~THYFKM33OSIS RESEARCH 13; 1017-1029 @Pergamon Press Ltd.1978. Printed
in Great Britain 0049-3a~P~78/1~01-1017 $02.00,!0
BINDINGOF PYRIDOXALPHOSPHATE TO HUMAN PLATELETS: ITS EFFECT ON PLATELET FUNCTION Kuchibhotla Subbarao+, Vijay V. Kakkar*, and Pankaj Ganguly Oepartment of Hematology, St. Jude Children's Research Hospital, Memphis, Tenn. USA and *Thrombosis Unit, King's College Hospital London, UK
(Received 2.6.1978; in revised form 17.9.1978. Accepted by Editor A.P. Fletcher)
ABSTRACT Pyridoxal phosphate, the coenzyme form of vitamin B6, exerted a concentration-dependent inhibition of epinephrine-induced platelet aggregation, -in vitro. A dose-dependent inhibition of [2-14C]-5-hydroxytryptamine release from platelets was also observed. Likewise, the coenzyme also blocked the ADP, thrombin and collagen-induced platelet aggregation and release. The fluorographs of platelets treated with pyridoxal phosphate and sodium borotritide indicated three predominently labeled platelet protein components of Mr = 170,000, 150,000 and 90,000. One of these polypeptldes was characterized as platelet glycoprotein I. The amount of tritium that was incorporated into these proteins showed a dependence on the concentration of the coenzyme and the label. The inhibitory effect of pyridoxal phosphate appears to be due to its specific binding to these platelet-surface proteins which may be involved in various platelet functions.
INTRODUCTION Pyridoxal 5'-phosphate (PALP), the biologically active coenzyme form of the vitamin B6 compounds, plays an important role in the metabolism of carbohydrates, fats and other proteins (1).
In addition to its normal
function as a cofactor, PALP also serves as a strong inhibitor of a variety of enzymes (2-5).
It interacts readily with certain protein
'To whom reprint requests should be sent.
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components of the red-cell membrane and thereby acts as a specific and relatively potent inhibitor of the anion-transporting system (6-9). Although the coenzyme represents more than 25% of all vitamin 8s compounds in human plasma (lO,ll), the effect(s) of PALP on platelet function is unknown.
Our earlier findings showed that PALP is a strong inhibitor
of ADP, thrombin, and collagen-induced platelet aggregation as well as blood coagulation both -in vitro and -in vivo (12,13). In this paper we provide evidence that the coentyme also blocks epinephrine-induced platelet aggregation and interacts with specific membrane components of the platelet surface that appear to play an important role in the physiological functions of human platelets. METHODS Epinephrine and PALP were obtained from'Sigma Chemical Co., London UK.
[2-"+C]-5'-Hydroxytryptamine (specific activity 57 mCi/mnol) and
tritiated sodium borohydride (NaBaH4) with a specific activity of 5 Ci/mmol were purchased from Amersham/Searle, Arlington Heights, Ill. Epinephrine and PALP solutions were prepared each day in 0.85% saline and 0.05M phosphate buffer-saline (pH 7.5), respectively.
The stock
solutions of both were kept in the dark at 4" throughout their use. Venous blood was obtained from normal healthy volunteers who had not taken any medication during the two weeks preceding blood collection. One volume of 4% sodium citrate was added to every nine volumes of blood, and the samples were then centrifuged at room temperature for 10 min at 300 x g.
The platelet count of isolated platelet rich-plasma (PRP)
was adjusted to 3OO,OOO/ul with autologous platelet-poor plasma. Platelet aggregation and serotonin release were measured according to a previously described method with epinephrine (0.5-2 PM) used as a stimulating agent (14).
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Two volumes of a washing solution containing 1% ammonium oxalate, 0.1 M KCl, 0.1% ethylene diamine tetraacetate (EDTA) and glucose, pH 7.5 was added to one volume of the PRP.
Platelets were then collected
by centrifugation of PRP at 800 x g for 15 min at room temperature. This washing procedure was repeated once and the platelet pellet was finally resuspended in calcium-free Tyrode buffer containing 5 mM glucose, pH 7.5.
