Potentiation of platelet aggregation by atrial natriuretic peptide

Life Sciences, Vol. 43, pp. 731-738 Printed in the U.S.A.

Pergamon Press

POTENTIATION OF PLATELET AGGREGATION BY ATRIAL NATRIURETIC PEPTIDE Alex L. Loeb and A d r i a n R.L. Gear

Departments of Pharmacology and Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 (Received in final form July 5, 1988)

Su,mmry Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (I pM-100 riM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 uM)- and ADP (2 or I0 uH)-induced aggregation. Maximum stimulation occurred between 10 to I00 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, a f f e c t e d p l a t e l e t a c t i v a t i o n and c y c l i c n u c l e o t i d e l e v e l s . Sodium uitroprusside ( I uM) i n h i b i t e d ADP, b u t n o t t h r o m b i n or epinephrine-induced aggregation. Both sodium n i t r o p r u s s i d e ( I uM) and ANP (I0 nM) increased cyclic G M P levels by 80% and 37Z, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77Z increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function. Atrial natriuretic peptide (ANP) causes natriuresis and the vasodilatiou of many vascular beds (1-4). Radioligand binding studies have identified ANP binding sites in a wide range of tissues, including vascular smooth muscle and endothelium, kidney, brain, adrenal and platelets (5-9). ANP is thought to act through these receptors, stimulating a particulate guanylate cyclase and increasing tissue levels of cyclic GMP (10-12). Using standard techniques, De Caterina et al. (13), were unable to demonstrate any significant ANP-mediated effects on human platelet aggregation induced by thrombin, arachidonate, epinephrine, collagen, ionophore A23187, platelet activating factor, serotonin, or a thromboxane analog. However, they did observe a small but significant inhibition of ADP-induced aggregation, but only with the highest dose of ANP used (400nM). In addition, ANP had no effect on thromboxane B 2 generation by platelets stimulated with ADP or epinephrine. 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc

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ANP Potentiates Platelet Aggregation

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To investigate possible effects of ANP on platelet function, we have used a new quenched-flow approach (14). Using this methodology, alterations in platelet function such as changes in shape and volume, rates of aggregation, and the biochemical events which occur within the first few seconds after activation, can be studied (14-17). The quenched-flow approach may be more sensitive than methods based on changes in light scattering for detecting platelet aggregation and employs flow conditions very close to those in blood vessels (14,18). Platelet aggregation, as measured by the disappearance of single unaggregated platelets from platelet rich plasma, occurs much faster than suggested by optical methods, the most common type of measurement (1922). After exposure of whole blood or platelets to aggregating agents such as ADP or thrombin, aggregation of single platelets begins within 1 second and is essentially complete by I0 seconds, a time when optical tracings may show little or no change. Since ANP can increase cyclic G M P levels in many cell types possessing receptors for the peptide (10-12), we also examined the effects of ANP on platelet cyclic nucleotide levels. The effects of ANP were compared to sodium nitroprusside, another agent known to affect platelet cyclic G M ~ Methods Chemicals Unless stated otherwise, (St. Louis, MO, USA).

