Naunyn-Schmiedeberg's Arch. Pharmacol. 310, 8 7 - 9 2 (1979) Naunyn-Schmiedeberg's

Archivesof Pharmacology �9 by Springer-Verlag 1979

Shape Change of Blood Platelets Brought About by Myelin Basic Protein and Other Basic Polypeptides

Andreas Laubscher 1, Alfred Pletscher 1, Conrad G. Honegger 1, and John G. Richards z

1 Department of Research, Kantonsspital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland 2 Research Division, F. Hoffmann-La Roche & Co. Ltd. CH-4002 Basel, Switzerland

Summary. Basic proteins and polypeptides (BPP) such as myelin basic protein (MBP), polyornithine (M.W. 40,000), polylysine and protamine, which are known to cause neuronal depolarization in the central nervous system, induced a shape change reaction in blood platelets of various species, including man. This reaction was not accompanied by platelet aggregation or marked alterations of 5-hydroxytryptamine release. Cyclic nucleotide levels were also unchanged. The shape change induced by polyornithine was in- hibited by heparin but not by antagonists of 5HT, catecholamines or 7-aminobutyric acid, substances which are known to have no effect on the MBP-induced neuronal depolarization. Other basic substances, e.g. low molecular weight polyornithine (M.W. 4,000), cytochrome c, spermine and spermidine, did not induce either platelet shape change or (as shown before) neuronal depolarization.

It is concluded, that 1) the shape change reaction of platelets seems to be a sensitive and simple means of detecting those BPP which induce functional changes in mammalian cells and 2) the use of platelets as models for neurons can be extended to include the action of BPP on the plasma membranes.

Key words: Myelin basic protein - Shape change - Blood platelets - Basic polypeptides - Depolar- ization.

extrude pseudopods and blebs (shape change reaction). This morphological transformation leads to an increase in light absorption of the platelet suspensions, which can be used to measure the shape change (Born et al., 1978).

Previous experiments indicated that the shape change reaction induced by 5HT was brought about by interaction of the amine with a specific receptor at the platelet surface. This receptor was affected by virtually the same agonists and antagonists as the neuronal 5HT- receptors in certain areas of the central nervous system (CNS). Also, various 5HT-antagonists showed a si- milar order of potency in both types of receptors. The 5HT receptors of the platelets have therefore been proposed as models for those of 5-hydroxytryptaminer- gic neurons (Graf and Pletscher, 1979; Laubscher and Pletscher, 1979).

In preliminary studies myelin basic protein (MBP) from nervous tissue induced a marked shape change reaction in blood platelets (Laubscher et al., 1979). MBP, like some other basic proteins and poly- peptides (BPP) also caused depolarization of neuronal cells in the CNS (Honegger et al., 1977; Gfihwiler and Honegger, 1979). The present experiments show that, in human platelets, the shape change inducing effect is limited to those BPP which also caused neuronal depolarization and that the mechanisms of BPP and 5HT in inducing the shape change reaction were different.

Introduction

When human blood platelets are incubated in plasma or artificial media and stirred with a magnetic stirrer, their form remains discoid as in the circulating blood. On addition of various agents, e.g. 5-hydroxytrypt- amine (5HT), the platelets assume a spheroid shape and

Send offprint requests" to A. Pletscher at the above address

Methods

Materials. MBP (average M.W. 18,300, i.p. > 10.6) was prepared from fresh bovine spinal cord using a slight modification i of the standard method (Deibler et al., 1972; Dunkley and Carnegie, 1974). Polyornithine (M.W. 40,000 and 4,000), polylysine (M.W. 13,000),

1 Omission of dialysis step after CM-Sephadex elution; Sephadex G-75 gel filtration with 0.1 M ammonium acetate pH 4.9 instead of 0.01 M HC1

0028-1298/79/0310/0087/$ 01.20

88 Naunyn-Schmiedeberg's Arch. Pharmacol. 310 (1979)

protamine (M.W. 5,600), spermine (M.W. 202) and spermidine (M.W. 145) were obtained from Sigma St. Louis, Mo., USA. In addition the following substances were used: Cytochromec (M.W. 13,000), ouabain, 5HT-creatinine sulfate (FlukaAG, Buchs, Switzerland), heparin and the receptor blockers spiroperidol, halope- ridol, mianserine, metergoline, phenoxybenzamine, propranolol, bicuculline (F. Hoffmann-La Roche & Co. Ltd., Basel, Switzerland), theophylline (Knoll A.G., Ludwigshafen, FRG). Dextran-10 was purchased from Pharmacia Uppsala, Sweden and the (1,2 3H)-5HT creatinine sulfate (specific radioactivity 26Ci/mmol) from New England Nuclear Corporation.