One ml of washed platelet suspension, containing
2-3 x log cells, was first treated with 1OU of neuraminidase (Behring Diagnostics, Somerville, N.J.)
for 30 min at 37", then with 5U of
galactose oxidase (Worthington Biochemicals, Freehold, N.J.) min, and finally with 0.1 mCi of NaB3H, for 15 min (15).
for 60
These platelets
were then solubilized in Laemnli buffer and the platelet proteins were separated by electrophoresis (16). Washed platelets (4 x lo8 cells) in 400 ~1 of calcium free Tyrode (pH 7.5) with 5 mM glucose were incubated for 15 min at 37" with varying concentrations of PALP dissolved in phosphate buffer-saline (pH 7.5).
After centrifugation at 800 x g for 10 min, the platelet
pellet was resuspended in 200 ~1 of Tyrode (pH 8.5) and incubated with 0.8 mCi (6 ug) of NaBsH, at 4" for 30 min.
The platelets were solubilized
with 200 ~1 of Laemnli buffer containing 5% sodium dodecyl sulfate and 1% 6-mercaptoethanol, and were then heated in a boiling water bath for 5 min (16).
Platelet proteins (about 50 pg/sample) were separated
by electrophoresis in 7.5% polyacrylamide slab gels (16).
Following
electrophoresis, the gels were stained with Coomassie blue (for protein identification) and impregnated with 2,5_diphenyloxazole for fluorography (17). RESULTS We previously showed that PALP is an effective inhibitor of ADP,
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thrombin and collagen-induced platelet aggregation as well as blood coagulation (12-13).
In this report, we show that PALP also inhibits
epinephrine-induced platelet aggregation (Fig. 1).
At a concentration
2 0.3 mM, PALP inhibited the "secondary" aggregation of platelets by epinephrine.
This inhibitory effect of PALP was dependent on the
concentration of the inhibitor and the preincubation time.
The release
of [2-14C]5-HT from platelets treated with epinephrine was also blocked
‘;;
80
-o\ 70 60 50
I
I min
i
FIG. 1 Inhibition by PALP of platelet aggregation induced by epinephrine (1 I.IM). To each 0.4 ml aliquot of platelet suspension (PRP), 0.1 ml of PALP (at a given concentration) was added and the samples were incubated for 15 min at 37°C. In the control samples, PALP was substituted with phosphate buffer-saline. The aggregation response of platelets to epinephrine induced platelet aggregation in the absence [l] and in the presence of 0.10 mM [2] and 0.3 mM PALP [3] is shown. The abcissa represents time.
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by PALP in a concentration-dependent manner (Table 1).
The inhibitory
effect of PALP on the aggregation and release induced by different agents could be due to binding of the coenzyme to specific surface components of platelets (12-13).
To identify such membrane proteins,
we fixed the PALP in a irreversible form to platelets by reduction with NaB3H,+, then solubilited the cells, separated the platelet proteins by electrophoresis, and determined by densitometry of the fluorographs, the extent of radioactivity associated with platelet proteins. TABLE 1 Effect of PALP Concentration on the Release of [2-14C]5-HT from Platelets Aggregating Agent
PALP Concentration (mM)
57 + 5.2 45 38 20 12
0 0.16 0.32 0.60 1.30
Epinephrine, 1 uM
5-HT Released (%)
Platelets labeled with [2-14C]5-hydroxytryptamine were mixed with varying concentrations of PALP and incubated for 15 min at 37". The release of [2-14C]5-HT from platelets was then determined after epinephrine was added. The results reported are the mean for five control and four PALP-treated platelets f 1 SE. The electrophoretic separation of platelet proteins yielded a large number of polypeptides with molecular weights that usually ranged from 10,000 to over 250,000 (Fig. 2. I).
The distribution of these proteins
remained unchanged when platelets were pretreated with PALP.
The
fluorographs of PALP and NaB3H,+ treated platelets showed about ten bands and three of which (+) were more intensely labeled.
