Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: A possible explanation for its specific PAF antiaggregating effects in human platelets

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 272, No. 2, August 1, pp. 466-475,1989

Inhibition of [3H]Platelet Activating Factor (PAF) Binding by Zn2+: A Possible Explanation for Its Specific PAF Antiaggregating Effects in Human Platelets’ DANIELE

NUNEZ,

Department of Biochtmistry,

RAJ KUMAR,

AND DONALD

J. HANAHAN2

University of Texas Health Science Center, San Antonio, Texas 78284-7760

Received January 17,1989, and in revised form March 14,1989

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC,,) was inversely proportional to the concentration of PAF present. The IC& values (in PM) for Zn2+ were 8.8 f 3.9,27 f 5.8, and 34 -+ 1.7 against 2, 5, and 10 nM inhibitory effects on rH]PAF, respectively (n = 3-6). Zn2+ exhibited comparable serotonin secretion and the I&, values (in PM) were 10 f 1.2,18 k 3.5, and 35 f 0.0 against 2,5, and 10 IIM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 PM), arachidonic acid (3.3 PM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within O-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of rH]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of rH]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC& value was 18 f 2 PM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2’, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2’ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with I& values being five- and sevenfold higher, respectively, than the IC,, for Zn’+, and with the I& values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the ICS0 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site. 0 1989 Academic Press. Inc.

Platelet activating factor (PAF)3 is a potent mediator which has been identified ’ Supported by Grant AQ-88’7 from the Robert A. Welch Foundation. ’ To whom correspondence should be addressed. 3 Abbreviations used: PAF, platelet activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine); ADP, adenosine diphosphate; Hepes, 4-(2-hydroxyethy)-1-piperazineethanesulfonic acid; BSA, bovine serum albumin; TNS, toluidinyl-Z-naphtalene sulfonate; DTT, dithiothreitol; EGTA, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid. 0003-9861/89 $3.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

chemically as l-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1,2). This phosphoglyceride is synthesized by various cells such as neutrophils, macrophages, basophils, platelets, and endothelial cells and has been shown to initiate a wide range of biological activities in vivo, on isolated organs such as heart, lung, smooth muscle, bronchi, liver, and on cells such as neutrophils, endothelial cells, and platelets (3-7). In platelets for example, while it is well recognized that Ca2’ plays an important role, in the agonist activity of PAF, the in466

ZINC INHIBITION

OF [$H]PLATELET

fluence of other cations in agonist-induced reactions has not been explored to any degree (8). Among the divalent cations present in significant levels in cells, Zn2+ has been found at relatively high concentrations in human platelets and in plasma (9, 10). Interestingly, Heyns et al. reported that Zn2+ at high concentration (over 200 PM) induced human washed platelet aggregation without secretion (11). By contrast, lower concentrations (l-25 j.&M) inhibited the aggregation and the secretion of dog platelets subject to collagen and epinephrine treatment (12). The same low range of concentrations of Zn2+ blocked rabbit washed platelet secretion induced by PAF or thrombin; however, Znzt showed only a very weak effect on the aggregation induced by PAF or thrombin (13). As a consequence of the above observations on the effects of Zn2’ on rabbit and dog platelet activation, a similar approach was extended to human platelets and to PAF receptor. This current investigation provides data on several facets of the activity of Zn2+ as a potent inhibitor of PAFinduced effects on human platelets. MATERIALS

AND METHODS

Chemicals and reagents. [aH]Serotonin (20 Ci/ mmol) and [5,6,8,9,11,12,14,15-3H]arachidonic acid (94.5 Ci/mmol) were purchased from New England Nuclear (Boston, MA); 32Pi (5 mCi/ml) was from ICN (Irvine, CA); l-O-[3H]octadecyl-2-acetyl-sn-glycero-3phosphocholine (80 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Bovine serum albumin (BSA) was from Miles Laboratories, Inc. (Naperville, IL). Human fibrinogen (Chemical Dynamics Corp., South Plainfield, NJ) was diluted in 0.9% saline solution to a final concentration of 20 mg/ml. 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine was purchased from Bachem (Bubendorf, Switzerland) and dispersed in BSA 0.125% saline solution. Unlabeled arachidonic acid was purchased from Sigma Chemical (St. Louis, MO), kept in chloroform/methanol (l/l, v/v) at -2O”C, dried under Nz, and then resuspended in 60% ethanol. ADP sodium salt and thrombin from Sigma were dissolved in 0.9% saline solution. Zinc chloride and cupric sulfate was from Fisher Scientific Co. (Fairlawn, NJ). Cadmium chloride was obtained from Sigma Chemical. Lanthanum chloride was purchased from Matheson Coleman &Bell Manufacturing Chemists (Norwood, OH). Bufers. The washing buffer for human platelets was composed of (mM): KCI, 2.6; NaCl, 137; NaHC03,

