Con A k 0.5 mM Ca” at the indicated MS+’ concentrations. (-1 + Cat+; (A-A) no added Ca++; (0) no added Ca++ or Con A.

PM. As shown in the upper panel of Figure 9, papaverine also inhibits the shape change reaction, but this is only a partial effect and its is obtained at concentrations which completely inhibit protein phosphorylation (50-100 PM). It is apparent from these results that papaverine exerts effects on shape change, protein phosphorylation and release, but that the effective concentrations needed are very different. This may indicate that each of the cellular processes initiated by Con A is influenced by cyclic nucleotides, but with different thresholds, with the late, calcium-sensitive release step having the greatest apparent sensitivity. [Theophylline and caffeine produce effects identical to those of papaverine, except that the half-maximal inhibitory concentrations are higher (0.5 mM and 1.5 mM, respectively) for inhibition of release.] Discussion It is clear from the present findings that phosphorylation of the 20,000 and 40,000 molecular weight proteins is not directly involved in the calcium-sensitive

Cell 1020

A.40K

PHOSPHORYLATION

1OOuM

PAPAVERINE

li I-

% n ‘2 X I 4

5HT

RELEASE

!5\

II IUT

lF

I 0 0.01 I

I

6

Figure 8. Effect Phosphorylation

I

5 of Zn++

1

on 3H-5-HT

LPAPAVE~INQ,

I

4 pZn*

3 Release

and

I

I 0.1

Figure 9. Effect and 40K Protein 40K

Protein

Labeled cells were treated with 100 gg/ml Con A f 0.5 mM Ca++ at the indicated Zn++ concentrations. Release and 40K protein phosphorylation were assessed as described in Experimental Procedures.

stage of release, nor is it a consequence of that stage. This follows from the observation that an essentially complete phosphorylation reaction can be achieved without any detectable secretion. It therefore seems that the specific phosphorylation occurs antecedent to those phases of release which appear to require the presence of extracellular calcium. This opens the question of which (if any) of the processes initiated by Con A and resulting in secretion actually involves the phosphorylation. The shape change is a reasonable possibility for such a process since first, Con A induces both shape change and phosphorylation in the absence of Ca++; second, both processes are apparently necessary for serotonin release; and third, microtubule inhibitors produce a covariance between shape change and phosphorylation. Furthermore, Adelstein and co-workers (Adelstein and Conti, 1975; Hathaway and Adelstein, 1979) have proposed that the 20K phosphoprotein is a light chain of myosin. As shape change is evidently a contractile process, and since phosphorylation of myosin light chain increases actomyosin ATPase activity (Adelstein et al., 1975), it would be reasonable to expect that phosphorylation of myosin light chain might be a

of Papaverine Phosphorylation

on Shape

\

I 10

I 100

uM Change,

3H-5-HT

Release

Labeled cells were preincubated with the indicated concentrations of papaverine for 5 min prior to addition of Con A. Shape change (A) was assessed as described in Experimental Procedures. The tracings have been offset vertically to allow easier comparison. NT is an untreated sample. whereas all other samples were injected with Con A to give 100 pg/ml. In the lower panel (B). 3H-5-HT release and 40K-specific phosphorylation are shown. The data are for samples treated with 100 pgB/ml Con A in the presence of 0.5 mM Cat+. Papaverine had no effect on release when Ca++ was omitted, although inhibition of 40K phosphorylation was seen under those conditions. (M) 3H-5-HT release: (O-0) 40K specific phosphorylation; (NT) 40K phosphorylation of an untreated sample.