all materials were obtained from Sigma

Platelets Platelet rich plasma (PRP) was obtained from human venous blood collected into acid citrate dextrose (ACD), as previously described (14). Washed platelets (PRPw) were prepared by diluting PRP with additional ACD (I:I0) to lower the pH, and adding prostacyclin (PGI2, 0.75 uM), and apyrase (4 ADPase units/ml). The suspension was centrifuged for 20 min at 350 g and resuspended in ACD containing apyrase (4 ADPase units/ml) to remove residual extracellular ADP. Following a second centrifugation (20 rain at 350 g), the platelet pellet was resuspended in HEPES-buffered Eagle's salts solution containing bicarbonate, human fibrinogen (0.5 mg/ml) and hirudin (0.05 U/ml). With this washing procedure, aggregation efficiencies are very close to those seen with PRP. Platelet recovery from whole blood was 96% for PRP and 85% for PRPw (17). Platelet A~re~ation The effect of ANP (rat, 28 amino acids, Peninsula Labs) on platelet aggregation was tested using a quenched-flow approach (14). As shown in figure I, separate syringes were loaded with the PRP, the inducer of aggregation, and the quenching aldehyde, and then mounted onto a variable speed syringe pump (Harvard). In control experiments, PRP and either thrombin (0.5 units/ml), epinephrine (15 uM) or ADP (2 uM) were mixed at a "T" junction and pumped through a '~eaction loop" (0.3 mm i.d. teflon tubing). The reaction was quenched by addition of glutaraldehyde (0.5% in saline) at a 2nd "T" junction. Glutaraldehyde fixes the state of platelet aggregation (14) and stops the reaction. At m a x i m u m pump speed, the residence time within the reaction loop was 1.5 seconds. Slowing the pump speed increased the PRPinducer interaction time in the reaction loop. All experiments were done at 37°C. Platelet aggregation was estimated by quantifying the disappearance of single, unaggregated platelets from the quenched reaction mixture. After appropriate dilution of the PRP mixture, coincidence-corrected counts of unaggregated platelets (singlets) were made using a resistive-particle counter (Particle Data, Elmhurst, IL) equipped with a 48 um orifice. Plotting the decrease in single platelets at each interaction time enables aggregation rates to be calculated. These rates were calculated using the initial slopes of the aggregation time course plots and are expressed as percent of single

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ANP Potentiates Platelet Aggregation

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platelets disappearing per second (14-18). Platelet sizes were derived from a computerized 128-channel pulse-height analyzer with reference to 2.02 um latex particles (Particle Information Services, Grants Pass, OR). Typical platelet sizes ranged from 6-7 f L

Platelets ( ~./- ANF) •"~,~3.~-,~e'~.',~,~;'J,':~¢~tl~-~ Reaction, ~¢ tube Inducer= : > ;~r "/ }

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Cyclic Nucleotides Cyclic nucleotide levels were determined in PRPw only. For these determinations, I0 ml PRPw were incubated for I0 min at 37 ° C before the e x p e r i m e n t s were begun. In a typical experiment, both before (control) and at defined intervals after drug addition, 0.8 ml PRPw was removed from the incubation and added to 0.089 m l I N HCl (final concentration, 0.I N HCl), i m m e d i a t e l y vortexed, put on ice and frozen at -20°C until assay. The extracts were thawed and refrozen two times to disrupt cellular material. This procedure was effective in stopping platelet cyclic nucleotide metabolism and extracting the nucleotides from the platelets. After centrifugation to remove platelet proteinss the cyclic nucleotide remaining in the supernatant was then analyzed by radioimmunoassay (New England Nuclear). The following procedure was used in every experiment to d e t e r m i n e the concentration of extracellular nucleotides present in the PRPw. An aliquot of PRPw was centrifuged at 15,000 x g for 5 min and the platelet poor supernatant acidified to give a concentration of 0 . 1 N HCL The cyclic nucleotide concentration in this platelet poor sample was typically 10% of the total in an identical aliquot of PRPw. This background value was then subtracted from the PRPw values to d e t e r m i n e the platelet cyclic nucleotide content, which is expressed as pmoles per 109 platelets. Stat~s~iqs Results are expressed as means ~ SEM. Differences b e t w e e n treatments were analyzed using the paired t-test, or for groups with more than one treatment, by Priedmann's test. Significance was accepted at the 0.05 level of significanc~

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ANP Potentiates Platelet Aggrega£ion

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Results E(fect of ANP on a22re2atiom The effect of ANP on the rate of human platelet aggregation was studied in the quenched-flow apparatus, as illustrated in figure I. ANP in doses from 1.0 pM to i00 nM, did not induce platelet aggregation by itself. To test the possibility that ANP might modulate the platelet response to known inducers of aggregation, PRP was pre-incubated with ANP at 37°C for 3 minutes prior to challenge with either thrombin (0.5 units/ml), epinephrine (15 uM) or ADP (2 uM). As shown in figure 2A-B, the % of single platelets remaining at 5 sec after epinephrine or thrombin treatment was 57% and 44%, respectively. ANP significantly potentiated the rate and extent of aggregation induced by both of these agents. While ANP did potentiate ADP-induced aggregation significantly, it was much less effective (data not shown). The ANP-induced potentiation of epinephrine-induced aggregation was dose-dependent. As shown in figure 3, A N P increased the initial rate of aggregation significantly, approximately doubling it. The m a x i m a l effect of ANP was seen at I0 pM (p<0.03, compared to controls). Aggregation was still faster than the control at I0 nM ANP.