Isolation of Platelets. Human blood was obtained from healthy donors by puncturing a cubital vein. Guinea pigs (Himalayan spotted, female, 600- 800 g) and Swiss rabbits (male, 2 - 3 kg) were bled under light ether anesthesia through a polyethylene cannula inserted in a carotid artery. The whole blood was collected in a plastic vial and mixed with 1/10Vol. 3.8 % trisodium citrate. 2H20. In rats (Ffillinsdorf breed of Wistar origin, male, 150-200g) blood was removed from the heart into a plastic syringe and anticoagulated in the same way. Platelet-rich plasma (PRP) was prepared by centri- fugation of the whole blood at 400 g for 10 min in plastic tubes. The platelets were isolated by means of dextran gradients, as described earlier (Graf et al., 1979), the PRP being layered on a gradient consisting of 4 ml 20 % dextran (bottom layer) and 7 ml 10 % dextran (upper layer) and centrifuged for 10 min. The platelets which banded between the two layers were removed and diluted with Tris-buffer 2 to give a final platelet concentration of 105 gl 1 (shape change) and approximately 5 x 104gl - I (5HT-release). The protein content of these suspensions was about 0.03 %. In other experiments PRP was diluted directly with Tris-buffer to give the same platelet concen- tration.

Ultramorphology. Suspensions of platelets in Tris-buffer were stirred and incubated with either 10 -6 M 5HT for I min, ]0 -6 M MBP for 3 rain, 10-7M polyornithine for 3 rain or solvent (H20) alone for 3 min (controls). They were then prefixed in an equal volume of 0.2 % glutaraldehyde in 0.1 M cacodylate buffer, pH7.3. After centri- fugation (1800g, 12rain), the pellets were left in contact with 3 ~ glutaraldehyde in the same buffer for 60min at room temperature and then cut into 1 mm cubes and fixed for a further 90 min under agitation. The pellets were stored in 0.1 M cacodylate containing 7 sucrose for 18- 24 h and then postfixed in 2 % osmium tetroxide in the same buffer for 1 h. These procedures were carried out at 4~ After dehydration in graded alcohol and embedding in Epon, ultra thin sections were prepared and stained with uranyl acetate and lead citrate.

Light Absorption. Platelet samples of 500 gl were preincubated either in PRP or Tris-buffer for 60 min at 37 ~ C. After addition of 5HT, BPP or polyamines the shape change was measured on an Elvi 840 aggregometer and recorded on a Rikadenki B361 recorder while the platelets were stirred at 1000 rpm with a magnetic stirrer. If there was a change in light absorption, its maximum, which was used in the calculations, usually occurred after 1 - 3 min. All the substances were added in a volume of 2.5-7.5 gl H20, ouabain, heparin and the receptor blockers (see materials) 1 min before the basic proteins. For details see Graf et al. (1979).

5HT-Release. Platelets were preincubated in plasma with 10 7 M 3H- 5HT for 30min at 37~ and isolated with a dextran gradient. Aliquots of 5 x 107 platelets were resuspended in 1 ml (final volume) Tris-buffer and incubated for 60 min at 37~ with 10-7M polyor- nithine or 10- 6 M MBP (final concentration). The BPP were added in a volume of 50 gl H20. Platelets incubated with H20 alone served as

2 Tris-buffer: 8.21 g NaC1 1-1, 0.57 g KC11-1, 1.01 g glucose 1-1, 0.93g tris (hydroxymethyl)-aminomethane 1-1, 3.80g trisodium citrate 2H20 1-1

controls. Thereafter, the platelets were separated on a millipore filter and their radioactivity counted as indicated earlier (Grafet al., 1979). 5HT release was calculated as the difference between the 3H-5HT content of platelets incubated with BPP and the 3H-5HT content of controls.