ACTIVATING

FACTOR

BINDING

467

12.0, NaH2P04, 0.42; MgC12, 1.0; glucose, 0.56; Hepes, 5.0. One volume of acid-citrate-dextrose (ACD) solution composed of citric acid 0.8% (w/v), trisodium citrate 2.2% (w/v), glucose 2.45% (w/v), was added to 9 vol of buffer pH 6.4 (Tyrode-Hepes-ACD (no Caa’)). The same buffer was used for platelet aggregation except that 1.3 mM Ca2+ was used instead of ACD and the pH was 7.4 (Tyrode-Hepes). Isolation of human platelets. The preparation of human washed platelets either unlabeled or labeled by [‘Hjserotonin, rH]arachidonic acid, or a2Pwas accomplished as described previously (14-16). Typically, 200 j&i %P diluted in 0.02 M HCl or 2 &i of rH]arachidonic acid dissolved in absolute ethanol were used for the labeling of 1 ml of the platelet suspension. Briefly, the platelets (3 X lo9 X ml-‘) were incubated in Tyrode-Hepes-ACD (no Ca’+), pH 6.4, with rH]arachidonic acid or a2P for 30 min at 37°C. Then the platelets were washed twice by centrifugation at 9009 for 10 min before the final resuspension in Tyrode-Hepes-ACD (no Ca*+), pH 6.4, to a final concentration of 3 X 109/ml. Measurement of platelet aggregation and [‘HIserobnin secretion. Fifty microliters of platelet suspension was added to a siliconized aggregometer cuvette with 450 ~1 of Tyrode-Hepes containing 0.2 mg fibrinogen/ml, 1.3 mM Caa+, and 0.25% BSA (final concentration). The platelets (3 X lO*/ml) were incubated for 1 min with stirring at 3’7°C at 900 rpm. Zn2+ or other cations dissolved in water were added 15 s before addition of the appropriate agonist. Platelet aggregation was measured by change in light transmission monitored by a Payton aggregation module (dual channel). Four minutes after addition of agonist, the samples were mixed with 25 ~1 of cold 1.5 M formaldehyde to stop the reaction and centrifuged at 12,000g for 2 min. The radioactivity of the supernatant was measured by liquid scintillation counting (Beckman LS 6800 liquid scintillation spectrometer) and the total [‘Hlserotonin secretion was obtained by treatment of intact labeled cells with Triton x-100 (final concentration, 0.2%). Values for [3H]serotonin secretion were calculated as described previously (16). Each of the secretion values in the absence of agonist was consistently below 5% of total serotonin secretion. Values for aggregation (%a) induced by agonists in the presence of Zn2+ or other cations were calculated by comparison with the control (agonist-induced aggregation in the absence of the indicated cation) and considered as 100%. In some experiments the inhibition percentage of agonist-induced aggregation and [3H]serotonin secretion in the presence of cation was calculated. An inhibition concentration curve was drawn in each series of experiments and an I& (concentration of cation that induced 50% inhibition) was determined. The means (SD) of the percentage of inhibition for each concentration of cation and the means