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FIG 2. Effect of ANP on epinephrine and thrombin-induced aggregation- ANPstimulated potentiation of A)epinephrine (15 uM) and B) thrombin (0.5 units /ml)-induced aggregation versus time. Aggregation is e x p r e s s e d a s a d e c r e a s e i n t h e p e r c e n t of f r e e , u n a g g r e g a t e d platelets (singlets) at each interaction time. ANP (I0 nM) significantly increased the rate at which platelets aggregated when compared to each inducer alone at each interaction time (*-p<0.05, paired t-test). These data are from 5 experiments.

Since this potentiation might be reflected by an activation of the platelet "shape change" prior to the induction of aggregation, we tested the effect of ANP on the platelet volume change induced by ADP. There is evidence that ADP or thrombin stimulation will induce a small increase in cell volume (23,24) while epinephrine does not. The increase has been documented by three techniques: resistive-particle sizing, platelet density, and m o r p h o m e t r i c analysis (23,24). Table I shows that while ADP caused a significant 9.7 % increase in platelet volume (6.41 to 7.03 fl), ANP (I0 nM) had no effect on this ADP-induced increase, or on resting platelet volumes. A similar lack of influence of ANP was found when thrombin was used as an inducing agent. In contrast, sodium nitroprusside, an inhibitor of A D P - i n d u c e d platelet aggregation, inhibited the ADP-induced volume increase by 59% (7.03 vs. 6.61 fl).

Vol. 43, No. 9, 1988

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ANP Potentiates Platelet Aggregation

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Effect of ANP concentration on e p i n e p h r i n e - i n d u c e d platelet aggregation. PRP w a s ~ r ~ i n c u b a t e d w i t h ANP at the concentrations shown for 3 min at 37vC before following the kinetics of epinephrine (15uM)-induced aggregatio~ The potentiation by ANP is expressed as a % of m a x i m a l effect on aggregation seen at 3 sec after platelet activatio~ The epinephrine-induced rate rose from 6.4 ~ 2.3 to 12.5 2.9 % singlets disappearing per secon~

TABLE I

Effect Of ANP And Sodium Nitroprusside On ADP-Induced Changes In Platelet Volume Situation

Platelet Volume (fl) Initial

Final

Control

6.41 _+ 0.11

7.03 _+ 0.09

ANP

6.53 + 0.18

7.00 + 0.11

SNP

6.36 _+ 0.09

6.61 _~ 0.I0

Washed platelets were incubated w i t h ANP (I0 nM) or sodium nitroprusside (SNP, I uM), and challenged w i t h 10 uM ADP. Resistive-particle volumes were measured before and after, and are s h o w n as fl ( m e a n _+ SEM). The d a t a are f r o m at least 4 experiment s •

735

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ANP Potentiates Platelet Aggregation

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Effects of ANP on vlatelet.cyclic nucleotides. Since ANP is thought to cause receptor-mediated increases in intracellular cyclic GMP (10-12), we compared the effects of ANP on platelet function with the actions of sodium nitroprusside, a compound known to increase platelet cyclic GMP levels (33). However, we found sodium nitroprusside (I uM) completely inhibited ADP-induced aggregation of PRPw, but not thrombin- or epinephrine-induced aggregation. To determine whether the A N P - i n d u c e d potentiation of aggregation was associated with changes in platelet cyclic nucleotide levels, cyclic GMP and cyclic AMP levels in PRPw were determined after stimulation with ANP or sodium nitroprusside. Both compounds induced significant increases in platelet cyclic GMP levels within I0 sec. Maximal increases were seen I rain after a d d i t i o n to P R P w (p<0.05), as s h o w n in F i g u r e 4. However, sodium nitroprusside also increased platelet cyclic AMP levels by 1.7 fold, while ANP t r e a t m e n t had no effect. For sodium nitroprusside-treated platelets, the time course of the cyclic nucleotide response was also different. At early time points (< 60 set), levels of both nucleotides were increased, while at I0 minutes, only cyclic GMP levels remained elevated (data not shown).

