Platelet prostaglandin D2 dehydrogenase in patients with myeloproliferative disorders

Prostaglandins Leukotrienes and Medicine 21: 207-220, 1986

PLATELET PROSTAGLANDIN D2 DEHYDROGENASE IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS Tateo Sugiyama, Minoru Okuma, Haruto Uchino The First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan (reprint requests to MO) ABSTRACT

NADP-linked 15-hydroxyprostaglandindehydrogenase for prostaglandin D2 (PGD2DH) transforms prostaglandin D2 (PGD2) to inactive 15-keto-PGD2. This enzyme activity was spectrophotometricallydetermined in the cytosol of platelets and platelet sensitivities to PGD2 were studied in patients with myeloproliferative disorders (MPD) as well as in normal subjects. Platelet sensitivities to exogenous and endogenous PGD2 were estimated by IC50 of added PGD2 for platelet aggregation and by the inhibitory effect of a specific thromboxane synthetase inhibitor (OKY-046) on collagen-induced aggregation, respectively. PGD2DH activities of MPD patients were significantly higher than those of normal subjects (p C 0.01). Although decreased sensitivity to exogenous PGD2 was detected in some MPD patients, they were not always associated with the increased enzyme activities. Furthermore, no specific correlation was found between PGD2DH activities and the inhibitory effects of OKY-046. Thus., PGD2DH seems to have little effect on the action of PGD2 against platelet aggregation in MPD patients and normal subjects. INTRODUCTION Human platelets peroxidize arachidonic acid (AA) to produce prostaglandin (PG) endoperoxides which are then converted to a number of other biologically active compounds including thromboxane A2 (TXA2), PGD2, PGE2 and PGF2a (1). Of these substances, only PGD2 is a potent inhibitor of human platelet aggregation (2-5) and has been suggested to play an important role in the negative regulation of platelet aggregation in vivo, since it is produced during aggregation of human platelets at sufficient concentrations to inhibit platelet aggregation (6-8). However, the role of PGD2 in platelet function has not been yet unequivocally understood. During the last few years, the metabolism and function of PGD2 in the circulatory system have been studied and a number of observations have 207

been reported (reviewed in ref. 9). Studies on the biosynthetic pathway of PGD2 in platelet-rich plasma (PRP) demonstrated that platelets lacked PGD2 synthetase activity and that PGD2 was generated from PGH2 by a plasma factor which was identified as serum albumin (8). Receptor binding studies with radioligand have confirmed that PGD2 exerts an antiaggregatory effect via the specific receptor on the platelet membrane, which is distinctly different from the common receptor for PG12 and PGEl (10,ll). Further, in regard to the biological inactivation of PGD2, an NADP-linked 15-hydroxyprostaglandindehydrogenase specific for PGD2 (PGD2DH) has been partially purified from the cytosol of human platelets (12) and it has been reported that this enzyme catalyzes the conversion of PGD2 to almost inactive 15-keto-PGD2 (12,13). In order to elucidate the significance of PGD2 in platelet functions, it should be of interest to study these metabolic processes of PGD2 in platelets of normal and pathological states, including myeloproliferative disorders (MPD). The present study deals with platelet PGD2DH activities and the possibility of the enzyme to regulate platelet-PGD2 interaction in MPD patients as well as in normal subjects. MATERIALS AND METHODS Blood collection and preparation of platelet suspensions Blood platelets from healthy donors and patients who had taken no drug known to interfere with platelet function for at least 2 weeks before venipuncture was studied. In the present invetigation, healthy subjects with hematocrit values > 35 % were regarded as "normal" (vide infra). Blood from "normal" donors were routinely collected in 0.1 volume of 3.8 % trisodium citrate from the antecubital vein, but the volumes of citrate added to MPD patients' blood were adjusted according to their hematocrit values (14). PRP and platelet-poor plasma (PPP) were prepared as previously reported (15). Informed consent was obtained from all patients and “normal” donors. Assay of PGD2DH activity The enzyme activity was spectrophotometricallydetermined in the cytosol of platelets by a slight modification of the method of Tokumoto et al. (13). All the following manipulations were performed at 4*C. PRP was mixed with 77 mMEDTA, pH 7.4 in a ratio of 9 : 1, and Centrifuged at 1,100 g for 30 minutes. The pellets were washed three times with 0.15 M NaCl solution containing 7.7 mMEDTA, pH 7.4. The washed platelet pellets were finally suspended in 10 mMpotassium phosphate buffer, pH 7.0, containing 0.1 mMdithiothreitol (1 - 2 x 109 platelets /ml) , and sonicated four times at minimal power for 15 seconds each with a Branson sonifier Model W185D. The sonicates were centrifuged at 105,000 g for 60 minutes. The resultant supernatant solution (cytoSo1 fraction) was stored in aliquots at -70°C and ured within 3 weeks a8 an enzyme preparation in the standard assay. The standard reaction mixture contained 0.1 M glycine-NaOH buffer, pH 9.5; 200 PM NADP, 200 IJM PGD2 (150 nmol dissolved in 7.5 ~1 of ethanol), and enzyme in a total