Cyclic Nueleotides. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine Y,5'-cyclic monophosphate (cyclic GMP) were measured in intact platelets. These were incubated in Tris-buffer in the same way as those used in the shape change experiments, except that theophylline was added to the incubation medium (final volume 1.2 ml) to give a final concentration of 10-3 M, and that the platelet suspension was not stirred. Three minutes after the addition of polyornithine or 5HT the reaction was stopped by removing 1 ml of the platelet suspension into 1 ml of perchloric acid 3 %. Cyclic nucleotides were extracted according to the assay protocol from Collaborative Research Inc., Waltham, Ma., USA, and determined by radioimmunoassay using the kit from Becton Dickinson Immunodiagnostics, Orangeburg, N.Y., USA.

Calculations. Statistical analysis of the results was performed using Student's t-test or with the aid of the formula for comparison of two proportions considering the continuity correction (Colton, 1974).

Results

Ultramorphology

Transmission electron microscopy of control platelets (Fig. l a) revealed a typical discoid shape, a smooth outline of the plasma membrane with occasional pits and craters and randomly distributed cytoplasmic organelles, such as the frequent e-granules and rather rare 5HT-granules. In contrast, the majority of 5HT- treated platelets had a spherical, often irregular form with extrusion of pseudopods or blebs (Fig. l b). In MBP- and polyornithine-treated platelets these differ- ences from controls were even more pronounced (Fig. l c, d). The subcellular organelles and canaliculi showed no marked changes from controls (Fig. 1 b - d) and platelet aggregation was virtually absent.

Light Absorption

Various BPP caused a marked increase in light absorp- tion in human platelets incubated in Tris-buffer, in- dicating the occurence of a shape change reaction. With the concentration causing a maximal effect, i.e. MBP (10-6M), polyornithine (10-TM) and polylysine 13,000 (3x 10-6M), the shape change reached its maximum after 2 - 3 min. Subsequently, the light ab- sorption decreased but was still elevated after 60 min. With 5HT the maximum shape change occurred within about I min and was completely reversed after 60 min (Fig. 2). The maxima obtained with MBP, polyor- nithine and polylysine were higher than that with 5HT (Fig. 2, Table 1).

The shape change reaction caused by these BPP, like that induced by 5HT, became more pronounced with rising concentrations of the substances, the ECso

A. Laubscher et al. : Shape Change of P/atelets by Basic Peptides 89

Fig. 1 a--d. Transmission electron micrographs of human blood platelets isolated on a dextran gradient, re-suspended in Tris-buffer and exposed to various compounds. Note the discoid shape of control platelets (a) and the shape change and pseudopods induced by 5-hydroxytryptamine (b), myelin basic protein (e) and polyornithine (d) Magnification: 9,450; bar = 1 gm

200 - MBP - Poly- Orni thin

1 0 0 -

5 0

c

,11 ,

5 0 -

i - II , ' ~ 2 ~ ,s 60 o i ~ 1 ~s 6'o M i n u t e s

Fig. 2. Shape change induced by 10-6 M myelin basic protein (MBP), 10-7 M polyornithine 40,000, 10 -6 M 5-hydroxytryptamine (5HT) and 3 x 10 6 M polylysine in human platelets. Typical experiments. The ordinate indicates the shape change in percent of that caused by 5HT ( = 100)

, ,11

Table 1. Shape change reaction induced by various substances in human platelets in protein-poor medium. ECs0 means the con- centration of a substance causing half the maximal effect. The maximal effect is indicated in percent of that obtained in each experiment with 5HT ( = 100). The figures are averages with SEM of 3 - 4 experiments each. PRP = platelet rich plasma; n.e. = no effect

Substance EC5 o (moi/1) Maximal effect

5-hydroxytrypt- 1.4 i-_ 0.1 x 10 -7 100 amine

Myelin basic protein 4.5 +_ 1.0 x 10-v* 165 • 7 id. in PRP 10 .5 n.e.

Polyornithine 40,000 1.8 • 0.3 x 10 -8* 194 • 18"* id. in PRP 8.3 • 3 . 9x10 -7 90 • 8

Polyornithine 4,000 3 x 10 -4 n.e. Polylysine 4.2 • 2 . 2 x 1 0 - 7 . * 134 • 9** id. in PRP 1.5 • 0.5 x 10 -5 79 • 21 Protamine 3.6 • 1 .0• .6* 79 _+ 13 Cytochrome c 10- 5 n.e. Spermine 10 .4 n.e. Sperm• 10 . 4 n.e.