These components
had apparent molecular weights (M,) of 170,000, 150,000 and 90,000 2 5,000 (Fig. 2. II).
After platelet surface glycoproteins were specifically
labeled by galactose oxidase and NaB3H4, the fluorograph showed an intensely labeled band (Fig. 2. III),
(&P-I of M, = 155,000
+_ 5,000
(15,18).
corresponding to a glycoprotein The location of a heavily
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labeled protein band observed in PALP-treated platelets (Fig. 2. II) was the same as that of GP-I
(Fig. 2. III).
This suggests that among
other components of platelets, PALP binds readily to plate let GP-I.
1
II
FIG. 2 Slab gel electrophoresis patterns of PALP-treated platelet proteins stained with Coomassie blue (I); and fluorographs of PALP/NaB3H,-treated platelets (II) and of NaB3Hk-treated platelets after oxidation with The patterns for several platelet samples galactose oxidase (III). prepared on different days, remained the same, but the position of platelet actin (A) varied slightly in relation to the length of the slab gel.
The densitometric scan of fluorographs of platelets treated with a constant PALP and varying amounts of NaB3H4 (0.3 to 3 pg) is shown in Fig. 3.
The labeling pattern again suggests that PALP interacted
strongly with three platelet proteins.
The amount of tritium incorporated
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A
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FIG.
3
Densitometric scans of fluorographs of platelets treated with constant PALP (1.6 mM) and different amounts of NaB3H,+: 0.3 ug (A): 1.5 Pg (B); 3 ug (C). The arrows represent the protein bands that are extensively labeled.
into these PALP-linked platelet proteins progressively increased with increasing amounts of NaB3H4 or PALP (Fig. 4).
When incubated with
PALP at -< 0.16 mM, platelets were not labeled in significant amounts (Fig. 4).
The fact that NaB3H4 alone failed to introduce a significant
amount of label into the platelet proteins, ruled out the possibility
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that platelets contained an appreciable amount of intraprotein Schiff bases.
The heavily labeled region at the bottom of the fluorograph
of platelets treated with PALP and NaB3H,+ or NaB3H, alone (Figs. 2 and 4) may be due to platelet lipids (19).
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n MIGRATION
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FIG. 4 Densitometric scans of fluorographs of platelets treated with 3 ug of NaBaH,,and different concentrations of PALP: 0 mM (A), 0.16 mM (B), 0.40 mM (C) and 1.6 mM (II).
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DISCUSSION The aggregation of blood platelets and clotting of fibrinogen are the two major hemostatic reactions that occur spontaneously at the site of vascular injury.
We previously reported that PALP is a
strong inhibitor of platelet function and blood coagulation (12,13). One of the major goals of this study was to determine if this inhibition was due to the binding of PALP to specific proteins on the platelet surface.
Previous studies had demonstrated the binding of PALP to
surface proteins of other cells (19,20).
We, indeed, found that PALP
interacted with about ten platelet proteins, (Fig. 2. II) of which three polypeptides with an apparent M, = 170,000, 160,000 and 90,000 were intensely labeled.
One of these heavily labeled protein components
with M, = 150,000 appears to be GP-I which has been proposed as a component part of thrombin receptor site in human platelets (21-23). The binding of PALP to GP-I may be an important finding because the coenzyme also inhibits thrombin-induced platelet aggregation and serotonin release (12,13).
It is of interest that only three out of several
platelet proteins had a remarkable affinity for PALP; this interaction appears to be specific because the inhibitory effect of PALP was evident even when the coenzyme (1.5 to 2 mM) was incubated with whole blood instead of PRP. Pyridoxal phosphate has been shown to be a potent inhibitor of the anion-transport system of red blood cells (6-9).
This transport
process was suggested to occur at a positively charged region on the membrane and presumed to involve an anion-binding protein of Mr = 95,000 (6-9).
The coenzyme has been shown to bind with this protein and block
the anion transport across the red-cell membrane (9).