468

NUNEZ, KUMAR,

of I&, were calculated from at least three separate preparations of platelets. Binding studitzs with [‘HJPAF. Human washed platelets were prepared as described above and resuspended at 3 X lo8 cells/ml in Tyrode-Hepes buffer, pH 7.4, containing 0.25% BSA and 1.3 mM Cazf. Then, Zn2+ and/or an excess of unlabeled PAF (1 PM) were added to 500 ~1 of platelet suspension 1 min before the addition of increasing concentrations of [sH]PAF (0.05 to 20 nM). After the cells had been incubated for 30 min at room temperature, the platelet suspension was diluted to 5 ml in cold Tyrode-ACD (no Ca’+), immediately followed by vacuum filtration through Whatman GF/C glass fiber as described (15, 17, 18). The filters were then washed twice with 5 ml of cold Tyrode-ACD (no Ca*+) and the radioactivity adsorbed to the filter was counted in 5 ml of Liquiscint (National Diagnostics, Manville, NJ). The radioactivity adsorbed to the filter without cells was subtracted from the values obtained in the presence of cells. Nonspecific binding was determined by including 1 PM unlabeled PAF in the assay mixture and specific binding was defined as the difference between total and nonspecific binding. A Scatchard plot (19) of concentration-dependent rH]PAF binding was analyzed by linear regression. In some experiments, different concentrations of Zn2+ or other cations were used (5 to 1000 pM) with a single concentration of [sH]PAF (1 nM). Binding of [3H]PAF was expressed as percentage of control binding of r3H]PAF in the absence of any added cations. [SHjPAF metabolism Platelets were incubated with different concentrations of E3H]PAF (0.5,1,5 nM) under binding experimental conditions at 20°C in the presence or absence of 100 PM Zn’+. Lipids were extracted by the Bligh and Dyer technique (20) and applied to a silica gel G TLC plate using a solvent system of chloroform/methanol/water (651351’7 v/v). Phospholipids were detected by TNS spray. The material contained in the individual spots was collected by scraping and extracted by the Bligh and Dyer technique (20) and the radioactivity in each extract was determined by liquid scintillation. Production of arachklonic acid and phosphatidic acid in human platelets. Human washed platelets (1.5 X 108,0.5 ml) labeled with [3H]arachidonic acid as described above were stirred in an aggregometer with 5 nM PAF or 0.1 U/ml thrombin for 2 min at 37°C. The reaction was stopped by the addition of 5 ml of chloroform/methanol (l/2, v/v) containing 0.2 N HCl and 5 mre EGTA. The phases were separated by the addition of an equal volume of chloroform and water and the lower phase was collected and dried under nitrogen. The neutral lipids were separated in a solvent system of petroleum ether/diethyl ether/acetic acid (50/5/l, v/v/v) using silica gel G plates. For “P-labeled platelets, lipids were extracted as described above and separated on HPTLC plates using a solvent system of chloroform/methanol/20%

AND HANAHAN

3aso .-6 ‘;g 60 & % ‘i; 6 ‘z= a

40

E

*O 0L

3.25

6.5

13

26

50

100

Zinc (PM)

FIG. 1. Effects of Zn2+ on human washed platelet aggregation induced by PAF. Washed human platelets (3 X 108/ml) diluted in Tyrode buffer, pH 7.4, with 1.3 InM Ca’+, 0.2 mg/ml fibrinogen, and 0.25% BSA were preincubated for 1 min at 37°C with stirring in an aggregometer. Znzf diluted in distilled water was added 15 s prior to PAF. In the presence of various concentrations of Zn’+, the percentages of inhibition of aggregation induced by 2 nM (O), 5 nM (A), or 10 nM (m) PAF, respectively, were determined. The percentage of inhibition was calculated by comparison with the control in the absence of Zn’+. Results are presented as means + SD of three separate experiments.

aqueous methylamine (3011815, v/v/v). After development in this solvent, the plates were sprayed with TNS reagent and visualized under ultraviolet light. Particular compounds were located by their comigration with authentic standards and then scraped into vials to determine the radioactivity by liquid scintillation counting. The standards used in the chromatogram of lipids extracted from [3H]arachidonic acid labeled platelets were monoglyceride, diglyceride, arachidonic acid, and triglyceride purchased from Serdary Research Laboratory Inc. (Port Huron, MG). In the case of the chromatogram of lipids extracted from q-labeled platelets, the standards used were phosphatidylinosito1 4,5-biphosphate, phosphatidylinositol 4-monophosphate, phosphatidylinositol, and phosphatidic acid which were purchased from Sigma Chemical (St. Louis, MO). RESULTS I. Zn” Inhibition Aggregatim

of PAP-Induced

As shown in Fig. 1, PAF-induced aggregation of human washed platelets was inhibited by Zn’+. The inhibition was proportional to the concentration of Zn2+ from 6.5 to 100 PM. At concentrations ranging from

ZINC INHIBITION

OF [3H]PLATELET

ACTIVATING

6.5 to 50 PM, Zn2+ inhibited the aggregation induced by 2 nM PAF with an IGo of 8 + 3.94 PM (n = 6). As the concentration of PAF increased, the inhibitory effects of Zn2+ decreased with I&, values of 27 -+ 5.77 and 34 f 1.73 PM against 5 and 10 nM PAF, respectively (n = 3). This inhibitory effect of Zn2+ on PAF-induced aggregation was independent of the concentration of external Ca2+ and Mg2+ (data not shown).

200

III.