key component of shape change. On the other hand, we have offered evidence that shape change can occur in the absence of the protein phosphorylation. Figure 9 shows that 50 PM papaverine inhibits phosphorylation down to untreated levels. It also shows that the same concentration of papaverine inhibits shape change to a much smaller extent, and therefore we would infer that the phosphorylation is not required for shape change. The experiments with colchicine and deuterium suggest further definition of the stage in the stimulusrelease sequence at which phosphorylation might occur. Colchicine disrupts microtubules and blocks tubule reassembly, while deuterium is thought to stabilize microtubules and facilitate reassembly. If indeed these are the modes of action of these compounds in our experiments, we would suspect that reorganization of cytoskeletal components is required for the phosphorylation reaction. White (1970) has presented

Blood Platelet 1021

Protein

Phosphorylation

ultrastructural evidence that microtubules are rearranged within the platelets upon treatment with some secretagogues (also see White and Gerrard, 1979). The effect of this rearrangement appears to be increased proximity between the dense serotonergic granules and the membrane system into which the contents are purportedly released. If this is the pro%ess affected by colchicine and deuterium, then the phosphorylation might be involved in some manner with the granule-membrane interactions. As a result of the work of Behnke (1970) and Nachmias, Sullender and Asch (19771, it is now considered possible that only one continuous microtubule coils around the granular zone of the platelet interior. Activation of shape change under such a model would not necessarily require disassembly, but rather a “tightening” of the coil, which would bring granules and membrane systems into closer proximity. There is good evidence for an interaction between microtubules and actomyosin filaments during platelet activation (White and Gerrard, 1979) such that if phosphorylation of these proteins (or at least of the 20K protein) represents actomyosin activation, then the effects of colchicine and D20 on phosphorylation might reflect this close association. One of the more intriguing observations of these experiments is the co-variance of the two proteins. Across the many treatments we have used, no instance has been found in which the two species of protein were differentially affected. This suggests that they share a common kinase/phosphatase system and/or the same cellular compartment. Preliminary experiments indicate that these are distinct peptides and that the lesser protein is not a degradative product of the larger band. In this context, it should be mentioned that the 40,000 molecular weight phosphoprotein, although functionally unidentified, has been purified by Lyons and Atherton (19791, who reported that it is a 47,000 molecular weight peptide with an isoelectric point of 6.8, and who rather conclusively showed that the protein is neither tubulin, actin nor the regulatory subunit of CAMP-dependent protein kinase. We are currently investigating similarities between this peptide and a 40,000 molecular weight phosphoprotein of brain (Browning, Bennett and Lynch, 1979a; Browning et al., 1979b). With regard to the role of extracellular calcium in the release of 5-HT, our experiments indicate that this cation exerts its effects via some process other than selective phosphorylation of platelet proteins. The calcium-sensitive stage of release was not accompanied by phosphorylation changes beyond those seen with Con A alone, and blocking this release with divalent cations was similarly without effect on phosphorylation. This leaves the questions first of how calcium does achieve the release of 5-HT, and second whether this conclusion might hold for other secretory systems. Since it is clear that most of the

ultrastructural processes associated with platelet release occur in the absence of calcium (hence in the absence of release), it follows that the calcium-sensitive release component is at a relatively late, possibly penultimate, stage to the release event. Although release of serotonin in the system we have described is sensitive to extracellular calcium, it is possible that shape change and phosphorylation are also calciumdependent processes which can utilize an intracellular calcium pool. It is clear, however, that even if this is so, extracellular calcium is required for serotonin release under the conditions of our assay, and that such an intracellular calcium pool is unable to support release. The generality of our findings to other release systems also remains a subject for future work. The platelet could represent an unusual situation, involving as it does relatively gross morphological transformations prior to the calcium-sensitive component of release. The calcium concentration range across which release is achieved, the relative potency of other cations in inhibiting calcium effects and the actions of microtubule modulators on calcium-dependent release all suggest, however, that the platelet mechanism is comparable to that used by other cell types. Experimental