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volume of 0.75 ml. The increase in absorbance at 415 nm was continuously followed by using a Shimazu spectrophotometer Model W200 (Shimazu, Kyoto.,Japan) in a 1 ml cuvette with a l-cm light path. The enzyme activity was calculated from the initial velocity of an increase in the absorbance based on the molecular extinction coefficient (46,500 M-1 cm-l) of 15-keto-PGD2 at pH 9.5 (13), and expressed as the amount (nmol) of 15-keto-PGD2 produced per min per mg of protein at 25*C. The protein concentration was determined according to Lowry et al. (16) with bovine albumin (BSA) as a standard. Measurement of NADP in platelet cytosol fraction NADP levels in the enzyme preparation were determined according to the fluorimetric assay method of Klingenberg (17) by the use of Shimazu fluorescence spectromonitor RF-SOOLC (Shimazu, Kyoto, Japan). Platelet aggregation study Aggregation studies on PRP were performed in a dual channel aggregometer linked to a pen recorder as previously described (15). Platelet counts were done by thrombocounter C (Coulter Diagnostics, Hialeak, Fla) and those in PRP were adjusted to 300 x lo3 platelets/p1 by autologous PPP. Platelet functions were studied within 3 hours of blood collection. To study platelet sensitivity to exogeneous PGD2, its inhibitory effects on collagen-induced aggregation and primary aggregation induced by ADP were observed. One minute after the addition of various concentrations of PGD2,to PRP, aggregation was induced with collagen (2 ng/ml) or ADP (1 pM). Platelet sensitivity to PGD2 was expressed in terms of IC50 (PGD2 concentration necessary to halve the aggregation response) in each aggregation. The maximal amplitude of the aggregation curve was used for quantification. In preliminary experiments with 9 "normal" women whose hematocrit values were between 42 % and 35 % (38.7 f 2.1, M + SD), IC50 values of PGD2 for collagen-induced platelet aggregation in PRP which were prepared from blood with adjusted volumes of citrate (14) (26.6 * 7.0 nM) were not significantly different (p > 0.5) from those values obtained from blood without such adjustment of citrate (28.8 t 7.3 nM). Based on these results, subjects with hematocrit values > 35 % were regarded as "normal" and no adjustment of citrate was done for their blood collection. In experiments with TX synthetase-inhibited platelets, individual PRP samples were incubated for 1 minute at 37'C with 0.1 mM OKY-046 [a specific TX synthetase inhibitor (18)], and aggregation was induced by the addition of collagen (2 pg/ml) or AA at the threshold concentration which was defined as the smallest amount of AA to induce 70 % increase in light transmission 3 minutes after the addition of AA to intact PRP. Individuals whose AA-induced aggregation was inhibited by OKY-046 were designated as "responders" and the others as "non-responders".

209

PROST.