In this
paper,
we have shown that PALP interacts strongly with a platelet protein component (M, = 90,000) which may be involved in anion transport.
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Others have shown that the release of serotonin by thrombin and calcium ionophore A23187 is blocked by PALP, presumably through an inhibition of the anion-transporting mechanism of human platelets (24).
Because
the coenzyme blocks the aggregation of platelets by a variety of agents, it is proposed that the interaction of PALP with platelet surface might inhibit a common step(s) that is crucial for the normal hemostatic function of human platelets. Since the coenzyrne is a phosphomonoester, it may not readily pass through biological membranes and consequently the Schiff base formation may be restricted to molecules on the outer membrane surface.
Indeed,
we found that the label was not associated with platelet actin, which is known to be located inside the platelet surface (Fig. 2. II).
The
labeling procedure is simple, rapid and does not cause any damage to the cells.
This method of labeling platelets with NaB3H,+ in
the presence of PALP might be applicable in studies to understand the topographic distribution of protein components on the platelet surface. ACKNOWLEDGMENTS This study was supported by project grant HL 16720, CORE grant CA 21763 from NIH (USA) and G973/756 from MRC (UK).
We thank Mr. H.R.
Bradford for his excellent technical assistance. REFERENCES 1.
Rodwell, V.W.
Metabolism of vitamin Bg.
D.M. Greenburg, editor. edition. 2.
In Metabolic Pathways.
Academic Press Inc.,
New York, 3rd
4: 411, 1970.
Anderson, B.M., Anderson, C.D., and Churchich, J.E. of glutamic dehydrogenase by pyridoxal 5'-phosphate. 5: 2893-2900, 1966.
Inhibition Biochemistry.
Vo1.13,No.6
3.
ON PLATELET
Johnson, G.A., and Deal, W.C. Jr.
FWiCTIOS
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Inactivation of tetrameric
rabbit muscle pyruvate kinase by specific binding of 2 to 4 moles of pyridoxal 5'-phosphate.
J. Biol. Chem. 245: 238-245,
1970. 4.
Blumenthal, K.M., and Smith, E.L.
Nichotinamide adenine dinucleotide
phosphate-specific glutamic dehydrogenase of Neurospora.
Isolation,
subunits, amino acid composition, sulthydryl groups and identification of a lysine residue reactive with pyridoxal phosphate and Nethylmaleimide. 5.
J. Biol. Chem. 248: 6002-6008, 1973.
Colombo, G., and Marcus, F.
Modification of fructose-l, 6diphosphatase
with pyridoxal 5-phosphate.
Evidence for the participation of lysyl
residues at the active site. 6.
Biochemistry -13: 3085-3091, 1974.
Cabantchik, Z.I., and Rothstein, A.
Membrane proteins related to
anion permeability of human blood cells.
1. Localization of
disulfonic stilbene binding sites in proteins involved in permeation. 7.
Membrane Biol, -15: 207-226, 1974.
Cabantchik, Z.I., and Rothstein, A.
Membrane proteins related to
anion permeability of human red blood cells.
II. Effects of
proteolytic enzymes on disulfonic stilbene sites of surface proteins. 8.
Membrane Biol. -15: 227-248, 1974.
Cabantchik, Z.I., Balshin, M., Breuer, W., Markus, H., and Rothstein, A.
A comparison of intact human red blood cells and
released and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes.
Biochim.
Biophys. Acta. 332: 621-633, 1975. 9.
Rothstein, A., Cabantchik, Z.I., and Knaut, P.
Mechanism of anion
transport in red blood cells: role of membrane proteins.
Fed.
Proc., -35: 3-10, 1976. 10.
Sauberlich, H.E., Canham,
J.E.,
Baker, E.M.
Biochemical assessment
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of the nutritional status of vitamin B6 in the human.
Am. J. Clin.
Nutr. 25: 629-642, 1972. 11.
Bain, J.A., and Williams, H.L. tissues and tissue fluids.
Concentrations of 8s vitamins in
In:
Inhibition in the nervous system
and gamma-aminobutyric acid, Roberts, E., Baxter, C.F., and Harreveld, A.V. editors.