* P < 0.01 versus 5HT ** P < 0.01 versus PRP

90 Naunyn-Schmiedeberg's Arch. Pharmacol. 310 (1979)

(concentration causing 50% of the maximal effect) being lowest with polyornithine (Table 1).

In platelets incubated in PRP the ECso ofpolylysine was about 35 times higher than in protein-poor medium and MBP in a concentration as high as 10-5 M did not induce a shape change reaction in PRP. Polyorni~hine also showed a higher ECso in PRP than in Tris-buffer, but the difference was not statistically significant (P > 0.05) due to a large standard error in PRP. In addition the maxima of the shape change reaction induced by polyornithine and polylysine were lower in PRP (Table 1).

Other Basic Substances

Among the other basic substances tested, only pro- tamine caused a shape change reaction. Its average maximum amounted to about 80 % of that induced by 5HT. In contrast, low molecular weight polyornithine (M.W. about 4,000), cytochrome c, spermine and sperm• had no effect even in relatively high con- centrations (Table 1).

Inhibitors of Shape Change

A variety of pharmacological agents in concentrations a b o v e 10-SM, e.g. 5HT-antagonists (metergoline, mianserine), neuroleptics (spiroperidol, haloperidol), adrenergic e- and B-blockers (phenoxybenzamine, pro- pranolol), the GABA-antagonist bicuculline and oua- bain did not inhibit the shape change caused by 10- 7 M polyornithine or 10 -6M MBP in human platelets. On the other hand, heparin (10 gg/ml) completely blocked the shape change reaction due to 10- 7 M polyornithine. The ICso (concentration causing 50 % inhibition of the maximal shape change induced by 1 0 - 7 M polyorni- thine) of heparin was 4.8 +_ 2.4 gg/ml (about 5 x 10- 7 M; 3 experiments).

Other Species

In other species, polyornithine also induced a shape change reaction in platelets isolated with a dextran gradient and incubated in protein-poor medium. The potency of the polypeptide in rats was about the same as that in man and significantly (P < 0.01) higher than that in rabbits. The ECso in guinea pigs showed large individual variations and was therefore not signif- icantly (P > 0.05) different from that in the other species (Table 2).

Release of 5HT

In human platelets prelabelled with 3H-5HT, MBP (10 -6 M) caused no release of the amine (0 + 1%;

4 experiments) compared to platelets incubated with the solvent alone, and polyornthine (10-7 M) had only a slight releasing effect (17 +_ 3%; 6 experiments; P < 0.01).

Cyclic Nucleotides

Polyornthine in a concentration of 10-VM did not increase the levels of either cyclic AMP or GMP in human platelets. Although 5HT showed no effect on the levels of cyclic AMP, it caused a marked rise in cyclic GMP which was enhanced with increasing con- centrations of the amine (Table 3).

Discussion

Our experimental technique allows a relatively sensitive measurement of the shape change reaction alone, in the absence of other major platelet alterations. In fact, the shape change caused by BPP was not accompanied by marked platelet aggregation, or release of 5HT, con- firming and extending our previous electronmicros- copic findings (Laubscher et al., 1979). However, the effects of BPP were more potent in platelets incubated in protein-poor buffer than in those suspended in PRP, possibly due to interaction of the BPP with plasma proteins in PRP.

It is known that BPP induce various changes in mammalian cells. For instance, basic polypeptides like polylysine caused platelet aggregation. Furthermore, BPP were taken up by pinocytosis and activated the

Table 2. Polyornithine induced shape change in platelets of various species. ECs0 means the concentration in mol/1 of polyornithine causing half the maximal effect. The figures are averages with SEM of 3 experiments

Species ECso

Man 1.8 • 0.3x10 -8 Guinea pig 3.1 • 1.2x 10 -v Rabbit 3.8 • 1.0x 10 -v Rat 1.9 _+ 0.4x 10 8

Table3. Action ofpolyornithine and 5-hydroxytryptamine (5HT) on cyclic nucleotides of human platelets. The values are averages with SEM of 3 experiments and are expressed in pmols/109 platelets

