Soluble guanylate cyclase of platelets: Function and regulation in normal and pathological states

Advan. Enzyme Regul., Vol. 32, pp. 35-56, 1992 Printed in Great Britain. All fights reserved

0065-2571/92/$15.00 ~) 1992 Pergamon Press plc.

SOLUBLE GUANYLATE CYCLASE OFPLATELETS: FUNCTION AND REGULATION IN NORMAL AND PATHOLOGICAL STATES I. S. SEVERINA Institute of Biologicaland MedicalChemistry,U.S.S.R. Academyof Medical Sciences, Moscow119832,U.S.S.R. INTRODUCTION Guanylate cyclase (EC 4.6.1.2) catalyzes biosynthesis of cyclic 3',5'guanosine monophosphate (cGMP) that is a powerful regulator of cell metabolism which significantly determines the functions of a cell. Guanylate cyclase exists in two forms: soluble and membrane-bound. It has been established now that the above forms are not only different proteins but also differently regulated enzymes (1). At the same time, the molecular mechanism of action of these enzymes has not been elucidated yet. Although the regulatory mechanism is far from being entirely clear, it is evident that guanylate cyclase is a very complex enzyme with a fairly fine and, apparently, unique mechanism of regulation. The great physiological significance of both forms of the enzyme makes it necessary to investigate each of these forms separately. The present paper is devoted to the soluble form of guanylate cyclase. The paper will consider the properties of guanylate cyclase from human and rat platelets which are directly connected with the endogenous regulation of the enzyme. The main emphasis will be on the role of heme in the functioning of guanylate cyclase, the role of guanylate cyclase in the regulation of platelet aggregation, as well as the guanylate cyclase activity and its regulation in rat heart and platelets in the case of acute ischemia of myocardium. A distinctive feature of soluble guanylate cyclases is the presence of heme, as a prosthetic group in the enzyme molecule (2). It is known that the immediate precursor of heme is protoporphyrin IX that has proved a strong activator of guanylate cyclase (3). The introduction of iron into the porphyrin ring leads to the formation of ferroprotoporphyrin IX, or heme, an inhibitor of guanylate cyclase (4). The sequence of reactions resulting in the formation of the guanylate cyclase holoenzyme, namely: protoporphyrin I X - iron- h e m e - guanylate cyclase, remains obscure. However, this system is essential to the endogenous regulation of guanylate cyclase. The role of heine in the functioning of soluble guanylate cyclase is thought to be mainly connected with the enzyme activation by nitric oxide (NO) and 35

36

I.S. SEVERINA

compounds that contain or form a free radical group NO. Therapeutic effect of vasodilators (such as glyceroltrinitrate, nitroprusside, etc.) is based on the activation of guanylate cyclase and accumulation of cGMP. As a result of interaction of a free radical group NO of these compounds with guanylate cyclase heme, the formation of the nitrosyl-heme complex and accompanying structural changes in the heme (4), guanylate cyclase activity gets stimulated. The important role of heme in the regulation of guanylate cyclase has acquired special significance after the discovery of Moncada (5) who established in 1987 the endogenous nature of NO. It is known that in endothelial cells, in response to the interaction of hormones and neurotransmitters (acetylcholine, histamine and others) with corresponding receptors, an endothelium derived relaxing factor (EDRF) is formed (6). In 1987 E D R F was identified as NO (5) formed from L-arginine (7) under the effect of L-arginine-NO-synthase. The vasodilating effect of E D R F is caused by cGMP accumulation which is the result of direct stimulation of guanylate cyclase (8). Today NO formation from L-arginine has been described, apart from endothelial cells (9), in other cells and tissues as well (10, 11). However, L-arginine-NO-synthase has so far been studied insufficiently. The question of the identity of E D R F and NO is not solved finally either. It is not excluded (12) that E D R F is a NO-containing compound. Meanwhile, there is no doubt that the physiological effect of NO is determined by guanylate cyclase activation due to nitrosylation of guanylate cyclase heme and cGMP accumulation. Thus, endogenous NO is a key factor of endogenous regulation of guanylate cyclase and elucidation of the role of heme in the guanylate cyclase functioning is connected both with the problem of endogenous regulation of guanylate cyclase, efficiency of most widely used nitrovasodilators and the problem of blood vessel tone as well. The nature of the bond between the heme and the protein guanylate cyclase molecule is obscure but lability of this bond has been proved. In the case of a decrease in pH of the medium (to pH 5), in the process of storage or purification of the enzyme, the heme can dissociate from the guanylate cyclase molecule, thereby causing a certain degree of heme deficiency of the enzyme (13). The strength of the heme binding in the guanylate cyclase molecule varies depending on the source of the enzyme (14, 15). There are no data indicating possible existence in tissues of soluble guanylate cyclase originally in the heme-deficient form. No doubt, heme deficiency of guanylate cyclase causes not only disorders in the endogenous regulation of the enzyme but also results in reduced efficiency of nitrovasodilators. MATERIALS AND METHODS Human platelets were isolated from the venous blood of donors as described (16). Rat platelets were obtained from the blood of decapitated