Pergamon Press, New York,
pp. 275-293,
1960. 12.
Subbarao, K., Kuchibhotla, J., and Green, 0.
Pyridoxine-induced
inhibition of platelet aggregation and release reaction. Part 13.
Circulation.
56: 77, 1977. II. -
Subbarao, K., Kuchibhotla, J., Ganguly, P., and Kakkar, V.V.
A
new physiological inhibitor of platelet function and blood coagulation. Biochem. Pharmacol. in press. 14.
1978.
Subbarao, K., and Kuchibhotla, J.
Adenosine 3', 5' diphosphate and
Effects on platelet function.
coenzyme A:
Biochem. Pharmacol. 27:
1193-1196. 1978. 15.
Gahmberg, C.G.
External labeling of human erythrocyte glycoproteins:
Studies with galactose oxidase and fluorometry.
J. Biol. Chem. -* 251.
510-515, 1976. 16.
Laemnli, U.K.
Cleavage of structural proteins during the assembly
of the head of bacteriophage T,,. Nature (Lond.) 227: 680-685, 1970. 17.
Bonner, W.M., and Laskey, R.A.
A film detection method for tritium
labeled proteins and nucleic acids in polyacmylamide gels.
Eur. J.
Biochem. 46: 83-88, 1974. 18.
Sidu, P., and Ganguly, P. platelets.
19.
Interaction of thrombin with mammaliam
Role of surface glycoproteins.
Fed. Proc. -37: 406, 1978.
Cabantchik, I.Z., Balshin, M., Breuer, W., and Rothstein, A. Pyridoxal Phosphate:
An anionic probe for protein amino groups
exposed on the outer and inner surfaces of intact human red blood
cells. 20.
J.
Biol. Chem. 250: 5130-5136, 1975.
Rifkin, D.B., Compans, R.W., and Reich, E.
A specific labeling
procedure for proteins on the outer surface of membranes.
J.
Biol. Chem. 247: 6432-6437, 1972. 21.
Ganguly, P.
Binding of thrombin to human platelets.
Nature (Lond.)
247: 306, 1974. 22.
Tollefsen, D.M., Feagler, J.R.,
Majerus, P.W.
thrombin to the surface of human platelets.
The binding of J. Biol, Chem. 249:
2646-2651, 1974. 23.
Ganguly, P, platelets.
Binding of thrombin to functionally defective A hypothesis on the nature of the thrombin receptor.
Brit. J. Haematology. -37: 47,51, 1977. 24.
Pollard, H.B., Goldman, T., Pozoles, C.J., Croutz, C.E., and Shulman, R.N.
Evidence for control of serotonin secretion from
human platelets by hydroxyl ion transport and owotic Proc. Nat. Acad. 74: 5295-5299, 1977.
lysis.
in Great Britain 0049-3a~P~78/1~01-1017 $02.00,!0
BINDINGOF PYRIDOXALPHOSPHATE TO HUMAN PLATELETS: ITS EFFECT ON PLATELET FUNCTION Kuchibhotla Subbarao+, Vijay V. Kakkar*, and Pankaj Ganguly Oepartment of Hematology, St. Jude Children's Research Hospital, Memphis, Tenn. USA and *Thrombosis Unit, King's College Hospital London, UK
(Received 2.6.1978; in revised form 17.9.1978. Accepted by Editor A.P. Fletcher)
ABSTRACT Pyridoxal phosphate, the coenzyme form of vitamin B6, exerted a concentration-dependent inhibition of epinephrine-induced platelet aggregation, -in vitro. A dose-dependent inhibition of [2-14C]-5-hydroxytryptamine release from platelets was also observed. Likewise, the coenzyme also blocked the ADP, thrombin and collagen-induced platelet aggregation and release. The fluorographs of platelets treated with pyridoxal phosphate and sodium borotritide indicated three predominently labeled platelet protein components of Mr = 170,000, 150,000 and 90,000. One of these polypeptldes was characterized as platelet glycoprotein I. The amount of tritium that was incorporated into these proteins showed a dependence on the concentration of the coenzyme and the label. The inhibitory effect of pyridoxal phosphate appears to be due to its specific binding to these platelet-surface proteins which may be involved in various platelet functions.