Procedures

Solutions for Platelet Suspension Medium A-O.1 2 M NaCI: 4.3 x 10m3 M KCI; 8.5 x 10m4 M MgCII; 3 X 10m3 M glucose; 1 X 1 O-’ M HEPES (pH 7.4). Medium BSame as medium A, but including creatine phosphate and creatine phosphokinase. 10 mM and 40 pg/ml, respectively. This medium is prepared from medium A immediately before use. Preparation of Platelets Washed rat platelets were prepared essentially as described by Bennett et al. (1979). Briefly, whole blood was collected into acidcitrate-dextrose and diluted with ‘h vol of medium A. The blood was then spun for 15 min at 200 x g,, in a Beckman microfuge equipped with a variable speed control and a stroboscopic tachometer. Plateletrich plasma was collected, layered over 30% BSA, centrifuged at 225 x g,,, for 5 min. then at 600 X g,.. for 3 min. Platelets were collected from above the BSA-plasma interface. pelleted at 600 x g,.. and resuspended in medium 8 at a cellular concentration of 5-8 x IO’ platelets per ml. Labeling of Platelets Platelet suspensions in medium B were incubated with 32P-orthophosphate at 1 mCi/ml for 30 min at 25’C. ‘H-5-hydroxytryptamine creatinine sulfate was added giving a serotonin concentration of 3 x 1O-’ M and 8.75 &i/ml. The platelet suspension was incubated for another 30 min. then spun at 600 x g,., for 3 min, and the supernatant was discarded. The platelets were then resuspended in medium B to a concentration of 5-8 X IO8 per ml. incubated with the various agents described in the text and assayed for serotonin release and protein phosphorylation. ‘H-5-HT Release Assay Typically, platelet suspensions were incubated with the specified additions in volumes of 100 ~1 for 5 min at 25’C. The incubations were initiated by adding labeled platelets to polyethylene microfuge tubes containing the specified additions, and were terminated by centrifugation at 11,000 X gmel for 15 sec. Aliquots of the supernatant fluid (25 pl) were removed immediately and transferred into liquid

Ceil 1022

scintillation vials. 3 ml ACS (Amersham) were added and the samples were counted in a Packard Tri-Carb scintillation counter. Protein Phosphoryfation Asssy In experiments where concomitant serotonin release was measured, protein phosphorylation was assayed by the following procedure. Immediately after initiating the reactions, each sample was split into 50 ~1 aliquots. After 5 min, one aliquot was assayed for 3H-5-HT release as described above. To the other sample, 50 pl of a 2X concentrated SDS stop solution was added. The 2X stop solution contained: 4.6% SDS, 10% P-mercaptoethanol, 20% glycerol and 0.126 M Tris-HCI (PH 6.8). The samples were heated at 50°C for IO-I 5 min and either frozen at -70°C or loaded directly onto polyacrylamide gels. 7.5-20% polyacrylamide gradient gels were run, stained, destained and prepared for autoradiography (Browning et al., 1979b). The dried gels were exposed to Kodak RP Royal XOmat film and the autoradiographic images were scanned with a Beckman model 25 spectrophotometer equipped with a gel scanning attachment. As described by Ueda, Maeno and Greengard (1973). a reliable method for sample comparison and quantification involves measurement of relative peak heights. At least ten bands in each sample were measured, and each band was expressed as the percentage of total protein-bound “P in that sample. Changes in the “specific” phosphotylation (% of total) therefore refer to effects that are in excess of any general effects on cellular protein phosphorylation. Since individual experiments were carried out on platelets from different animals, and because of slight day-to-day variations in gel electrophoretic patterns, the percentage of total phosphorylation that a particular protein represents under a given set of conditions varies among the experiments presented here. Within a given experiment, however, the 40K band’s percentage of total is reproducible to the nearest percentage point. Shape Change Analysis A modification of the turbidimetric method of Born and Cross (1963) was used to evaluate the morphological changes associated with platelet shape change. A Perkin-Elmer Model 124D Double Beam Spectrophotometer was modified so that 100 ~1 of a Con A solution could be rapidly injected into a siliconized cuvette containing 400 pl of a platelet solution. Changes in light transmittance, measured on the absorbance scale, were evaluated at 600 nm relative to an untreated platelet solution. Both test and reference platelet solutions remained unstirred and were at a concentration of l-2 x 10’ cells per ml. The relatively low cell concentration allows for a more sensitive photometric aggregation

response and reduces (Born, 1970).

