Effect of metabolic inhibitors on thrombin-induced membrane potential changes in washed human platelets

THROMBOSIS RESEARCH 24; 299-306, 1981 0049-3848/81/220299-08$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

EFFECT OF META.BCLI(: INHIBITOR ON THRCXBIN-INDUCED MEMBRANE POTENTIAL CHANGES IN WASHED HUMAN PLATELETS

Sheryl M. G. Sepersky and Elizabeth R. Simons Department of Biochemistry Boston University School of Medicine Boston, MA 02118

(Received 14.9.1981; in revised form 20.10.1981. Accepted by Editor G.A. Jamieson)

ABSTRACT Platelet stimulation by thrombin or other agonists is influenced by the metabolic status of the platelet, i.e., hy the available supply of metabolic ATP. We have shown earlier that such stimulation by thrombin is accompanied by a dose-dependent depolarization of the platelet transmembrane potential and by a decrease in the transmembrane pH gradient. We have now studied the effect of metabolic inhibitors on the membrane potential changes as assessed with the fluorescent cation 3, 3'-dipropylthiodicarbocyanine. Preincubation of platelets with either 2-deoxy-D-glucose or antimycin A leads to a partial depolarization of the resting platelet's membrane. These pre-incubated platelets then exhibit a decreased membrane potential change upon a-thrombin stimulation. It is known that the platelet continuously replenishes its glycogen. The availability of such energy stores may be responsible for the restoration of approximately 20% of the thrombin response which we have observed after 3 hours of incubation with antimycin A. The intracellular glycogen does not appear to play a major role in the early platelet response to thrombin which is unimpaired 30 minutes after the completion of gel-filtration, the time at which the intracellular glycogen levels are at their lowest. These studies indicate that the thrombin-induced platelet membrane potential change, like other steps in platelet activation, depends upon maintenance of continuous metabolic function.

Key words: thrombin.

Metabolism, membrane potential, platelet activation (or response),

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INTRODUCTION The basic platelet reaction initiated by platelet aggregating agents comprises the sequential steps of induction',transmission, and execution (shape change, aggregation and release) (1) which require the maintenance of a minimal energy level (2-5). A glucose-containing platelet suspension medium can satisfy the energy requirements for ATP synthesis for a period of time -ex vivo (6). Almost 80% of the glucose transported into the platelet is metabolized by anaerobic glycolysis while most of the remaining 20% processed is through the Krebs cycle and oxidative phosphorylation by the limited number of platelet mitochrondria (7,8). Energy is also stored in the platelet in the form of glycogen and of fatty acids, but the extent to which their utilization contributes to the energy required for the platelets' stimulus response has not yet been delineated (9). When one of the two major ATP-generating sequences is blocked, the others can compensate somewhat (1,2,4,10), but not always sufficiently to sustain optimal platelet function -ex vivo (lO,!.l). When two pathways are inhibited simultaneously, the entire platelet response to stimuli is severely impaired (12,13). Since the platelet can store as much glycogen as skeletal muscle on a gram for gram basis, glycogenolysis contributes substantially to the glycolytic flux (8,14), but cannot preserve normal ATP stores if tbe respiratory chain is uncoupled by antimycin A (AA) (15) or if glycolysis is blocked by 2-deoxy-D-glucose (2-DOG) (16). In the presence of both metabolic inhibitors, the required continuous ATP production is thus blocked and the platelet's response to stimuli appears to decrease progressively, paralleling the decreased intracellular ATP level (10). It has been demonstrated that the process of glyconeogenesis is continuous within the platelet even when the rate of glycogen degradation increases during platelet stimulation (7,14,17). The platelet exhibits stimulus-response coupling as shown by dose-dependent changes in transmembrane potential and pH gradient paralleling serotonin release (18-22) as well as in calcium release and in non-metabolic ATP secretion (22). The involvement of its metabolic processes in the stimulusresponse coupling has hence been of interest. We have therefore used transmembrane potential changes, an early and readily quantitated effect (l8-2L) to investigate the metabolic dependence of stimulus-response coupling in the thrombin-platelet reaction. MATERIALS AND METHODS Platelet Preparation: Blood, drawn by peripheral venipuncture from normal human volunteers. was added to 3.8% sodium citrate in a ratio of 10 to 1 and centrifuged at 126 x g for 10 minutes at ambient temperature. The platelets were prepared from the resultant platelet-rich plasma (PRP) as previously described (18) by gel-filtration in glucose-free Tyrodes' buffer containing apyrase: 0.14 M NaCl, 12 mM NaHC03, 2.7 mM KCl, 1.0 mM MgC12, and 0.5% bovine serum albumin at pH 7.35. The eluted gel-filtered platelets (GFP) were diluted to a final concentration 55 x lo6 per ml after which stock solutions of the metabolic agents were added to final concentrations of 2 x 10s5 M antimycin A (in ethanol, final ethanol concentration 0.2% (V/V)), 5.6 mM 2-deoxy-D-glucose, or 5.6 mM D-glucose (stocks in 0.15 M NaCl). The time elapsed between venipuncture and the incubation with inhibitors was 1 hour. Reagents: Bovine a-thrombin was purified chromatographically from topical bovine thrombin (Parke-Davis) using the method of Lundbland (23,24). Its activity is expressed in fibrinogen clotting units (23).