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

37

animals according to (17). E D T A was used as the anticoagulant (0.1 M and 0.2 M for human and rat platelets, respectively, with an EDTA-blood ratio of 1:10) (v/v). The suspension of washed platelets in 50 mM Tris-HCl buffer, pH 7.6, containing 0.2 mM dithiothreitol (DTF) was sonicated at 0°C for 20 sec on a MSE - - 5-78 sonicator (UK), and then centrifuged at 105,000 x g for 1 hr. Supernatants were chromatographed on DEAE-cellulose 52 (Whatman, UK). Two columns (2 ml each) were equilibrated with 50 mM Tris-HCl buffer (pH 7.6), containing 0.2 mM D T r . Each supernatant (2-4 mg by protein) was applied to the column. After washing the column with 50 mM Tris-HCl buffer (pH 7.6), guanylate cyclase was eluted with the same buffer, containing 0.22 M NaCI. The enzyme activity was assayed by the method of Murad (18). The samples (final vol 150/~1) contained 50 mM Tris-HCl buffer (pH 7.6), 1 mM GTP, 4 mM MgCI2, 4 mM creatine phosphate, 20 mg (120-160 units) of creatine phosphokinase, 10 mM theophylline and the 105,000 x g supernatant or the guanylate cyclase preparation after DEAE-cellulose chromatography (10/~g or 2/zg, by protein, respectively). Some additives were used if necessary. The amount of cGMP formed in the course of enzymatic reaction (10 min at 37°C) was determined by radioimmune assay using the cGMP RIA Kit (Amersham, UK). In the experiments with human platelets, the activating effect of sodium nitroprusside (final concentrations 0.1 mM and 0.25 raM, with 105,000 x g supernatant or with the guanylate cyclase preparation after DEAE-cellulose chromatography, respectively) was determined after its preincubation with the enzyme for 45 min at 0°C. Carnosine (13-alanyl-L-histidine) was used at the final concentration of 0.1 mM and preincubated with guanylate cyclase for 10 min at 0°C before the substrate or sodium nitroprusside were added. For the determination of cGMP content in platelets, the platelet-rich plasma (PRP) was used. PRP was obtained after centrifugation of human blood samples containing 1/10 vol of 3.8% sodium citrate at 450 g for 10 min at room temperature, 4 ml of PRP were centrifuged at 1500 g for 3 min and the platelet pellet was suspended in 0.4 ml of 4 mM EDTA. After sonication and extraction in a boiling-water bath for 5 rain, the samples were centrifuged at 3000 x g for 15 rain; the supernatant was assayed for cGMP concentration. The protein was determined by the method of Lowry (19). The platelet aggregation was studied with healthy donors. Platelet aggregation in PRP was induced by ADP and studied according to the turbidimetric method of Born (20). The appropriate platelet count in PRP was adjusted with platelet-poor plasma (PPP). PPP was obtained by centrifugation of PRP at 650 x g for 30 min. Aggregation was expressed as the percentage fall in optical density after adding ADP. The difference in

38

I.S. SEVERINA

optical density between PRP and PPP was arbitrarily set at 100% (21). The following reagents were used in the study: GTP sodium salt (Fluka, Switzerland), Dq-'F (Serva, Germany), sodium nitroprusside (Chemapoi, CSFR), carnosine ([3-alanyl-L-histidine) and protoporphyrin IX (Fluka, Switzerland). Other reagents and salts were from Sigma (USA). RESULTS

AND

DISCUSSION

Role of Heme in the Guanylate Cyclase Functioning Earlier, when we compared the capacity of soluble guanylate cyclase preparations from different sources to be activated by sodium nitroprusside (22), we failed to reveal any stimulating effect of this compound on rat platelet guanylate cyclase. In the meantime, 20-30-fold nitroprussideinduced activation of the enzyme from human platelets and rat heart was noted (22). Because of the lack of information concerning rat platelet guanylate cyclase we studied this enzyme in more detail. Possible heine deficiency of guanylate cyclase from rat platelets was confirmed by purification studies. The 105,000 x g supernatants of human and rat platelets were chromatographed on D E A E cellulose in 50 mM Tris-HCl buffer (pH 7.6), with subsequent elution of the enzyme with 0.22 M NaCl. II

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FIG. 1. Chromatography on DEAE-cellulose of 105,000 x g supernatants of human platelets (A) and rat platelets (B) using stepwise elution with 0.22 M NaCI. Elution buffer: 50 mM Tris-HCl (pH 7.6) containing 2 x 10-4 M Dq'T. Abscissa - - fraction number; ordinate - protein profile (/zg/ml) - - continuous line; specific activity of guanylate cyclase preparation in the presence of Mg 2+ (pmoi/min/mg protein) - - dotted line.

39

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

Figure I shows that the profiles of stepwise elution of human (Fig. 1A) and rat (Fig. 1B) platelet guanylate cyclase are actually identical and contain 2 protein peaks (I and II); only peak II possesses guanylate cyclase activity. In both cases the activity is represented by a fairly sharp peak. In experiments with human platelets, the total guanylate cyclase activity (with Mg 2÷) eluted from the column amounts to 171 _ 14%, whereas with rat platelets, this value was 87 + 10% (the activity of guanylate cyclase (with Mg 2÷) applied to the column was taken as 100%). The overall protein amount eluted from the column was 95-100% in both cases. The increase (1.5-2-fold) of total guanylate cyclase activity eluted from the column in human platelet experiments in the result of D E A E chromatography indicated the removal of some endogenous inhibitor. As can be seen from Table 1, this inhibitor is really in the inactive protein peak I. The addition of the inhibitor to the active fraction of peak II inhibits its activity by 70%. The guanylate cyclase preparation obtained loses about 80% capacity to be activated by sodium nitroprusside, though this capacity is restored upon the addition of the "inhibitory" fraction (peak I). In the experiments with rat platelets no increase in the total elution activity has been observed in peak II; however, peak I does not possess the inhibitory properties and rat platelet guanylate cyclase is not activated by sodium nitroprusside either before or after DEAE-cellulose chromatography (see Table 1). Hemecontaining proteins are known to have a distinctive absorption maximum in the 420-430 nm region (the Soret band). Figure 2A demonstrates that the absorption spectrum of the 105,000 x g supernatant of human platelets contains the absorption maximum in the region of 415 nm. The latter maximum disappears from the spectrum of the guanylate cyclase

TABLE I. EFFECT OF PROTEIN PEAK I ON THE ACTIVITY OF HUMAN AND RAT PLATELET GUANYLATE CYCLASE AND THE ENZYME ACTIVATION BY SODIUM NITROPRUSSIDE Source of enzyme

Specific activity of enzyme (with Mg2+), % Eluate after DEAEcellulose chromatography