Zn2’ Inhibition of Platelet Aggregation Induced by PAF in the Absence of Albumin

The inhibitory effects of Zn2+ described above were studied on platelets stimulated by PAF solubilized in BSA. It has also been shown that Zn2+ could bind to albumin (26). In order to rule out that Zn2+ could inhibit PAF effects by affecting the interaction of PAF with albumin, the assays were also performed with PAF solubilized in 50% ethanol, in the absence of BSA in the reaction system. Under these conditions, the I&,, value for Zn2+ against aggregation induced by 0.5 nM PAF was 7.7 + 5.9 PM (n = 3), comparable to the I& values reported above in the presence of BSA. These results suggested that the inhibition by Zn2+ could not be explained by an effect on the interaction of PAF with BSA since the same pattern of inhibition was observed in the absence or presence of this protein. IV

Behavior of Zn2’ Alone on Human Washed Platelet Aggregation

At concentrations which inhibited PAFinduced platelet activation (from 5 to 100

BINDING

469

150

E ” 100 B 8 50

II. Eflect of Zn” on PAF-Induced Serotonin Secretion

Comparable to the effect of Zn”+ on PAFinduced aggregation, the inhibitory effect of Zn2+ on PAF-induced serotonin secretion was also dose dependent and decreased as the concentration of PAF increased. The ICsO values were 10 + 1.15,18 f 3.46, and 35 + 0.0 /IM against 2, 5, and 10 nM PAF, respectively (n = 3). Even at a high concentration of Zn2+ (100 PM), the secretion was not totally inhibited (approximately 90% inhibition) (data not shown).

FACTOR

0

A

2. Effect of Zn2+ on human platelet aggregation and [aH]serotonin secretion induced by thrombin (0.05 U/ml), ADP (5 PM), arachidonic acid (3.3 pM), and PAF (10 nM). Columns A and B represent, respectively, the percentage of aggregation and secretion induced by these agonists in the presence of Zn2+ (100 PM). The percentages of aggregation and secretion were calculated by comparison with the control in the absence of Zna+ and considered as being 100%. Aggregation and secretion were performed as described in Fig. 1 and under Materials and Methods. Results are presented as means + SD of three separate experiments. The control values of secretion (% of total secretion) for ADP (5 PM), AA (3.3 PM), PAF (10 nM), and thrombin (0.05 U/ml) were 6 + 5,27 + ‘7,36 + 2, and 69 f 2 (n = 3). The control values of aggregation (7% of total aggregation) for the same concentrations of ADP, AA, and PAF, and thrombin were 35 + 25,35 f23,78f6,and88kll(n=3). FIG.

PM), Zn2+ alone (without

agonists) had no aggregating activity. As described previously (12), at concentrations greater than 200 PM, Zn2+ triggered a delayed and a very slow aggregation pattern with more than 10 min needed to reach a plateau. This Zn2+-induced aggregation was not accompanied by serotonin secretion (data not shown). T/: Speci&cit~ of Inhibitory Efect of Zn’+ on PAF-Induced Platelet Aggregation and Secretion

At a high concentration of Zn2+, e.g., 100 aggregation and secretion induced by ADP (5 PM), arachidonic acid (3.3 PM), and thrombin (0.05 U/ml) were not inhibited (Fig. 2). Of interest, even a small potentiation of the effects of ADP and arachidonic acid was observed. By contrast, PM, platelet

470

NUNEZ,

KUMAR,

L

AND

HANAHAN

triggered a specific reversal of the aggregation induced by PAF (data not shown). VII. Eflects of Other Cations on Platelet Activation

t t t t ‘r FIG. 3. Influence of time of addition of ZnZf on disaggregation of platelet aggregates induced by PAF. Zn’+ (100 &M) was added at the times indicated by the open arrows after the addition of 5 nM PAF, indicated by the closed arrows.

the same concentration of Znzf inhibited almost completely the aggregation and the secretion induced by a high concentration of PAF (10 nM). VL Kinetics of Inhibitory Efects of Zn2’ on PAF-Induced Platelet Aggregation and Secretion The inhibition of aggregation and secretion induced by PAF was not dependent on the time of prior incubation of Zn2+ with platelets (between 15 and 600 s). In fact, Zn2+ clearly affected aggregation and secretion induced by addition of PAF (data not shown). On the other hand, Zn2+ (100 PM) added between 15 and 120 s after the addition of PAF not only blocked further platelet aggregation but could reverse PAF-induced platelet aggregation (Fig. 3). Furthermore any additional [3H]serotonin secretion induced by PAF was also inhibited by Zn2+ (data not shown). These latter effects of Zn2+ on aggregation and secretion decreased with time. Thus, approximately 2 min after the addition of PAF, aggregation became irreversible and insensitive to the addition of Zn2+ (Fig. 3). The inhibition of aggregation and secretion by Zn2+ added after PAF was proportional to the concentration of Zn2+ (data not shown). Zn2+ added after ADP, arachidonic acid, and thrombin did not affect platelet aggregation indicating that Zn2+