E

Radioimmunoassay for TXB2 The amount of TXB2 was measured on unextracted plasma by a radioimmunoassay kit according to the Instruction Manual. After 0.1 mM indomethacin was added to PRP samples which had been stirred for 3 minutes in the presence of AA at the threshold concentration, plasma samples for the assay were prepared by centrifuging them at 12,000 g for 2 minutes at 4'C and stored at -3O'C until analyzed. Statistical analysis The Student's t-test was used to evaluate statistical significances. Correlation was estimated by linear regression analysis using the leastsquares method. Patients and "normal" controls Thirty-eight patients with MPD were studied, including 14 with chronic myeloid leukemia (CML), 11 with essential thrombocythemia (ET) and 13 with polycythemia vera (PV). They were 26 - 84 (53 t 15, M f SD) y.o. and consisted of 18 men and 20 women. Diagnosis was determined as previously described (15). Sixty-eight "normal" volunteers [32 men between 20 and 62 (36 + 13) y.o. and 36 women between 20 and 63 (33 + 16) y.0.1 were also studied as normal controls. Materials ADP, BSA, NADP, D-glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma; AA from Nakarai Chemicals, Kyoto, Japan; indomethacin from Sumitomo Chemicals, Osaka, Japan; collagen from HormonChemie, Muenchen, West Germany. PGD2 and OKY-046 were donated by One Central Research Institute, Osaka, Japan. TXB2 r3H] RIA Kit was purchased from New England Nuclear, Boston, MA. All other chemicals were of reagent grade. RESULTS Some properties of PGD2DH from the cytosol of human blood platelets were first examined. The enzyme activity was stable at -7O'C for at least 3 weeks and lost by boiling the cytosol for 5 minutes. The formation of 15-keto-PGD2 was proportional to the enzyme amount up to 1.4 mg (data not shown) and dependent upon concentrations of PGD2 and NADP (Fig. 1,2). Some extent of the activity was detected even if no NADP was added (Fig. 2), probably because of NADP originated from the platelet cytosol which contained 0.14 + 0.04 nmol (n = 4) NADP/mg protein. The optimal pH for the enzyme reaction was around 9.5. The reaction product showed a maximal absorption at 415 nm at pH 9.5 and the absorption disappeared by acidification of the reaction mixture to pH 3 with 1 N HCl and reappeared at alkaline pH values.

210

PGD:!

(JJM)

Figure 1. Effect of PGD2 concentration on 15-keto-PGD2 formation. The incubation was carried out with 0.72 mg of the enzyme preparation in the presence of varying PGD2 concentrations as indicated.

^a E

0.02

.c E > :

5 N %

4_

0.0’

0 ti; Y

II, , 1

2

3

4

5

10

200

NADP added tiMI

Figure 2. 15-keto-PGD2 formation as a function of NADP added. Enzyme activity was assayed with 0.86 mg of the enzyme preparation in the presence or absence of NADP added as indicated.

211

0.06 7s E ;: .E L g C

0.05

0.04

cu 8 n

0.03

b yzi

0.02

LA r 0.01

I

I

CML

PV

ET

Figure 3. PGD2DH activities of platelets in MPD patients. Dotted area, normal range (M 5 2SD, n = 29).

PGD2DH activities of platelets from 29 "normal" subjects and from 38 MPD patients were 0.026 4 0.007 and 0.033 ?:0.012, respectively, and the difference between them was significant (p < 0.01). When a normal range of the enzyme activity was defined as mean t 2SD, 8 of 38 patients showed increased activity, while no patients showed decreased one (Fig. 3). There was no sex difference in the enzyme activities of "normal" subjects and/or MPD patients (data not shown). The clinical and laboratory data together with PGD2DH activities and IC50 of PGD2 in collagen-induced aggregation of the 8 patients with increased PGD2DH activities are summarized in Table 1. The patients were 34 - 81 y.o. and six of them were women. Of the 8 patients, 6 had elevated platelet counts. Decreased platelet sensitivity to PGD2 was found in 2 of the 3 patients where IC50 of PGD2 could be examined. Two patients had a bleeding tendency, and 1 had a thrombotic episode. In the 30 patients with normal enzyme activities, 4 had episodes of cerebral thrombosis, 2 had erythromelalgia, 2 had myocardial infarction, and 3 had nasal bleeding.

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Table 1.

Case

Age

No.