Extent of activation by sodium nitroprusside 105,000 x g supernatant

Eluate after DEAEcellulose chromatography

- peak I

+ peak I

- peak I

- peak I

+ peak II

Human platelets

100

33 +_ 5

Rat platelets

100

90 _+ 17

25 _+ 4 (100%) no activation

5 -- 0.9 (20%) no activation

16.8 +_ 3.2 (67%) no activation

40

I.S. SEVERINA

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FIG. 2. Spectra of 105,000 x g supernatants of human platelets (A) and rat platelets (B) before and after chromatography on DEAE-cellulose. (A) 105,000 x g supernatant - - 0.58 mg/ml (1); Peak I (inhibitory) - - 0.15 mg/ml (2); Peak II (guanylate cyclase preparation) - - 0.58 mg/ml (3). (B) 105,000 x g supernatant - - 0.45 mg/ml (I); Peak I - - 0.15 mg/ml (2); Peak II - - (guanylate cyclase preparation) - - 0.28 mg/ml (3). Abscissa - - wavelengths (nm); ordinate - - optical density.

preparation after chromatography on DEAE-cellulose (peak II) but can be observed in the "inhibitory" fraction (peak I). These findings indicate the heme nature of the endogenous inhibitor and heme deficiency of the enzyme preparation obtained. Investigations of the exact nature of this endogenous regulator are now in progress. Our studies with rat platelet guanylate cyclase showed the absence of absorption in the region of the Soret band both in the 105,000 x g supernatant and protein peaks I and II (Fig. 2B). The obligatory condition, however, is that rat platelets should be isolated from plasma free from contamination with erythrocytes. Thus, the failure of stimulating rat platelet guanylate cyclase by sodium nitroprusside as well as the absence in the spectra of the original 105,000 x g supernatant of rat platelets and protein peaks I and II of the absorption maximum in the region of the Soret band (Fig. 2B) allow the conclusion that, unlike human platelet guany|ate

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

41

cyclase, the enzyme from rat platelets is present in these cells in the heme-deficient form from the very beginning. Absence of the inhibitory action of hemoglobin on basal rat platelet guanylate cyclase activity (17) and a failure to reveal a stimulation of the enzyme by nitroprusside on hemoglobin addition (17) suggest that in contrast to the commonly accepted view, heme is not a prosthetic group of rat platelet guanylate cyclase. Hence, rat platelets cannot be used as a model for studying the action of NO and NO-containing compounds on platelet guanylate cyclase. In view of the revealed original heme deficiency of rat platelet guanylate cyclase, in a series of experiments with the 105,000 x g supernatant of human platelets (donors) we paid attention to an extremely low degree of sodium nitroprusside-stimulated activation of guanylate cyclase (which was only about 5-fold instead of the usual 25-fold); this insignificant activation degree was not decreased after DEAE-cellulose chromatography of the supernatant. Moreover, the total guanylate cyclase activity (with Mg2+) eluted from the column did not exceed that applied to the column. The above data evidenced the original heme deficiency of human platelet guanylate cyclase in this supernatant. In order to check this supposition, we analyzed the results of our study on the effect of carnosine on the functioning of human platelet guanylate cyclase. Carnosine (13-alanyl-L-histidine) is a water-soluble antioxidant (23), which owing to its antioxidant properties is widely used as a therapeutic drug preparation, efficiently applied in the case of inflammatory processes, cataract, for healing wounds, etc., i.e., when as the result of lipid peroxidation, the integrity of cellular membranes is disrupted (24, 25). A characteristic feature of soluble guanylate cyclase is the capacity of the enzyme for being activated by free radicals. The latter are presumed to be endogenous regulators of guanylate cyclase activity. The study of the effect of carnosine on human platelet guanylate cyclase showed (26) that carnosine at a concentration which does not affect the basal activity of the enzyme strongly inhibits (approximately by 70%) activation of guanylate cyclase by sodium nitroprusside (26). Meanwhile, carnosine does not affect the insignificant nitroprusside-induced activation of a heme-deficient guanylate cyclase preparation (26). It also does not produce an inhibitory effect on guanylate cyclase stimulation by protoporphyrin IX (26). It was found that the inhibitory effect of carnosine was caused by its interaction with guanylate cyclase heme. Furthermore, the activating action of nitroprusside on guanylate cyclase was postulated to involve two effects: (i) an insignificant, nonspecific, heme independent rise in activity (possibly due to oxidation of labile SH-groups of the enzyme) and (ii) activity stimulation caused by the interaction of a free radical group NO of nitroprusside with guanylate cyclase heme and the formation of the nitrosyl heme complex. Thus, carnosine appeared to be a specific inhibitor of

42

I.S.

SEVERINA

TABLE2. EFFECTOF CARNOSINEON THE ACTIVATIONOF HUMANPLATELET GUANYLATECYCLASEBY SODIUMNITROPRUSSIDE Enzyme

Extent of activation

105,000 x g supernatant

DEAE-cellulose eluate

control

carnosine (1 mM)

control

carnosine (1 raM)

29 + 1.0 (100%) 6.0 + 0.6 (100%)

10.1 + 1.6 (34%) 4.8 + 0.8 (80%)

4.1 + 0.9 (100%) 5.3 ___0.3 (100%)

4.4 + 1.0 (105%) 5.0 + 0.5 (94%)

heme dependent guanylate cyclase activation by nitroprusside and can be used for evaluating the degree of the enzyme saturation with heme (26). Table 2 gives comparative data on the effect of carnosine upon nitroprusside-induced activation of guanylate cyclase of human platelet 105,000 x g supernatants differing in the intensity of the stimulating effect as well as enzyme preparations obtained after DEAE-cellulose chromatography of these supernatants. As can be seen from Table 2, in our experiments with the 105,000 x g supernatant with an insignificant degree of sodium nitroprusside-induced activation of guanylate cyclase (6-fold instead of conventionally observed 29-fold), the activation degree was not decreased after chromatography of the supernatant on DEAE-cellulose. The above-mentioned low stimulating effect also remained unchanged upon the addition of carnosine. The absence of the inhibitory effect of carnosine on guanylate cyclase stimulation by nitroprusside provides supporting evidence that this activation is hemeindependent and indicates the original heme deficiency of the enzyme in these supernatants. Thus, there are people whose platelet guanylate cyclase has low sensitivity to NO and NO-containing compounds.