Cd2+, CL?, and La3+ were tested for their ability to influence the aggregation and [3H]serotonin secretion induced by PAF and thrombin (Table I). In contrast to Zn2+, the concentrations of Cu2+ or La3+ inhibiting 50% aggregation induced by 10 I’IM PAF were significantly above 100 PM (I& = 250 f. 14 and 967 +- 251 PM (n = 3)). These latter cations at lower concentrations also inhibited aggregation induced by 0.025 U/ ml thrombin (I&,, = 33 +- 2 and 263 f 42 PM for Cu2’ and La3+ respectively). However, Cd2+ at concentrations between 100 and 500 PM inhibited only PAF-induced platelet aggregation (I&,, = 160 + 40 PM (n = 3)) whereas thrombin-induced aggregation was affected only in presence of higher concentrations (I(& = 717 & 189 @M (n = 3)); the same inhibitory properties of these latter cations were observed on serotonin secretion induced by PAF and thrombin (Table I).

TABLE

I

INHIBITORYCAPABILITY OF CdZf,Cu2+, ON PLATELET AGGREGATION AND Inhibition

(IC,pM)

Aggregation Cation

Thrombin

PAF

Cd’+

16Ok

La3+

967 f 251

40

Chz+

250f

14

717 _+ 189 263 f 42 33f 2

ANDL~~+ SECRETION

Secretion PAF 483 f 208 700 f 397 16Ok 42

Thrombin 630+345 212k 33 38-t 4

Note. After preincubation of human platelets with different concentrations of Cd’+, Cu2+, or La3+ for 1 min, the aggregation and [3H]serotonin secretion induced by 10 nM PAF or 0.025 U/ml thrombin were assayed as described in Fig. 1. The I&, was determined as the concentration of cations that inhibited platelet aggregation and secretion to 50% of the control. The latter was performed in the absence of the indicated cation. Each value represents the mean -+ SD from three separate experiments.

ZINC INHIBITION

OF [3H]PLATELET

ACTIVATING

FACTOR

471

BINDING

These results suggested that Zn2+ and unlabeled PAF affect a similar site(s) or reaction in preventing the rH]PAF specific binding to human platelets. b. Scatchard plot of [‘HPAF binding to human washed platelets. Scatchard analy-

[3H] PAF,nM

FIG. 4. Effect of Zn2+ on rH]PAF binding to human washed platelets. Washed platelets were suspended in Tyrode-Hepes buffer, pH 7.4, containing 0.25% BSA, 1.3 mM Ca’+. Zn2+ and/or an excess of unlabeled PAF were added to 500 ~1 of platelet suspension 1 min before the addition of increasing concentrations of [3H]PAF (0.05 to 2.6 nM). After incubation of the cells for 30 min at room temperature, the suspensions were filtered and the radioactivity bound to platelets was counted and calculated to dpm by the standard procedure described under Materials and Methods. (0) Control; (A) 100 FM Zn’+; (0) 1 pM unlabeled PAF; (U) 100 jtM Zn’+ and 1 jtM unlabeled PAF added together. Results are presented as means + SD of three separate experiments performed in duplicate.

sis demonstrated at least two types of [3H]PAF binding to human platelets (Figs. 5A and 5B). One binding of high affinity and lower capacity was linear with a Kd value of 0.36 nM and a calculated 349 putative site receptors per platelet (n = 3). This binding was considered specific and associated with the agonist biological response (Fig. 5B). The other type of binding was unsaturable with an infinite number of binding sites and represented nonspecific binding (Fig. 5A). The addition of 20 PM Zn2+ to platelets led to a decreased affinity of the binding sites for PH]PAF. The convergence of Scatchard plots in the absence and presence of 20 ~.LMZn2+ at a high concentration of PH]PAF suggested that 20 f.&MZn2+

VIII. Inhibitory Eflects of Zn” on [‘HjPAF Binding to Human Washed Platelets a. General observations. As shown in Fig. 4, the total binding of [3H]PAF to human washed platelets increased with the concentration of [3H]PAF without reaching a plateau. rH]PAF nonspecific binding increased linearly with the concentration of [3H]PAF in the presence of an excess of unlabeled PAF (1 PM). The [3H]PAF specific binding defined as the [3H]PAF total binding minus the rH]PAF nonspecific binding was saturable. It reached a plateau near 0.65-l nM [3H]PAF (data not shown). rH]PAF binding in the presence of Zn2+ (100 j.&M) was inhibited, but increased linearly with the increased concentration of [3H]PAF in a manner similar to that described in the presence of an excess of unlabeled PAF. When Zn2+ (100 PM) and unlabeled PAF (1 PM) were added together, no additive inhibitory effects were observed on [3H]PAF binding.