INTRODUCTION Pyridoxal 5'-phosphate (PALP), the biologically active coenzyme form of the vitamin B6 compounds, plays an important role in the metabolism of carbohydrates, fats and other proteins (1).
In addition to its normal
function as a cofactor, PALP also serves as a strong inhibitor of a variety of enzymes (2-5).
It interacts readily with certain protein
'To whom reprint requests should be sent.
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components of the red-cell membrane and thereby acts as a specific and relatively potent inhibitor of the anion-transporting system (6-9). Although the coenzyme represents more than 25% of all vitamin 8s compounds in human plasma (lO,ll), the effect(s) of PALP on platelet function is unknown.
Our earlier findings showed that PALP is a strong inhibitor
of ADP, thrombin, and collagen-induced platelet aggregation as well as blood coagulation both -in vitro and -in vivo (12,13). In this paper we provide evidence that the coentyme also blocks epinephrine-induced platelet aggregation and interacts with specific membrane components of the platelet surface that appear to play an important role in the physiological functions of human platelets. METHODS Epinephrine and PALP were obtained from'Sigma Chemical Co., London UK.
[2-"+C]-5'-Hydroxytryptamine (specific activity 57 mCi/mnol) and
tritiated sodium borohydride (NaBaH4) with a specific activity of 5 Ci/mmol were purchased from Amersham/Searle, Arlington Heights, Ill. Epinephrine and PALP solutions were prepared each day in 0.85% saline and 0.05M phosphate buffer-saline (pH 7.5), respectively.
The stock
solutions of both were kept in the dark at 4" throughout their use. Venous blood was obtained from normal healthy volunteers who had not taken any medication during the two weeks preceding blood collection. One volume of 4% sodium citrate was added to every nine volumes of blood, and the samples were then centrifuged at room temperature for 10 min at 300 x g.
The platelet count of isolated platelet rich-plasma (PRP)
was adjusted to 3OO,OOO/ul with autologous platelet-poor plasma. Platelet aggregation and serotonin release were measured according to a previously described method with epinephrine (0.5-2 PM) used as a stimulating agent (14).
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Two volumes of a washing solution containing 1% ammonium oxalate, 0.1 M KCl, 0.1% ethylene diamine tetraacetate (EDTA) and glucose, pH 7.5 was added to one volume of the PRP.
Platelets were then collected
by centrifugation of PRP at 800 x g for 15 min at room temperature. This washing procedure was repeated once and the platelet pellet was finally resuspended in calcium-free Tyrode buffer containing 5 mM glucose, pH 7.5.
One ml of washed platelet suspension, containing
2-3 x log cells, was first treated with 1OU of neuraminidase (Behring Diagnostics, Somerville, N.J.)
for 30 min at 37", then with 5U of
galactose oxidase (Worthington Biochemicals, Freehold, N.J.) min, and finally with 0.1 mCi of NaB3H, for 15 min (15).
for 60
These platelets
were then solubilized in Laemnli buffer and the platelet proteins were separated by electrophoresis (16). Washed platelets (4 x lo8 cells) in 400 ~1 of calcium free Tyrode (pH 7.5) with 5 mM glucose were incubated for 15 min at 37" with varying concentrations of PALP dissolved in phosphate buffer-saline (pH 7.5).
After centrifugation at 800 x g for 10 min, the platelet
pellet was resuspended in 200 ~1 of Tyrode (pH 8.5) and incubated with 0.8 mCi (6 ug) of NaBsH, at 4" for 30 min.
The platelets were solubilized
with 200 ~1 of Laemnli buffer containing 5% sodium dodecyl sulfate and 1% 6-mercaptoethanol, and were then heated in a boiling water bath for 5 min (16).
Platelet proteins (about 50 pg/sample) were separated
by electrophoresis in 7.5% polyacrylamide slab gels (16).