Guanylate Cyclase and Platelet Aggregation Heme deficiency of guanylate cyclase is essential not only for exhibiting vasodilating properties of nitric oxide (NO) and NO-containing drug preparations, but also for manifestation of the antiaggregation action. Earlier cGMP was thought to promote platelet aggregation (27). However, it has now been found that guanylate cyclase activators that increase cGMP level in platelets produce the antiaggregation effect (28). The significance of these findings has become especially evident after revealing the endogenous nature of nitric oxide (NO), its capacity for activating platelet guanylate cyclase and manifesting the antiaggregation properties (6, 29). Nevertheless, the role of guanylate cyclase in this process remains

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

43

unclear. Therefore, we decided to explore its involvement using human platelets as a model. Our task was to analyze how guanylate cyclase functions in aggregating platelets, whether any changes take place in guanylate cyclase activity and its regulation in the course of aggregation/disaggregation of platelets. For this purpose, we used a model of ADP-induced reversible aggregation of human platelets (donors) in vitro within a wide ADP concentration range (from 0.5 to 10/zM) at the platelet concentration in plasma of 2.5x108 platelets per ml. Figure 3 shows that platelet aggregability increased with the increase in the concentration of the inducer but in each case had a reversible character, i.e., once the aggregation maximum was achieved, disaggregation followed. It is noteworthy that the higher the aggregation, the more time is needed to achieve the maximal aggregation level (Fig. 3). In the analysis of the effect of aggregation on platelet guanylate cyclase activity and its regulation, we used platelets at the peak of aggregation (Fig. 3) and the comparison was made with control platelets that were not treated with ADP (30). Figure 4 demonstrates that elevation of platelet aggregability is accompanied by increase in nitroprusside-induced guanylate cyclase activation and decrease in basal activity. The reversible phase of ADP-induced aggregation in vitro showed that the maximum aggregation (Fig. 3) is followed by platelet disaggregation. Therefore, we studied the dynamics of changes in the basal guanylate cyclase activity and its capacity

80.

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FIG. 3. Dependence of platelet aggregation on A D P concentration in plasma. Abscissa - - time (rain) after A D P addition; final A D P concentration (p,M) is designated near corresponding aggregograms; ordinate - - platelet aggregation (%).

44

I.S. SEVERINA 200

150

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FIG. 4. Changes in the basal guanylate cyclase activity and the enzyme activation by sodium nitroprusside in platelets during aggregation. (1) Guanylate cyclase activation by sodium nitroprusside, regression factor -- 0.90. (2) Basal guanylate cyclase activity (with Mg2+), regression factor -- 0.89. Abscissa -- platelet aggregation (%); ordinate -- changes in guanylate cyclaseparameters (% of control).

for activation in the process of platelet aggregation-disaggregation, i.e., using enzyme preparations isolated from platelets at different levels of aggregation. Figure 5 shows the results obtained with 5/zM A D P causing 80% reversible aggregation (Fig. 5, curve 5). Immediately after the A D P addition, the response of guanylate cyclase to the added nitroprusside begins to increase and in several min achieves its maximum, exceeding 2-2.5-fold of the initial value at this point (Fig. 5, curve 3). After that the reverse process follows and by the 10-12th min the original level of guanylate cyclase aggregation is restored. The basal guanylate cyclase activity also changes: first the activity is observed to fall, with the minimal value at a point corresponding to the maximum of nitroprusside-induced activation of the enzyme (Fig. 5, compare curves 4 and 3), then activity becomes normalized and returns to the initial value (Fig. 5, curve 4). The decrease in the basal activity revealed in our experiments (see Fig. 4) does not seem, at first sight, to correspond to the literature data so far reported about elevation at this period of intraplatelet c G M P level. However, we too confirmed a rise in the cGMP level in the course of aggregation. As can be seen in Figure 5 (curve 1), the induction of aggregation is accompanied by the enhancement of c G M P content which reaches its maximum (exceeding the original level 3.5-fold) at a point corresponding to the maximum of the curve of guanylate cyclase activation by sodium nitroprusside, with the subsequent decrease in the c G M P content to the initial level. It should be noted that

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

45

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FIG. 5. Dynamics of changes in the cGMP level (1), guanylate cyclase heme deficiency(2), guanylate cyclase activation by sodium nitroprusside (3) and basal guanylate cyclase activity (4) at 80% reversible platelet aggregation induced by 5 p,M ADP (aggregogram-- curve 5). Abscissa -- time (min) after ADP addition; ordinate - - changes in the measured parameters (% of the initial value). The results of a typical experiment are presented. Each point is the mean of 2 or 3 parallel determinations.

activity of c G M P dependent phosphodiesterase during aggregation does not change (31). We also found that the phosphodiesterase activity in control platelets, in platelets at the peak of aggregation, and after the process was normalized remained the same (data not shown). Thus, the reported results support the existing notion that c G M P elevation in tissues is mainly due to guanylate cyclase activation. The dynamics of change in the basal guanylate cyclase activity, its ability for activation and c G M P content in platelets in the course of aggregation/disaggregation (see Fig. 5), are identical at all levels of reversible aggregation. However, it should be mentioned that the proportionality between the increase in the degree of reversible aggregation and the enhanced capacity for guanylate cyclase activation by sodium nitroprusside (as shown in Fig. 4) is preserved merely within the limits up to 40-50% reversible aggregation. Within this range, the aggregation peak coincides with maximum guanylate cyclase activation. In