L-

0

IO 203040

50 60 700090

fmol Bound

100

200

300

per 1.5 xlO*Platelets

FIG. 5. Scatchard plot of 13H]PAF binding to human washed platelets in the absence (0) and presence of 20 (+) and 100 pM Zn2+ (m). Experimental conditions were as described in Fig. 4 and under Materials and Methods. Thus, 500 ~1 of platelet suspension was preincubated with or without Zn2+ for 1 min before the addition of various concentrations of PAF (0.1 to 20 nM). After a 30-min incubation at room temperature, the total binding of [‘H]PAF to the cells was measured (A). Each point is the mean value of duplicate assays of three separate experiments. The Scatchard plot of the specific binding of [3H]PAF is presented in B. The data were analyzed by linear regression.

472

NUNEZ,

KUMAR,

AND HANAHAN

so-[3H]PAF and/or [3H]alkylacylphosphocholine was not detected under the experimental conditions of the binding studies, in the presence or absence of Zn2+ (data not shown).

1 0

5

IO

50 100 Co1mls Q.LM)

500

1000

FIG. 6. Concentration-dependent inhibition of rH]PAF binding by Zn2+, Cd’+, and &a+. The [3H]PAF binding study was performed as described in Figs. 4 and 5 except that a single concentration of rH]PAF (1 nM) and different concentrations of Zn2+ (O), Cd2+ (0), Cu2+ (A) (5 to 1000 pM) were used. The rH]PAF binding in the presence of these cations was expressed as percentage of the binding of r3H]PAF in the absence of the indicated cation. Each point is presented as the mean + SD of three separate experiments performed in duplicate.

did not reduce the number of binding sites. Also, 100 PM Zn2+ prevented the specific binding of rH]PAF without affecting the nonspecific binding of TH]PAF. (Fig. 5A). c. Concentration-dependent inhibition of [‘HIpAP binding by Znzf: Comparison with other cations. The effect of various concentrations of Zn2+, Cd2+, Cu2+, or La3+ on [3H]PAF binding was investigated and the results are given in Fig. 6. Zn2+, Cd’+, or Cu2+ effectively inhibited the binding of 1 nM rH]PAF in a dose-dependent manner whereas La3+ had no effect. The decrease in 1 nM [3H]PAF binding reached a plateau at 4’7 t- 6,50 f 7, and 57 f 1% (n = 3) of control in the presence of 80-100 PM Znzt, 500 PM Cd2+, and 1000 PM Cu2+. The value of rH]PAF binding in the presence of 1 ,uM unlabeled PAF was 48 & 7% of control (n = 3) and was considered to be representative of the nonspecific binding of rH]PAF. The I&, of Zn2f was 18 +- 2 PM (n = 3). The I& values for Cd’+ and Cu2+ were, respectively, 92 t- 34 and 347 +- 159 PM (5- and 19fold higher than ICW for Zn2+). These results showed that Zn2+ was the most effective of the three cations in inhibiting PAF binding. IX Metabolism of [‘KJPAF In agreement with previous studies (17, 18), metabolic conversion of rH]PAF to ly-

X Eflects of Zn” on Phoaphatidic Acid and Arachidonic Acid Formation Induced by PAF and Thrombin As shown in Table II, 5 nM PAF did not trigger an obvious increase in free arachidonic acid. However, the same concentration of PAF stimulated the formation of phosphatidic acid and this stimulation was inhibited by 100 I.LM Zn2+. On the other hand, a threshold concentration of thrombin (0.1 U/ml) was capable of stimulating the formation of free arachidonic acid and phosphatidic acid; these effects of thrombin were not inhibited by 100 PM Zn2’ (data not shown). These results indicated that Zn2+ could have a specific effect on the production of phosphatidic acid induced by PAF. DISCUSSION

The present study shows quite dramatically that Zn2+ at concentrations between 1 TABLE II EFFECTSOFZ~'+ONTHEFORMATIONOFPHOSPHATIDIC ACID AND FREE ARACHIDONIC ACID AFTER STIMULATIONOFHUMANPLATELETSBY PAF