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Antimycin A (AA), Z-deoxy-D-glucose (2-DOG), adenosine S'triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADP+), and bovine serum albumin were purchased from Sigma Chemical Company. Hexokinase and glucose6-phosphate dehydrogenase were obtained from Calbiochem Behring Corp., and the cyanine dye, 3,3'-dipropylthiodicarbocyanine (diS-C3-(S)), was the kind gift of Dr. Alan Waggoner. Assay for Membrane Potential Chances: Fluorescence measurements were made on a Perkin Elmer 650-10s spectrofluorimeter equipped with a stirring apparatus and a thermostatted cuvette holder. Membrane potential changes were monitored as previously described, using diS-C3-(5) as a probe (18-21) and expressed as (F-Fo)/Fo, where F is the fluorescence level at one minute after stimulation and F, is the initial haseline fluorescence intensity of the unstimulated platelet suspension. Assay for Glycogen Content: Platelet glycogen was determined by a modification of the method of Schwartz and Rall (25). The measurements were made on platelets hydrolyzed in 1.0 ml 30% (w/v> YOH for 30 minutes. The digest was incubated at 4OC for at least 1 hour after the addition of 1 drop of saturated Na2S04 and 1.5 ml of ice-cold 100% ethanol. After centrifugation at 3500 x g at 4OC for 10 minutes, the precipitate was dried and then hydrolyzed in 1 M HCl at 100°C for one hour, neutralized, and lyophilized. Fluorimetric determinations of the glycogen hydrolyzed to glucose in the redissolved samples were performed by a coupled hexokinase-glucose-6-phosphate dehydrogenase assay (26). A 100 ul aliquot of the sample was added to 1.0 ml of a reaction buffer containing 50 mM Tris (hydroxymethyl) aminomethane, pH 7.5, 10 mM MgC12, 3 x 10Y4 M ATP, 8 x 10q5 M NADP+ and .0025 U/ml glucose-6phosphate dehydrogenase. At zero time 3 ul of a 100 IU/ml stock solution of hexokinase was added. The reaction was allowed to proceed for 20 minutes at 25OC and the formation of NADPH followed by the increase in its fluorescence intensity at 460 nm upon excitation at 340 nm. Glycogen content is expressed as nanograms of glucose equivalents per 55 x 106 platelets. RESULTS Gel-filtered platelets (GFP) were incubated at 37OC in the presence of either D-glucose, antimycin A (AA), 2-deoxy-D-glucose (2-DOG), D-glucose plus antimycin A, or 2-deoxy-D-glucose plus antimycin A, compounds which by themselves do not alter the fluorescence of cell-free 2 x 10V6 M diS-C3-(5). An aliquot of these platelet suspensions equilibrated at 3i'OCin the presence of 2 x 10-6 M diS-C3-(5) exhibited, as we have previously demonstrated, a baseline fluorescence F, which depends upon the membrane potential of the resting platelets. This resting value (approx. 45.5% of that exhibited by dye in buffer), was unaltered by the presence of glucose, but was increased (corresponding to a depolarization of the membrane) by pre-incubation with either 2-DOG of AA (Table I). The effects of these two inhibitors, when used simultaneously, do not appear to be additive. When 8 yM digitonin, a concentration high enough to penneabilize the plasma but not the organellar membranes (27,26), was added to these platelets, release of diS-C3-(5) occurred. When the probe had reequiiibrated (within 30 sec.j, the new fiuorescence intensity F-",ig ('TableIj was similar in glucose and in 2-DOG-preincubated plateiets, but 12% iower in those exposed to AA.

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FIG. 1 Thrombin-induced DiS-C3-(5) fluorescence changes in gel-filtered platelet suspensions incubated with 5.6 x 10-3X glucose (@-+I, glucose plus 2 x 10q5Y AA @*-*A>, AA (O***O), 5.6 x lo-311 2-DOG (O--O), and 2-DOG plus AA (m--m).

INCUBATION

TIME

(min.)

FIG. 2 Total glycogen isolated from gel-filtered platelets, measured as glucose. Additions at zero incubation time: 5.6 x 1@-3M glucose (w ) , glucose Plus 2 x 10-5~ &I. (A.--A), AA (o...o), 5.6 x 10m3M 2-DOG (o-o), and 2-DOG plus

AA (H--=1.

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Table I Effect of Metabolic Inhibition on the Membrane of Unstimulated Platelets Average No platelets (n=4) Glucose (n=7) Z-DOG (no glucose) (n=3) AA (no glucose) (n=4) AA (with glucose) (n=3) AA + Z-DOG (n=3)

56.5 25.7 34.3 31.4 29.1 34.3

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Potential

Average F$dig(a)

47.0 48.7 42.7 42.2 41.5

+ 0.8 + 1.3 + 1.1 + 2.0 +- 2.3

(a) mean of indicated number (n) of experiments + standard deviation. Fo : Baseline (equilibrium) fluorescence intensity. %dig = New equilibrium fluorescence intensity attained within 30 seconds of addition of 8uM digitonin.