Following
electrophoresis, the gels were stained with Coomassie blue (for protein identification) and impregnated with 2,5_diphenyloxazole for fluorography (17). RESULTS We previously showed that PALP is an effective inhibitor of ADP,
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thrombin and collagen-induced platelet aggregation as well as blood coagulation (12-13).
In this report, we show that PALP also inhibits
epinephrine-induced platelet aggregation (Fig. 1).
At a concentration
2 0.3 mM, PALP inhibited the "secondary" aggregation of platelets by epinephrine.
This inhibitory effect of PALP was dependent on the
concentration of the inhibitor and the preincubation time.
The release
of [2-14C]5-HT from platelets treated with epinephrine was also blocked
‘;;
80
-o\ 70 60 50
I
I min
i
FIG. 1 Inhibition by PALP of platelet aggregation induced by epinephrine (1 I.IM). To each 0.4 ml aliquot of platelet suspension (PRP), 0.1 ml of PALP (at a given concentration) was added and the samples were incubated for 15 min at 37°C. In the control samples, PALP was substituted with phosphate buffer-saline. The aggregation response of platelets to epinephrine induced platelet aggregation in the absence [l] and in the presence of 0.10 mM [2] and 0.3 mM PALP [3] is shown. The abcissa represents time.
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by PALP in a concentration-dependent manner (Table 1).
The inhibitory
effect of PALP on the aggregation and release induced by different agents could be due to binding of the coenzyme to specific surface components of platelets (12-13).
To identify such membrane proteins,
we fixed the PALP in a irreversible form to platelets by reduction with NaB3H,+, then solubilited the cells, separated the platelet proteins by electrophoresis, and determined by densitometry of the fluorographs, the extent of radioactivity associated with platelet proteins. TABLE 1 Effect of PALP Concentration on the Release of [2-14C]5-HT from Platelets Aggregating Agent
PALP Concentration (mM)
57 + 5.2 45 38 20 12
0 0.16 0.32 0.60 1.30
Epinephrine, 1 uM
5-HT Released (%)
Platelets labeled with [2-14C]5-hydroxytryptamine were mixed with varying concentrations of PALP and incubated for 15 min at 37". The release of [2-14C]5-HT from platelets was then determined after epinephrine was added. The results reported are the mean for five control and four PALP-treated platelets f 1 SE. The electrophoretic separation of platelet proteins yielded a large number of polypeptides with molecular weights that usually ranged from 10,000 to over 250,000 (Fig. 2. I).
The distribution of these proteins
remained unchanged when platelets were pretreated with PALP.
The
fluorographs of PALP and NaB3H,+ treated platelets showed about ten bands and three of which (+) were more intensely labeled.
These components
had apparent molecular weights (M,) of 170,000, 150,000 and 90,000 2 5,000 (Fig. 2. II).
After platelet surface glycoproteins were specifically
labeled by galactose oxidase and NaB3H4, the fluorograph showed an intensely labeled band (Fig. 2. III),
(&P-I of M, = 155,000
+_ 5,000
(15,18).
corresponding to a glycoprotein The location of a heavily
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labeled protein band observed in PALP-treated platelets (Fig. 2. II) was the same as that of GP-I
(Fig. 2. III).
This suggests that among
other components of platelets, PALP binds readily to plate let GP-I.
1
II
FIG. 2 Slab gel electrophoresis patterns of PALP-treated platelet proteins stained with Coomassie blue (I); and fluorographs of PALP/NaB3H,-treated platelets (II) and of NaB3Hk-treated platelets after oxidation with The patterns for several platelet samples galactose oxidase (III). prepared on different days, remained the same, but the position of platelet actin (A) varied slightly in relation to the length of the slab gel.
The densitometric scan of fluorographs of platelets treated with a constant PALP and varying amounts of NaB3H4 (0.3 to 3 pg) is shown in Fig. 3.
The labeling pattern again suggests that PALP interacted
strongly with three platelet proteins.