Additions Controls %I’+ lc@~M PAF: 5 IIM PAF, 5 nM + %I’+, 100PM

Arachidonic acid (dpm/4.5 x IO*) 251(277-226) 294 (484-105) 338 (383-294) 217

(27-157)

Phosphatidic acid (dpmI4.5 X l@) 359 (502-216) 417 (328-506)

1193 (1398-988) 447 (434-460)

Note. The platelet suspension labeled with r3H]arachidonic acid or with 32P, as described under Materials and Methods, was stirred in an aggregometer with 5 nM PAF for 2 min at 37°C. The incubation was stopped by addition of solvent and lipids were extracted and separated by thin-layer chromatography (see Materials and Methods). Free [3H]arachidonic acid and [32Plphosphatidic acid levels were determined after the 2-min aggregation period. The results are shown as the means of two separate experiments. The values of each experiment are indicated within the parentheses.

ZINC

INHIBITION

OF

[3H]PLATELET

and 100 PM is a specific inhibitor of human washed platelet aggregation and secretion induced by PAF (1 to 10 nM) in the presence of 1.3 mM Ca2+. The human washed platelets were very sensitive to Zn2+ with 50% inhibition at less than 10 pM Zn2+ on both aggregation and secretion induced, for example, by 2 nM PAF. The inhibition of aggregation and secretion was proportional to Zn2+ concentration. No inhibitory action toward aggregation and serotonin secretion induced by ADP, arachidonic acid, and thrombin was detected in the presence of 100 pM Zn2+. In actual fact, a potentiation of these latter agonist effects was observed. The inhibition of the aggregation and the secretion induced by PAF in the presence of Zn2+ could be overcome by the increasing concentration of PAF, suggesting a competition phenomenon between the interactions of PAF and Zn2+ with the platelet. The same inhibitory potency of Zn2+ against PAF action on human platelets was observed in the absence and presence of albumin in the reaction system. Hence, an effect of Zn2+ which bound to albumin (26) on the interaction of PAF with this protein (40) could not be responsible for the observed inhibition. Of considerable interest, introduction of Zn2+ within O-2 min after the addition of PAF not only blocked further platelet aggregation and [3H]serotonin secretion but also caused platelet disaggregation. This indicated that the process which aggregated platelets after the addition of PAF could be interrupted by the addition of Zn’+. However, if Zn2+ was introduced 2 min after the addition of PAF, the aggregation was irreversible and insensitive to this cation. The platelet disaggregation could be triggered by inhibition of the exposure of the fibrinogen binding site on the platelet surface as previously observed after the addition of PAF antagonists (38). These effects of Zn2+ on human platelets contrasted sharply with those observed on rabbit platelets (13). In this latter species Zn2+ had quite different effects on response of platelets to PAF and was not as specific since secretion induced by thrombin was also inhibited; furthermore, no platelet disaggregation was detected after the addition of Zn2+. Other different characteris-

ACTIVATING

FACTOR

BINDING

473

tics between the two species have been previously described; for example, it has been shown that the inhibitory potency of several PAF antagonists in rabbit platelet membranes differed from that in human platelet membranes (39). This specific inhibition by Zn2+ against PAF action on human platelets indicated that Zn2+ could possibly affect the binding of PAF to the membrane. No inhibition of PAF-induced aggregation was seen in platelets preincubated with Zn2+ and then washed and treated with PAF, suggesting that Zn2+ interacted with the surface of platelet membrane (data not shown). Furthermore, analysis of rH]PAF binding indicated that Zn2+ between 10 and 100 pM inhibited the binding of 1 nM [3H]PAF to platelets in a concentration-dependent manner. The concentration of Zn2+ which inhibited the specific binding of 1 nM [3H]PAF by 50% (18 + 2 j&M) reduced the aggregation induced by 2 nM PAF by 80%) indicating that the blocking of specific binding by Zn2+ could contribute substantially to the prevention of platelet activation by PAF. A Scatchard plot of [3H]PAF binding in the presence of different concentrations of rH]PAF showed that [3H]PAF bound to at least two different types of sites in human platelets. The apparent Kd value of the higher affinity and lower capacity site was approximately 0.36 nM, the number of sites was calculated to be 349 (n = 3), and this binding site(s) was considered to be specific (putative receptor). The other type of binding was nonsaturable with an infinite number of binding sites and was considered to be nonspecific. Interestingly, 20 pM Zn2+, repeatedly in three different experiments, caused a decrease in affinity of [3H]PAF specific binding to platelets, without a clear reduction of the number of sites. This decrease of the affinity of [3H]PAF specific binding to platelets and the nature of the inhibition by Zn2+ which could be overcome by increased concentrations of PAF indicates that these two components act on the same receptor site or on two contiguous sites. Unlabeled PAF, 1 PM, or Zn2+, 100 pM, totally prevented [3H]PAF specific binding. No evident addition of their effects on rH]PAF total binding was observed when 1 pM unlabeled PAF and