the likelihood

of contact-induced

Preparation for Scanning Electron Microscopy Platelets in medium A were either left untreated or were incubated at 25°C for 2 min with 50 pg/mi Con A. whereupon glutaraldehyde (0.4 M in medium A) was added to give a final concentration of 0.1 M. Afler 30 min at 4°C the cell suspensions were allowed to settle for 90 min onto glass coverslips which had been treated with 5 mM polyI-lysine (molecular weight -3000). After the coverslips were washed 3 times with 0.1 M potassium phosphate (pH 7.21, the solvent was exchanged for serially increasing concentrations of ethanol in H?O. then freon 113 in ethanol. Critical point drying in halocarbon 13 was followed by gold coating using a Hummer II sputterer (Technics). The samples were examined in a Hitachi S-500 scanning electron microcope. Reagents “P-orthophosphoric acid was purchased from ICN and ‘H-5-hydroxytryptamine creatinine sulfate from New England Nuclear. Con A, BSA (30% sterile solution, B grade) and HEPES were from Calbiochem. Phosphocreatine. poly-I-iysine. coichiclne and papaverine, along with the enzymes phosphoryiase B. creatine phosphokinase, giyceraldehyde-3-phosphate dehydrogenase (all from rabbit muscle) and cytochrome C (horse heart) were purchased from Sigma. Recrystallized acryiamide was from Scientific Chemical Co. (Huntington

Beach, California). N.N.methylene from BioRad. Zinc Acetate was ExP Water Gap, Pennsylvania). D20 was tilled over EGTA. All other reagents

bisacryiamide and TEMED were grade from Heico, Inc. (Delaware purchased from ICN. then rediswere analytical grade or better.

Acknowledgments This research was supported by a grant to W. F. 6. from the Muscular Dystrophy Association. The manuscript was typed by Darlene Thomp son. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

August

7. 1979

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Adelstein. R. S.. Daniel, J. L.. Conti, M. A. and Anderson, W., Jr. (1975). Platelet myosin phosphorylation: studies on the kinase, substrate, and effect of phosphorylation. In Proteins of Contractile Systerns, E. N. A. Biro, ed. (Amsterdam: Elsevier/North Holland). pp. 177-l 66. Behnke. 0. (1970). Microtubules Exp. Pathol. 9, l-92.

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Belville, J. S.. Bennett, W. F. and Lynch, G. (1979). A method for investigation of the role of calcium in shape change, aggregation and serotonin release by rat platelets. J. Physiol.. in press. Bennett, W. F.. Belviile. J. and Lynch, G. (1979). Calcium dependent serotonin release from blood platelets: a model system for neurosecretion. Neuroscience 4, 1203-I 209. Borisy, G. G. and Taylor, E. W. (1967). The mechanism colchicine. Binding of colchicine-3H to cellular protein. 34, 525-533. Born, G. V. R. (1970). Observations on the change platelets brought about by adenosine diphosphate. 467-511. Born, G. V. R. and Cross, M. J. (1963). platelets. J. Physiol. 168, 178-l 95.

of action of J. Cell Biol.

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of blood

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Dunwiddie, T. V. and Lynch, G. (1979). The relationship between extracellular calcium concentrations and the induction of hippocampal long-term potentiation. Brain Res. 169, 103-l 10. Gillespie, E. (1975). tion. Ann. NY Acad.

AMP, calcium,

and secre-

Greengard. P. (1979). Cyclic nucleotides. phosphorylated and the nervous system. Fed. Proc. 38, 2208-2217.

proteins,

Hasiam, R. J. phorylation of constituents. Biophys. Res.

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