The amount of tritium incorporated
OS PLATELET
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A
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FIG.
3
Densitometric scans of fluorographs of platelets treated with constant PALP (1.6 mM) and different amounts of NaB3H,+: 0.3 ug (A): 1.5 Pg (B); 3 ug (C). The arrows represent the protein bands that are extensively labeled.
into these PALP-linked platelet proteins progressively increased with increasing amounts of NaB3H4 or PALP (Fig. 4).
When incubated with
PALP at -< 0.16 mM, platelets were not labeled in significant amounts (Fig. 4).
The fact that NaB3H4 alone failed to introduce a significant
amount of label into the platelet proteins, ruled out the possibility
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that platelets contained an appreciable amount of intraprotein Schiff bases.
The heavily labeled region at the bottom of the fluorograph
of platelets treated with PALP and NaB3H,+ or NaB3H, alone (Figs. 2 and 4) may be due to platelet lipids (19).
C
n MIGRATION
DISTANCE -
FIG. 4 Densitometric scans of fluorographs of platelets treated with 3 ug of NaBaH,,and different concentrations of PALP: 0 mM (A), 0.16 mM (B), 0.40 mM (C) and 1.6 mM (II).
c)?j PLATELET
FLYiCTTI)S
lL72j
DISCUSSION The aggregation of blood platelets and clotting of fibrinogen are the two major hemostatic reactions that occur spontaneously at the site of vascular injury.
We previously reported that PALP is a
strong inhibitor of platelet function and blood coagulation (12,13). One of the major goals of this study was to determine if this inhibition was due to the binding of PALP to specific proteins on the platelet surface.
Previous studies had demonstrated the binding of PALP to
surface proteins of other cells (19,20).
We, indeed, found that PALP
interacted with about ten platelet proteins, (Fig. 2. II) of which three polypeptides with an apparent M, = 170,000, 160,000 and 90,000 were intensely labeled.
One of these heavily labeled protein components
with M, = 150,000 appears to be GP-I which has been proposed as a component part of thrombin receptor site in human platelets (21-23). The binding of PALP to GP-I may be an important finding because the coenzyme also inhibits thrombin-induced platelet aggregation and serotonin release (12,13).
It is of interest that only three out of several
platelet proteins had a remarkable affinity for PALP; this interaction appears to be specific because the inhibitory effect of PALP was evident even when the coenzyme (1.5 to 2 mM) was incubated with whole blood instead of PRP. Pyridoxal phosphate has been shown to be a potent inhibitor of the anion-transport system of red blood cells (6-9).
This transport
process was suggested to occur at a positively charged region on the membrane and presumed to involve an anion-binding protein of Mr = 95,000 (6-9).
The coenzyme has been shown to bind with this protein and block
the anion transport across the red-cell membrane (9).
In this
paper,
we have shown that PALP interacts strongly with a platelet protein component (M, = 90,000) which may be involved in anion transport.
1026
O?i PLATELET
FLXCTION
vo1.13,xo.6
Others have shown that the release of serotonin by thrombin and calcium ionophore A23187 is blocked by PALP, presumably through an inhibition of the anion-transporting mechanism of human platelets (24).
Because
the coenzyme blocks the aggregation of platelets by a variety of agents, it is proposed that the interaction of PALP with platelet surface might inhibit a common step(s) that is crucial for the normal hemostatic function of human platelets. Since the coenzyrne is a phosphomonoester, it may not readily pass through biological membranes and consequently the Schiff base formation may be restricted to molecules on the outer membrane surface.
Indeed,
we found that the label was not associated with platelet actin, which is known to be located inside the platelet surface (Fig. 2. II).
The
labeling procedure is simple, rapid and does not cause any damage to the cells.
This method of labeling platelets with NaB3H,+ in
the presence of PALP might be applicable in studies to understand the topographic distribution of protein components on the platelet surface. ACKNOWLEDGMENTS This study was supported by project grant HL 16720, CORE grant CA 21763 from NIH (USA) and G973/756 from MRC (UK).
We thank Mr. H.R.
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