(yr)

1 2 3 4 5 6 7 8

35 44 38 34 45 48 64 81

Clinical and laboratory data of patients with increased PGD2DH activities

Sex

F F F M F F F M

Diagnosis

CML CML CML CML CML

ET PV PV

Blood 1) PGD2DH platelets (x 103/ill)

274 557 572 2,070 1,534 1,694 1,650 304

0.052 0.054 0.041 0.043 0.062 0.062 0.048 0.050

1)2) IC50

Histor Bleed- tic;:-

(nM)

ing

N.D. N.D. 62.0 N.D. TIA N.D. 30.0 N.D. Purpura 56.0 Gingival bleeding

1) Normal ranges ( M f 2SD): PGD2DH = 0.026 + 0.014 (n = 29); IC50 = 19.6 + 18.2 (men, n = 20), 28.5 + 16.6 (women, n = 24) 2) IC50 for collagen-induced aggregation; N.D., not done

Table 2 shows sex difference in platelet sensitivities to PGD2 in terms of Ic50 for platelet aggregation. In collagen-induced aggregation, men showed significantly higher sensitivity than women, whereas there was no significant sex difference in the sensitivity of primary aggregation induced by ADP. No significant difference by age was found both in men and in women (data not shown).

Table 2.

Platelet sensitivity to PGD2 in normal men and women

IC50 (nM) of PGD2 for aggregation induced by Subjects Collagen

ADP

Men

20.258.5 [35.8+13.4 (32)]

11.6k4.6 [28.0+3-O (13)]

Women

1) 30.6k9.3 [33.0+15.7 (36)]

13.4k4.0 [23.0+3.0 (13)]

Each value denotes M f SD. [ 1, age W; ( 1) p < 0.001 vs. corresponding value of men

213

1, n

0

50

150

100

200

300

200

300

lC50 Wvl)

0

50

150

100 IC50

(nM)

Figure 4. PGD2DH activities and inhibitory effects of PGD2 on collageninduced platelet aggregation (Ic5G) in MPD patients (0) and normal subUpper panel, men; bottom panel, women; dotted area, normal jects (X). ranges (M f 2SD).

To investigate the role of PGD2DH activity in platelet sensitivity to exogenous PGD2, we studied the enzyme activity and IC5G of PGD2 for collagen-induced platelet aggregation in 15 "normal" subjects as well as in 18 MPD patients (Fig. 4). Platelets from 11 patients had high IC5G (i.e. resistance to PGD2), but only 3 of them had increased enzyme activities. No significant correlation was found between the enzyme activity and platelet sensitivity to exogenous PGD2 in MPD patients and “normal" subjects.

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Platelet sensitivity to PGD2 in collagen-induced aggregation was decreased in 9 of 10 "non-responders",whereas it was decreased in 1 of 19 "responders" (Fig. 5). Over 95 % of TX synthetase activities of platelets was inhibited by 0.1 mM OKY-046 in "non-responders" as well as in "responders", and IC50 of OKY-046 for TX synthetase activities were 4.6 + 0.5 nM (n = 4) in the former and 4.5 + 2.3 pM (n = 4) in the latter. No significant correlation was found between PGD2DH activities and inhibitory effects of OKY-046 on collagen-induced aggregation in platelets of "normal" subjects and MPD patients (Fig. 6).

r

??

?? 0

0

Re4parder Nonresponck Respc&r Nonresponder

Figure 5. Inhibitory effects of PGD2 on collagen-induced platelet aggregation (IC50) in "responders" and "non-responders" of MPD patients (0) and normal subjects (0). Left panel, men; right panel, women.

215

Figure 6. PGD2DR activities and inhibitory effects of OKY-046 on collagen-induced platelet aggregation in MPD patients (0) and normal subjects r = -0.176. (0).