46

I.S. SEVERINA

case of a further increase in the extent of reversible aggregation, for example at 80% reversible aggregation, as shown in Fig. 5, the maximal guanylate cyclase activation remains at a point corresponding to approximately 50% of reversible aggregation. This is followed by normalization of guanylate cyclase parameters, despite the fact that 80% reversible aggregation has not reached its maximum yet. Apparently, a rise in the degree of reversible aggregation above 50% (in vitro conditions) causes the process to become gradually irreversible, when the protective functions of the guanylate cyclase system are exhausted. Indeed, the same changes in guanylate cyclase parameters (with the maximum of changes at 40-50% aggregation) were also observed in the case of development of irreversible aggregation (when l0 p.M ADP was used) (data not shown), i.e., changes in the functioning of platelet guanylate cyclase during aggregation occur at the earliest stages of the process. If guanylate cyclase activation is the response of the cell directed to the decrease of ADP-induced platelet aggregation, preincubation of platelets with nitroprusside prior to the ADP addition should lead to the weakening of aggregation, right down to its entire prevention. Our data demonstrated that at the peak of 50% reversible aggregation, a fall in the basal activity (from 182 + 14 to 119 + 9 pmol cGMP/min/mg protein) with concomitant rise (from 9.9 + 0.7 to 16.2 + 1.1) in the degree of sodium nitroprusside-induced guanylate cyclase activation, was observed. In platelets whose aggregation was prevented by nitroprusside (0.1 mM nitroprusside was introduced 3 min before the addition of ADP) the latter parameters of platelet guanylate cyclase did not differ from the control ones and equaled 196 + 15 and 9.4 + 0.4 for the basal activity and the activation degree, respectively. Nitroprusside not only prevents aggregation but also promotes disaggregation. Figure 6 demonstrates that the addition of nitroprusside at the peak of 50% reversible aggregation enhances platelet disaggregation. The antiaggregational action of nitroprusside increases with the elevation of its concentration and becomes maximal at 1 × 10-4 M (see Fig. 6). The maximal rate of nitroprusside-induced disaggregation is observed at the time corresponding to the maximal increase in the degree of guanylate cyclase activation by sodium nitroprusside (data not shown). Thus, the efficiency of the disaggregation process is greatly dependent on the activity and reactivity of platelet guanylate cyclase. As mentioned above, heme deficiencyof guanylate cyclase is accompanied by lowering of responsiveness of the enzyme to sodium nitroprusside. The more heme deficiency is expressed, the lower the stimulating effect of nitroprusside, and vice versa. The enhancement of nitroprusside-induced guanylate cyclase activation upon the addition of hemoglobin (about 1.5-1.8-fold) (22), was presumed to be associated with the additional formation of the nitrosyl-heme complex at the expense of hemoglobin, its transfer to guanylate cyclase, thus compensating for its heine deficiency

SOLUBLE G U A N Y L A T E CYCLASE OF PLATELETS

80

47

SNP

60 40 20

lO-4M

lO-SM

FIG. 6. Effect of sodium nitroprusside on ADP-induced platelet aggregation. Abscissa - time (min) after ADP addition; ordinate - - platelet aggregation (%). Reversible aggregation (2.5 x 108 platelets/ml, 2/zM ADP). The arrows indicate the time of the addition of sodium nitroprusside at concentrations designated near the corresponding aggregograms. The results of 4 experiments of the same type are presented.

(32). Studies on the hemoglobin effect upon guanylate cyclase activation by sodium nitroprusside in the course of platelet aggregation revealed changes in the efficiency of hemoglobin action. As can be seen in Figure 5, during platelet aggregation, the stimulating effect of hemoglobin gets lower, then is entirely lacking at the time corresponding to the maximal nitroprusside-induced activation of the enzyme, and is restored afterwards. Thus, one can suppose that the initiation of aggregation is accompanied by increasing the saturation of the enzyme with heme which may account for the enhancement of the ability of the enzyme to be activated by sodium nitroprusside. A similar heme-dependent mechanism of guanylate cyclase activation underlies the enzyme stimulation by endogenous NO (8). The formation of NO from L-arginine was recently shown in human platelets (33). Meanwhile, the mechanism of guanylate cyclase activation by protoporphyrin IX and arachidonic acid is known (26, 34) not to involve heme. Moreover, the stimulating effect of arachidonic acid was reported (34) to be higher in the case of heme-deficient guanylate cyclase. Therefore, we compared the effect of L-arginine, protoporphyrin IX and arachidonic acid with that of sodium nitroprusside on the functioning of human platelet guanylate cyclase during platelet aggregation. It is clear from Figure 7 that the induction of aggregation is followed by an increase in the degree of guanylate cyclase activation not only by nitroprusside but by protoporphyrin

48

I.S. SEVERINA

150m

to

i 140" 130' 120" 1t0'

! 2 3 4

100' TimeafterADPadditionImln)

FIG. 7. Dynamics of change in guanylate cyclase activation by sodium nitroprusside (1), protoporphyrin IX (2), arachidonic acid (3) and L-arginine (4) at 45% reversible aggregation induced by 1 /.LMADP. The activating effects of sodium nitroprusside (0.1 mM) and arachidonic acid (6 ~M) were determined after their preincubation with the enzyme (separately) at 0°C for 45 and 30 min, respectively. In similar experiments with protoporphyrin IX (5 /~M) and L-arginine (1 mM), no preliminary incubation with guanylate cyclase was needed. The samples were then incubated for 15 min at 37°C. The amount of cGMP formed was determined as indicated in Methods. Abscissa - - time (min) after ADP addition; ordinate - - changes in guanylate cyclase activation (% of the initial value). The mean values of 3 experiments of the same type are given.

IX, arachidonic acid, and L-arginine as well. The dynamics of changes in the degree of activation with the latter stimulants is the same as in the case of sodium nitroprusside (compare curves 2, 3, 4 with curve 1, Fig. 7). The maxima of guanylate cyclase activation with the above compounds coincide in time and intensity with the peak of nitroprusside-induced guanylate cyclase activation. Later on, the intensity of guanylate cyclase stimulation with each of the activators falls to the initial level. Thus, once the ADP-induced aggregation is initiated, guanylate cyclase becomes more sensitive to all enzyme activators used irrespective of the involvement of the guanylate cyclase heme in the mechanism of the enzyme activation. The results provided evidence that the initiation of aggregation induces guanylate cyclase activation, promotes the accumulation of cGMP and

49

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

inhibition of aggregation. In other words, guanylate cyclase acts as the negative feedback mechanism to control platelet aggregation and mediates a signal to disaggregation. The molecular mechanism of cGMP involvement in the process of platelet aggregation is so far unclear. Figure 8 represents the proposed hypothetical scheme of likely sites of cGMP action as a regulator of platelet aggregation. The scheme has been developed on the basis of literature data and our own results but is somewhat simplified, because the regulatory role of cGMP was considered without taking into account its relationship to other secondary messengers. At the same time this scheme was very convenient for our studies. As follows from the Figure: cGMP suppresses the release of arachidonic acid by inhibiting phospholipase A 2 activation and thus prevents the formation of thromboxanes A 2 and B 2. The latter are powerful stimulators of Ca 2÷ release and cause platelet activation and aggregation. cGMP impedes the formation of 1,2-diacylglycerol and inositoltriphosphate through the inhibition of phospholipase C activation. 1,2-Diacylglycerol is a potent activator of protein kinase C which

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_ ,

,

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~I IMaWJzmR I I

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FIG. 8. Model summarizing the possible role of platelet soluble guanylate cyclase and cGMP

in regulation of platelet aggregation.