Substance Cyclic AMP Cyclic GMP

Controls 19.8 + 0.9 2.3 • 0.2 5HT10 6M 17.7 + 1.0 9.0 • 1.0" 5HT 10 -5 M 16.9 + 2.1 30.6 • 3.4* Polyornithine 10 .7 M 17.7 + 0.9 2.1 • 0.2

* P < 0.01 versus controls

A. Laubscher et al. : Shape Change of Platelets by Basic Peptides 91

pinocytotic uptake of other proteins by mammalian cells, including neurons (Ryser, 1968; Gr6ttum, 1969; Jenkins et al., 1971; White, 1972; Massini et al., 1974; Hadley and Trachtenberg, 1978; Taketomi and Kuramato, 1978). The present experiments show that BPP can also induce a shape change reaction in platelets. Thereby, not all the BPP are active. Their activity probably depends on their molecular size and conformation; as can be seen, low molecular weight polyornithine is much less active than the high mole- cular weight form, and cytochrome c, a non-random coil basic protein, is inactive. A dependence of the effects of BPP on their molecular weight has also been reported in other experimental systems (Ryser, 1968; Taketomi and Kuramato, 1978).

The results suggest that the shape change reaction induced by BPP is not mediated by the 5HT receptors of the plasma membrane; the time course of the shape change caused by BPP differs from that due to 5HT, and the maximal effects of BPP are higher. Furthermore, strong inhibitors of the 5HT-induced shape change (e. g. metergoline, spiroperidol) (Graf et al., 1979; Laubscher and Pletscher, 1979) are not inhibitors of the action of BPP, and heparin, an inhibitor of the shape change caused by polyornithine is not an inhibitor of that due to 5HT (unpublished results). In addition, BPP do not seem to act via release of 5HT into the medium, since MBP in concentrations which induce shape change have no effect on the 5HT content of the platelets. Finally, polyornithine, unlike 5HT, does not cause an increase in the level of cyclic GMP in platelets. However, the role of this nucleotide in the shape change reaction remains to be clarified (Agarwal and Steiner, 1976; Haslam et al., 1978).

The exact mechanism of BPP in inducing shape change reactions is not fully clear. BPP whose platelet aggregating effect is decreased by the acid mucopoly- saccharide heparin (Taketomi and Kuramato, 1978) are thought to attach themselves to the plasma mem- branes and so decrease their negative surface charge. A similar interaction between BPP and the membrane might also initiate the shape change reaction. This idea is supported by our finding that heparin decreases the shape change induced by polyornithine. However, the succession of events leading to the transition of the platelets from a discoid to a spheroid shape remains to be elucidated.

As can be seen, the shape change reaction in platelets is induced only by those BPP, e.g. MBP, polyornithine 40,000, polylysine and protamine, which have been reported to cause neuronal depolarization. Other basic substances, such as cytochrome c, spermine and spermidine, have no effect on either type of cell. Moreover, the shape change due to MBP is not antagonized by haloperidol, propranolol, phenoxybenz-

amine or bicuculline, substances which also have no effect on MBP-induced neuronal depolarization (Hon- egger et al., 1977; G/ihwiler and Honegger, 1979; unpublished). These results suggest that platelets could also be used as models for the action of BPP on neurons. The use ofplatelets might, for instance, help to elucidate the mode of action of substances causing neuronal depolarization. As an example, ouabain, an inhibitor of Na+-K+-ATPase present in neurons and platelets, caused neuronal depolarization (Gfihwiler and Honegger, 1979) but does not induce a shape change reaction in platelets. This indicates that the shape change induced by BPP can hardly be due to an inhibition of Na+-K+-ATPase and that the mech- anisms of the depolarizing effects of ouabain and MBP in neurons are probably different. Furthermore, the shape change reaction of platelets might be used as a simple method for identifying those BPP which cause neuronal depolarization and to screen for substances inhibiting the membrane effects of BPP. Of interest in this connection is the speculation that some of the neurological manifestations in the course of de- myelinating disorders, e. g. multiple sclerosis, are due to a direct action of MBP on neuronal membranes (Gfih- wiler and Honegger, 1979).

Acknowledgements. We thank Miss Brigitte Gieux and Miss Martha Handschin for skilful technical assistance and Dr. Martin Oberholzer for statistical advice.

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Received June 6/Accepted September 13, 1979