474

NUNEZ,KUMAR,AND

100 /IM Zn2+ were used in the assay, suggesting that 100 pM Zn2+ did not influence the nonspecific binding of [3H]PAF to human platelets. The activity and specificity of Zn2+ toward PAF-induced platelet activation and PAF binding to platelet were compared with those of other cations (Cu2+, La3+, Mg2+, Ca2+). In contrast to Zn2+, the concentrations of Cu2+, La3+, or Cd2+ inhibiting 50% aggregation induced by 10 nM PAF were well above 100 PM. Cd2+ and CL?+ prevented the rH]PAF binding but to a much lesser extent compared to the effects of Zn2+. Nevertheless, this indicated perhaps a common mechanism in the modulation of r3H]PAF binding by these three latter cations. The [3H]PAF binding was shown to be independent of the presence of Mg2+ and Ca2+ in the range of concentrations tested (0 to 0.01 M) (data not shown). This absence of an influence of Ca2+ on [3H]PAF binding to whole platelets has been previously reported (17,21). However, Hwang (25) has reported a potentiation of rH]PAF binding to human platelet membranes by Ca2+ and Mg2+. The effects of these cations on membranes cannot be compared to those on intact platelets since receptors on the cytosolic side of the membrane cannot be excluded. The inhibition by Zn2+ on PAF-induced platelet aggregation was shown to be independent of the concentrations of external Ca2+ suggesting the absence of interaction between Zn2+ and Ca2+ in the inhibition process. In conclusion, compared to the effects of other cations, it appears that there is high degree of selectivity of Zn2+ as an inhibitor of PAF effects. The binding of an agonist to a cell such as a platelet is considered to involve the stimulation of specific phospholipases A2 and C. The importance of these events in platelet activation has been well reported (8). However, experiments on the release of arachidonic acid showed that PAF was unable to stimulate phospholipase A2 in human platelets as described previously by Crouch and Lapetina (37). On the other hand, PAF triggered the formation of phosphatidic acid which results from the phosphorylation of diacylglycerol by a diacylglycerolkinase. Diacylglycerol is a in-

HANAHAN

variant product of phospholipase C. The production of phosphatidic acid could be considered an indicator of phospholipase C involvement. Zn2+, 100 PM, could block the formation of phosphatidic acid induced by PAF. This Zn2+ inhibition of PAF-induced of phosphatidic formation was probably indirect and related to the inhibition of PAF binding since no inhibition of thrombin-induced formation of phosphatidic acid was seen in the presence of Zn2+ (data not shown). The influence of Zn2+ on PAF interaction with platelet binding sites is not surprising and can be supported by some relevant observations. It has been observed that Zn2+ regulates the binding of steroid hormones and neurotransmitters to their receptors (28-33). These inhibitory or stimulatory effects of Zn2+ were reported to involve receptor affinity and/or number of binding sites and also to be reversed by DTT and other thiol-reducing reagents. In human platelets, Zn2+ promotes the binding of the coagulation factors high-molecular-weight kininogen and factor XI (34). These effects were observed with the same concentrations of Zn2+ which inhibited the binding of PAF to human platelets. Different reactivities of the human platelet to Zn2+ could occur depending on the type of receptors on the platelet membrane. Of potential relevance to the PAF activation of platelets, Findik and Presek (35) reported a stimulation of protein tyrosine kinase activity of human platelet membranes by a high concentration of Zn2+ (1 ITIM). Hence, different metabolic effects could be exerted by Zn2+ in platelets depending on the concentration of this cation. Finally it is pertinent to note that Berg (36) suggested a specific structure for the zinc binding domains from transcription factor IIIA and related proteins. These domains, or “zinc fingers,” may well have a role in the behavior of zinc in the current study. It is interesting to note that the concentrations of Zn2+ required to inhibit PAFinduced activation of human platelets is close to the level of Zn2+ found in the plasma, i.e., 10 to 20 PM. The findings reported here strongly suggest that Zn2+ could be an important physiological modu-

ZINC

INHIBITION

OF [3H]PLATELET

lator of PAF activity on platelets and perhaps other cells. These actions could have importance in thrombotic and inflammatory pathologies.

ACTIVATING

FACTOR

16. SUGATANI,

BINDING

J., AND HANAHAN,

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