50

I.S. SEVERINA

phosphorylates platelet proteins (20 kDa and 40 kDa), causes platelet activation with subsequent aggregation. Inositoltriphosphate which increases the intracellular Ca 2+ concentration also causes platelet activation and aggregation. Thus, by inhibiting 1,2-diacylglycerol and inositoltriphosphate formation, cGMP inhibits platelet aggregation. cGMP inhibits the formation of phosphatidic acid which is readily formed in platelets from 1,2-diacylglycerol under the action of 1,2-diacylglycerol kinase. Phosphatidic acid acts as an ionophore: it provides the release of intracellular Ca 2+, causes platelet activation and their subsequent aggregation. In other words, cGMP prevents the disintegration of phospholipids, including inositol-containing phospholipids, and inhibits platelet activation and aggregation via a common mechanism of inhibition of Ca 2÷ accumulation. Indeed, in preliminary experiments we have shown intraplatelet Ca 2÷ elevation during ADP-induced platelet aggregation, which was lacking if the platelets were treated with sodium nitroprusside prior to ADP addition (data not shown). The scheme presented in Figure 8 suggests a possible mechanism of enhancing the capacity of guanylate cyclase to be stimulated by various activators during ADP-induced platelet aggregation (see Fig. 7). The increased disintegration of inositol phospholipids during aggregation, accumulation of Ca 2÷ and 1,2-diacylglycerol led to the stimulation of protein kinase C activity and intensification of phosphorylation processes. It is very likely that platelet guanylate cyclase may be among the substrates of protein kinase C (35). Phosphorylation of guanylate cyclase is known to be accompanied by the enzyme activation (36). One can suppose that guanylate cyclase phosphorylation is one of the first responses of the cell to the initiation of aggregation directed to cGMP elevation and inhibition of aggregation. Thus, the results indicate that changes in the functioning of platelet guanylate cyclase and the ability of platelets for aggregation are interrelated. Consequently, platelets characterized by the increased aggregability should differ from normal platelets by modifications in the functioning of guanylate cyclase. A rise in platelet aggregability accompanies a variety of human diseases. We have chosen patients with diabetes mellitus as the model for our investigations. Earlier many authors noted a high level of spontaneous aggregation in vivo for this type of disease and the increased sensitivity of such platelets to inducers of aggregation in vitro (37, 38). However, the investigations in vitro referred to the irreversible stage of aggregation. In our studies, we concentrated our attention on the reversible phase of ADP-induced aggregation. We studied sensitivity to ADP-induced aggregation as well as activity of

SOLUBLE G U A N Y L A T E CYCLASE OF PLATELETS

51

platelet guanylate cyclase and its regulation in platelets of healthy donors and patients with type I and type II diabetes mellitus (21). It was shown (21) that platelets of diabetic patients are 1.62-fold (for type I) and 2.33-fold (for type II) more sensitive to ADP than normal platelets. A decrease in the basal guanylate cyclase activity and the reduced capacity of the enzyme for activation by sodium nitroprusside and protoporphyrin IX (39) were also noted (see Table 3). As can be seen in Table 3, the above-detailed decreases in the guanylate cyclase parameters are expressed more in the case of type II diabetes than for type I diabetes. In other words, differences have been revealed in the activity of platelet guanylate cyclase and its regulation not only between healthy donors and diabetic patients but which is especially important between different types of diabetes mellitus. In this connection, the directed effect on guanylate cyclase could be used for lowering the increased ability of platelets for aggregation. The presented data permit us to consider platelet guanylate cyclase as a protective mechanism in the way of the aggregation development. The

TABLE 3. H U M A N PLATELET GUANYLATE CYCLASE ACTIVITY AND THE ENZYME ACTIVATION BY SODIUM NITROPRUSSIDE AND PROTOPORPHYRIN IX IN 105,000 x g SUPERNATANTS OF PLATELETS FROM HEALTHY DONORS AND PATIENTS WITH TYPE I AND TYPE II DIABETES MELLITUS Extent of activation by

Subjects

ADP concentration needed for 50%aggregation* (~,M)

Donors

2.05

208 + 12 (12)$

11.34 + 0.91 (12)

4.93 + 0.55 (5)

Type I diabetes

1.3

140 + 10 (8)

6.38 __+0.29 (8)

3.04 + 0.10 (4)

Type II diabetes

0.9

105 + 7 (6)

3.59 + 0.37 (6)

1.96 + 0.16 (4)

Activity of guanylate cyclase with Mg 2+ (pmol cGMP/min/mg)

sodium nitroprusside (0.1 raM)

protoporphyrin IX:~ (5 ~M)

*Platelet aggregation in PRP was induced by ADP (see Methods) and was performed with healthy donors of both sexes with normal body weight (mean 71 kg) and aged 22 to 45 years, as well as diabetic patients of both sexes with normal body weight (mean 69 kg) who had been diabetic for a period ranging from 5 to 20 years. The patients were in the state of compensation. Their ages were in the range of 20 to 45 years for type I and 24 to 45 years for type II diabetes. tThe number of subjects is given in parentheses. ~No preliminary incubation of the enzyme with protoporphyrin IX was needed.

52

I.s. SEVERINA

regulatory role of cGMP manifested at the earliest stages of aggregation opens the possibility of directed pharmacological effect on platelet guanylate cyclase for normalizing the pathologically increased platelet aggregability and preventing their spontaneous aggregation.