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200

150

100

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FIG 4. Sodium nitroprusside and ANP effects on platelet cyclic nucleotide~ Sodium nitroprusside (SNP, I uM) and ANP (10 riM) were incubated with washed platelet~ After 60 set, aliquots were acidified to extract platelet cyclic nucleotides, as described in the methods. Control samples were obtained i m m e d i a t e l y prior to drug addition. Basa~ levels of cyclic GMP and cycli~ A M P were 1.6 + 0.6 pmol/10 ~ platelets and 12.5 ~ 1.0 pmol/10 platelets, respectively. * significant increase compared to control (p<0.05). These data are from 4 experiment~ DISCUSSION

ANP has been reported to bind to receptors and to act at many target sites within the cardiovascular system, including the brain, kidney: vessel wall, and the adrenal (5-7). Although binding sites for the peptide have been reported on platelets (8-9), previous studies have revealed no physiological or functional platelet response to ANP. However, the receptor has been found to be closely associated with, and possibly might be the same protein as the particulate form of guanylate cyclase in other tissues (25-27). It has been proposed that there are two types of ANP receptors, only one of which is coupled to guanylate cyclase (27,28). We feel that at least some of the

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ANP Potentiates Platelet Aggregation

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platelet ANP receptor is coupled to guany late cyclase, since our present findings suggest that ANP can effect platelet function, and that this action can be correlated with an increase in platelet cyclic G M P levels. Recent evidence (29) has demonstrated that platelet particulate guenylate cyclase may in fact be as active as the soluble enzyme. De Caterina et al. (13) have reported that ANP did not influence platelet function. These investigators, however, used standard, optical aggregation techniques, which will not detect rapid changes in platelet function. The quenched-flow approach, coupled to single-particle counting is, on the other hand, capable of detecting changes in platelet function which occur within the first few seconds following platelet activation (14-17). This approach monitors the disappearance of single platelets as they aggregate (14,19). Optical techniques depend upon the light scattering changes caused by aggregation of small aggregates, and is therefore an inherently less sensitive system for following the earliest phases of platelet aggregation (18,22). Our finding that ANP potentiates thrombin-, epinephrine-, and ADP-induced aggregation is the first report of a functional effect of the peptide on platelets. The potentiation was dose-dependent, with a maximal effect on the rate of aggregation occurring near I0 pM ANP, a dose similar to that described for maximal receptor binding (8). Because ANP receptor activation has been associated with increased cyclic G M P l e v e l s in t a r g e t tissues, we also t e s t e d the effect of s o d i u m nitroprusside, a substance known to increase platelet cyclic GMP levels, on the shape change and aggregation induced by ADP. In contrast to ANP, sodium nitroprusside inhibited the ADP-induced shape change as well as ADP-induced aggregation. These opposite effects may be explained not by any effect of cyclic GMP per se, but by a sodium nitroprusside-induced increase in cyclic AMP. Using washed platelets and correcting for extracellular nucleotide, we found that sodium nitroprusside, but not ANP, induced a 77% increase in cyclic AMP levels. Both sodium nitroprusside and ANP significantly increased cyclic GMP levels. These findings suggest that the cyclic AMP increase accounts for the sodium nitroprusside induced inhibition of platelet function and that the ANP-induced potentiation of platelet aggregation may have been due to the increased accumulation of cyclic GMP with no change in cyclic AMP. We speculate that one possible cause of confusion within the literature as to the role of cyclic GMP in platelet function (30-35) has been the simultaneous increase in cyclic AMP and GMP when nitrovasodilators are used as agonists. In conclusion, we have shown that pre-treatment of human platelets with ANP will induce a dose-dependent potentiation of platelet aggregation This effect of ANP may be due to an elevated level of cyclic GMP within the platelet. Acknowledgements: We would especially like to thank Ms. Diana Freas and Mr. Jack Spears for their excellent technical assistance. This study was supported in part by the following grants from the NIH: HL-27014, HL-37629, H~07193.

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