Platelets equilibrated with metabolic inhibitor(s) and fluorescent probe were then subjected to stimulation by 0.025 U/ml a-thrombin injected remotely by microsyringe. The fluorescence was monitored continuously and increased linearly for the first 30-60 seconds indicating a constant initial rate of depolarization (18). In the presence of one of the metabolic inhibitors, the extent of stimulatability decreased with increasing incubation time (Figure 1), but remained 5-10% of the controls for up to 2% hours after the inhibitor had been added. It decreased more rapidly when both inhibitors were present simultaneously. Although the availability of glucose during AA incubation did not noticeably alter the early AA-induced inhibition of the platelet thrombin response (~2.5 hours) an apparent recovery of 20% of the initial response after longer (3% to 4) hour incubation was noted. Figure 1 is representative of four similar experiments. The glycogen content of washed platelets, already lower than that of the corresponding number of platelets in PRP by approximately 37-52% (8), decreased further, attaining a minimum level %4_5 minutes after the completion of gel-filtration (105 minutes after venipuncture) (Figure 2). This minimum is less pronounced (700 to 1,000 ng/glucose equivalents/55 x lo6 platelets) if the gel-filtration is conducted in the presence rather than the absence of glucose (data not shown on Figure 2) but both preparations exhibit a regeneration of glycogen stores to the same extent by 255 hours total elapsed time. DISCUSSION Normal platelet metabolism must supply sufficient energy from the maintenance of basal function in the resting state as well as provide a sufficient level of metabolic ATP immediately available upon activation. This energy is derived from circulating glucose _in vivo and requires a glucose-containing buffer _ex vivo (6,8,10,28,29). Gel-filtration or centrifugation utilize additional energy which can be derived by an enhanced rate of glycogenolysis (2,10,14,28-31). Although platelets can derive energy from fatty acids (9), the extent to which these replenish ATP stores exhausted by separation processes or platelet activation is not yet known. The enhanced glycogenolysis is corroborated by our finding that the platelet glycogen content decreased during the hour long gel-filtration process regardless of the availability of

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exogenous glucose (data not shown). Glycogen breakdown continued for about 45 minutes even if exogenous glucose was added after gel-filtration (Figure 2) These observations are compatible with glycogenolysis as a mechanism of preserving function of metabolic integrity and of maintenance of transmembrane gradients in the unstimulated platelet. The contribution of fatty acid degradation to these mechanisms remains ill defined. It has been reported that the capacity of the platelet to respond to a specific stimulus (e.g.: a-thrombin) decreases in the presence of glycolytic and respiratory inhibitors or during stressful preparative procedures (1,8, 10,28-31). Our results corroborate these findings. Furthermore, they allow us to conclude that incubation for over 1 hour with 2-DOG decreases the diS-C3-(5) uptake (i.e., the quenching, since internalized probe does not fluoresce) by 33%, corresponding to a proportional decrease in the resting platelet's membrane potential (18). Preincubation with AA decreases it by only 22%. However, the reasons for these changes are clearly different, as shown by the digitonin-treated aliquots. After 2-DOG or glucose incubation the same amount of probe release occurs, while less is released from the cytoplasm of AA treated platelets, implying that less prohe is available to it. Since AA uncouples the respiratory chain while 2-DOG does not (15), the trapped probe (12% of that released in the absence of AA) is probably associated with the mitochrondria. Stimulation by o-thrombin leads to a smaller depolarization in inhibitor treated than in control platelets. The effect of AA or 2-DOG on this response far exceeds that on resting platelets (70% or more vs. 22 to 33%). These studies of stimulus-response coupling evaluate only the initial linear rate of. that response (18) and not the eventual maximal value attained. We have no information on the latter as yet. It should be noted that the rate of response by a platelet to thrombin stimulation is comparable in an AA or in a 2-DOG treated platelet, but slower Thus, the rate of response of inhibitorin each than in control platelets. treated platelets does not necessarily depend upon the mode of action of the It is however possible that this observation may be metabolic inhibitor. explained by the fact that 2-DOG and AA each block a metabolic pathway responsible for approximately half of the energy stored as ATP (7,8) but produced in different cellular compartments. It is apparent that the availability of glycogen, while necessary for ex vivo maintenance of the resting transmembrane potentials, does not affect -Utilization of that energy store does not the thrombin response directly. correlate temporally with platelet-thrombin response, and the store is still high (SO%) after long incubation when thrombin response has virtually been eradicated (Figures 1 and 2). We do not know how fatty acid availahility affects our findings. We conclude that while the role of the rapid transmembrane potential changes associated with thrombin stimulation of the basic platelet reaction sequence is not yet elucidated,:their dependence upon a continuous exogenous energy source is demonstrated by this study. ACKNOWLEDGEVENT We thank Dr. Alan Waggoner of Amherst College for his kind gift of These studies were diS-C3-(5), and John C. Whitin for valuable discussions. supported in part by NIH grants HL 15335 and T 32HL 7501.

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