Guanylate Cyclase in Myocardial Ischemia The interrelationship between the decrease in activity and ability for activation of platelet guanylate cyclase and the enhancement of platelet aggregability prompted our studies on the functioning of soluble guanylate cyclase in the case of myocardial ischemia in rats (40, 41). It should be noted that the role of soluble guanylate cyclase at pathological states has not been greatly studied. There exists only fragmentary indications of the increased guanylate cyclase activity in lymphocytes in some types of leukemia, in the case of proliferative processes in epidermis, at hypertrophy of myocardium (42-43). The acute ischemic damage of rat myocardium was induced by the ligation of the coronary artery under the control of the electrocardiogram. Since disorders in the enzyme activity in the organ are frequently manifested in blood elements, including platelets, we studied the activity of soluble guanylate cyclase and its regulation both in the ischemic zone of the left ventricle of the heart and platelets of rat 15 min and 24 hr after surgery. The results are summarized in Table 4. As can be seen from Table 4 the guanylate cyclase activity is already significantly decreased 15 min after surgery. In the heart the decrease amounted to 44% of normal values and 18% in the platelets. In 24 hr, guanylate cyclase activity in the ischemic zone of the heart continues decreasing insignificantly (to 30%), whereas in platelets there appears to be a tendency to restoration of the enzyme activity (to 30% of normal, in comparison with 18% detected 15 min after the surgery). Activation of rat myocardial guanylate cyclase by sodium nitroprusside falls to 50% of normal values in 15 min and disappears completely in 24 hr (see Table 4). Thus, in the case of the acute myocardial ischemia, the changes in guanylate cyclase activity in heart and platelets have been found to be synchronous, with the more sharply expressed changes being noted in platelets. The fall in guanylate cyclase activity in the case of the acute myocardial ischemia apparently can be connected with intensification of free radical processes occurring at the earliest stages of the pathological development, while the sharply reduced capacity of myocardium guanylate cyclase to be activated by sodium nitroprusside is probably due to the loss of the heme of guanylate cyclase, whose splitting is promoted by the lowered pH of the medium which is characteristic of the pathology in question. It is

SOLUBLE GUANYLATE CYCLASE OF PLATELETS

53

TABLE 4. GUANYLATE CYCLASE ACTIVITY AND ENZYME ACTIVATION BY SODIUM NITROPRUSSIDE IN HEART AND PLATELETS OF RATS AT MYOCARDIAL ISCHEMIA Rats with myocardial ischemia 15 rain after surgery 24 hr after surgery

Intact rats

Source of enzyme 105,000 X g supernatant of heart* 105,000 x g supernatant of platelets

Activity (pmol cGMP/ mirdmg)

Extent of activationt

Activity Activity (pmol Extent (pmoi Extent cGMP/ of cGMP/ of min/mg) activationt min/mg) activationt

8.0 + 0.9 (100%)

10 ::t:0.8 (100%)

3.5 + 0.5 5.1 + 0.4 2.4 + 0.8 No (44%) ( 5 0 % ) ( 3 0 % ) activation

15.2 + 3.2 (100%)

No activation

2.9 + 0.4 No 5.1 + 1.08 No (18.1%) activation (33.5%) activation

*Soluble guanylate cyclase was isolated from rat heart according to (17) by centrifugation of 10% tissue homogenate at 105,000 x g for 1 hr. The homogenate was prepared using the left ventricle from the hearts of intact rats and the ischemic zone of the left ventricle from the hearts of rats with myocardial ischemia. The 105,000 x g supernatants of rat heart and platelets used were 100/~g and 20/zg by protein per sample, respectively. tGuanylate cyclase was preincubated with 0.1 mM sodium nitroprusside for 45 rain at 0°C. For the enzyme assay see Methods. essential that the above results have revealed a high sensitivity of platelet guanylate cyclase to pathological changes occurring in the myocardium at the earliest stages of the pathological process and indicated the possibility of early detection of such disorders using this enzyme. SUMMARY C h r o m a t o g r a p h y of 105,000 x g supernatants of h u m a n and rat platelets on DEAE-cellulose yielded identical elution profiles containing 2 protein fractions (peaks I and II). Only p e a k II was found to possess guanylate cyclase activity. In the spectrum of the 105,000 x g supernatant of h u m a n platelets the absorption m a x i m u m was specified at 410 nm (the Soret band) which disappeared from the spectrum of the active protein fraction (peak II) but was detected in the nonactive fraction (peak I). The enzyme preparation was obtained in the heme-deficient form. In the experiments with rat platelets, the Soret band was absent from the corresponding spectra and the enzyme was not activated by sodium nitroprusside; i.e., in soluble guanylate cyclase of rat platelets, unlike the generally accepted notion, the h e m e is not a prosthetic group of the enzyme.