DISCUSSION 15-hydroxyprostaglandindehydrogenase is the key enzyme in biological inactivation of most prostaglandins (19). Although two types of 15hydroxyprostaglandin dehydrogenase i.e., NAD-ldnked and NADP-linked ones, are present in mammalian tissues, these enzymes act mainly on PGE and PGF but poorly or not at all on PGD (20,21). Recently, an NADP-linked 15hydroxyprostaglandin dehydrogenase specific for PGD2 (i.e. PGD2DR) was demonstrated from swine brain (22) and human blood platelets (12). In the present study, we have determined the activities of PGD2DH in platelets of "normal" subjects and FfPDpatients, and examined the significance of this enzyme in platelet aggregation, particulary in its sensitivity to PGD2. As shown in preliminary experiments, the properties of the enzyme activity under the standard assay condition using the cytosol fraction of platelets were similar to those of partially purified PGD2DH (12). The reaction product showed a maximal absorption at 415 nm at pH 9.5 and the absorption intensity was dependent on pH values. These spectral properties suggested that the reaction product was identical with l5-keto-PGD2

216

as described previously (12,13,22). We found that PGD2DH activities of MPD patients were significantly higher than those of "normal" subjects. When a normal range of the enzyme activity was defined as mean + 2SD, 8 of 38 patients showed increased activities. As the conversion of PGD2 to inactive 15-keto-PGD2 could be enhanced in the patients with increased enzyme activities, it may be speculated in such patients that the inhibitory effect of PGD2 on platelet aggregation is decreased and that thereby platelet function is enhanced. Although decreased platelet sensitivity to added PGD2 in collagen-induced aggregation was detected in 11 of 18 patients examined, these patients were not always associated with the increased enzyme activities. Futhermore, in collagen-induced aggregation, men showed significantly higher sensitivity than women, but no sex difference was found in the enzyme activities. Thus, no significant correlation was found between the enzyme activity and platelet sensitivity to exogenous PGD2 in MPD patients and/or "normal" subjects. Our results that platelet sensitivities to PGD2 were decreased in 9 of 10 "non-responders" and in only 1 of 19 "responders" are in favour of the concept that PGD2 generated in PKP by the activation of TX synthetaseinhibited platelets plays an important role for the inhibitory effect of TX-synthetase inhibitors on platelet function (23,24). Thus, possible effects of endogenous PGD2 on platelet aggregation was studied by use of the inhibitory effects of OKY-046 (a specific TX synthetase inhibitor) on collagen-induced platelet aggregation. As no significant correlation was found between the PGD2DH activity and the efficacy of the TX synthetase inhibitor, no significant effect of the PGD2DH activity on the action of endogenous PGD2 against platelet aggregation was suggested in our experimental system. Although we detected increased activities of PGD2DH in some patients with MPD, the enzyme activity seemed to have little effect on the action of exogenous and/or endogenous PGD2 against platelet aggregation in these patients. Whether this abnormality in these patients have any clinical significance or could be related to hyperactive platelets possibly leading to thrombosis is unknown, since patients with increased enzyme activities were associated with no specific complication. In MPD patients, a number of qualitative platelet abnormalities including those of some enzyme activities have been reported (reviewed in ref. 25,26), but these abnormalities are not always found to be associated with clinical manifestations (25). The platelet abnormalities may not be identical in all cases, and the basic defects that underlie the abnormalities in platelet function in MPD still remain to be determined. It was demonstrated that 20 of 23 MPD patients exhibited the diminished sensitivity to PGD2 (27) and that in these patients, the platelet receptor for PGD2 was selectively reduced in number (28). We confirmed that many of MPD patients showed a resistance to PGD2 by a different method from that of the previous reports (27,28), but this resistance did not correlate with the PGD2DH activity. Whether the resistance to PGD2

217

that we detected in our patients is accounted for by the abnormality of platelet receptors for PGD2 was not studied. In conclusion, PGD2DH activities of platelets in MPD patients were significantly higher than those in "normal" subjects. However, the enzyme activity seemed to have little effect on the action of exogenous and/or endogenous PGD2 against platelet aggregation in MPD patients and "normal" subjects. The significance of PGD2DH in the platelet function remains to be elucidated. ACKNOWLEDGEMENTS We are indebted to Dr. Masahiro Miki, Dr. Masaro Tashima and Dr. Shigeki Sensaki for referring the patients to us for investigation, and to Dr. Takao Shimizu and Dr. Shu Narumiya for valuable discussions on the determination of PGD2DH activities. This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant from the Sankyo Research Foundation for Life Sciences. REFERENCES 1. Marcus A J. The role of lipids in platelet function: with particular

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