54

I.S. SEVERINA

It was shown that carnosine (13-alanyl-L-histidine), a water-soluble antioxidant, inhibits guanylate cyclase activation by sodium nitroprusside. This inhibitory effect is caused by the interaction of carnosine with the guanylate cyclase heme and can be used for evaluating the degree of saturation of the enzyme with the heme. ADP-induced aggregation of human platelets (donors) is accompanied by a fall in the basal guanylate cyclase activity (with Mg 2÷) and the enhancement of the enzyme stimulation with sodium nitroprusside, protoporphyrin IX, arachidonic acid and L-arginine with simultaneous cGMP elevation in platelets. A hypothetic scheme of the regulatory role of cGMP in platelet aggregation is proposed. In the experiments with the acute myocardial ischemia of rats, 15 min after the surgery a sharp fall in the platelet guanylate cyclase activity accompanied by a decrease in the enzyme activity in the ischemic zone of the left ventricle of heart took place. The results provided evidence of the high sensitivity of platelet guanylate cyclase to pathological changes occurring in the myocardium at the earliest stages of the development of pathology. REFERENCES 1. J. TREMBLAY, R. G E R Z E R and P. HAMET, Cyclic GMP in cell function, Advan. Second Messenger and Phosphoprotein. Res. 22, 319-385 (1988). 2. R. G E R Z E R , E. BOHME, F. HOFMANN and G. SCHULTZ, Soluble guanylate cyclase purified from bovine lung contains heme and cupper, FEBS Lett. 14, 71-74 (1981). 3. L. J. IGNARRO, K. S. WOOD and M. S. WOLIN, Activation of purified soluble guanylate cyclase by protoporphyrin IX, Proc. Natl. Acad. Sci. U.S.A. 79, 2870-2873 (1982). 4. L . J . IGNARRO, K. S. WOOD and M. S. WOLIN, Regulation of purified soluble guanylate cyclase by porphyrins and metalloporphyrins; a unifying concept, Advan. Cycl. Nucl. Prot. Phosphor. Res. 17, 267-274 (1984). 5. R. M. J. PALMER, A. G. FERRIGE and S. MONCADA, Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor, Nature 327, 524-526 (1987). 6. R. F. F U R C H G O T and J. V. ZAWADSKI, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373-376 (1980). 7. R . M . J . PALMER, D. S. ASHTON and S. MONCADA, Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature 333, 664-666 (1988). 8. A. MULSCH, E. BOHME and R. BUSSE, Stimulation of soluble guanylate cyclase by endothelium-derived relaxing factor from cultured endothelial cells, Eur. J. Pharmacol. 135, 247-250 (1987). 9. R . M . J . PALMER and S. MONCADA, A novel citrulline forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells, Biochem. Biophys. Res. Commun. 158, 348-352 (1989). 10. M . A . MARLETYA, P. S. YOON, R. IYENGAR, C. D. LEAF and J. S. WISHNOK, Macrophage oxidation of L-arginine to nitrite and nitrate: Nitric oxide is an intermediate, Biochemistry 27, 8706-8711 (1989).

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11. M. PALACIOS, R. G. KNOWLES, R. M. J. PALMER and S. MONCADA, Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands, Biochem. Biophys. Res. Commun. 168, 802-809 (1989). 12. P. M. MYERS, R. L. MINOR, Jr., R. G U E R R A , Jr., J. N. BATES and D. G. HARRISON, Vasorelaxant properties of endothelium derived relaxing factor more closely resemble S-nitrosocystein than nitric oxide, Nature 345, 161-163 (1990). 13. P. GRAVEN and F. DeRUBERTIS, Requirement for heme in the activation of purified guanylate cyclase by nitric oxide, Biochim. Biophys. Acta 745, 310-321 (1983). 14. R. GERZER, F. HOFMANN and G. SCHULTZ, Purification of a soluble sodium-nitroprusside-stimulated guanylate cyclase from bovine lung, Eur. J. Biochem. 116, 479-486 (1981). 15. S. TSAI, R. ADAMIK, V. MANGANIELLO and M. VAUGHAN, Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide, Biochem. J. 215, 447-455 (1983). 16. Y . Y . CHIRKON, I. A. TYSHCHUK, N. N. BELUSHKINA and I. S. SEVERINA, Guanylate cyclase from human blood platelets, Biochemistry (USSR) (Engl. Transl.) 52, 821-828 (1987). 17. V. P. MIROSHNICHENKO, O. G. BUSYGINA and I. S. SEVERINA, Similarity and variations in properties of the guanylate cyclase soluble forms from rat heart and thrombocytes, Voprosy Meditsinskoi Khimii (USSR) 4, 60--66 (1989). 18. H. J. BRANDWEIN, J. A. LEWICKII and F. MURAD, Reversible inactivation of guanylate cyclase by mixed disulfide formation, J. Biol. Chem. 256, 2958-2962 (1981). 19. O . H . LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265-275 (1951). 20. G. V. R. BORN, Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature 194, 927-929 (1962). 21. Y. Y. CHIRKOV, I. A. TYSHCHUK and I. S. SEVERINA, Guanylate cyclase in human platelets with different aggregability, Experientia 46, 697-699 (1990). 22. I. S. SEVERINA, Activation of guanylate cyclase and the regulatory role of cyclic 3' ,5'-guanosine monophosphate, Biochem. lnternat. 17,265-278 (1988). 23. A . A . BOLDYREV and S. E. SEVERIN, The histidine-containing dipeptides, carnosine and anserine: distribution, properties and biological significance, Advan. Enzyme Regul. 30, 175-194 (1990). 24. K. NAGAI and T. SUDA, Realization of spontaneous healing function by carnosine, Meth. Find. Expl. Clin, Pharmacol. 10, 497-507 (1988). 25. A. A. BOLDYREV, A. M. DUPIN, A. Y. BUNIN, M. A. BABIZHAEV and S. E. SEVERIN, The antioxidative properties of carnosine, a natural histidine-containing dipeptide, Biochem. lnternat. 15, 1105-1113 (1987). 26. I. S. SEVERINA and O. G. BUSYGINA, Effect of carnosine on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX, Biochem. lnternat. 22, 455--465 (1990). 27. A. D. GOLDBERG, M. K. HADDOX, S. E. NICOL, D. B. GLASS, C. H. SANFORD, F. A. KUEHL and R. ESTENSEN, Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the Yin Yang hypothesis, Advan. Cycl. Nucl. Res. 5, 307-330 (1975). 28. B . T . MELLION, L. J. IGNARRO, E. H. OHLSTEIN, E. G. PONTECORVO, A. L. HYMAN and P. J. KADOWlTZ, Evidence for the inhibitory role of cGMP in ADP-induced human platelet aggregation in the presence of NO and related compounds, Blood 57, 946-955 (1981). 29. U. FOSTERMANN, A. MULSCH, E. BOHME and R. BUSSE, Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries, Circ. Res. 58, 531-538 (1986). 30. Y . Y . CHIRKOV, N. N. BELUSHKINA, I. A. TYSHCHUK and 1. S. SEVERINA, Changes in human platelet guanylate cyclase activity during ADP-induced aggregation, Bull. Exptl. Biol. Med. (USSR) 2, 152-154 (1991). 31. A. J. BARBER, Cyclic nucleotides and platelet aggregation. Effect of aggregatory

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