0021-9i2x/92/i503-0i21$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright c 1992 by The Endocrine Society
Vol. 75, No. 3 Printed m U S.A
3,3’,5’-Triiodothyronine Inhibits Human Platelet Aggregation HIROYA MASAKI*, MITSUSHIGE NISHIKAWA, MASAYOSHI YOSHIMURA, NAGAOKI TOYODA, NOR10 YOSHIKAWA, AND MITSUO INADA Second Department
of
Internal
Medicine,
Kansai
Medical
Collagen-Induced
MASAYA URAKAMI, YASUKIYO MORI,
University,
Moriguchi
City, Osaka 570, Japan
ABSTRACT To clarify further the activity of rTcl, we examined the effect of rT:? on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rTa, T,, Ta, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rTij inhibited by 50% collagen-induced platelet aggregation was 30 f 4 (mean ? SE) Fmol/L. rT:$, T,, and T:] did not
differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT:, inhibited collagen-induced phosphorylation of the 20. kilodalton protein (myosin light chain) in platelets. In contrast, rTrl did not inhibit 12-0-tetradecanoylphorbol13-acetate (TPA)or thrombin-induced platelet aggregation and inhibited only minimally TPAinduced 40-kilodalton protein phosphorylation. These results suggest that rTn inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT:+ (J Clin Endocrinol Metab 75: 721-725,1992)
I
any drug for at least 2 weeks before venipuncture. The platelet rich plasma (PRP) was obtained from the supernatant after centrifugation (250 X g for 10 min at 22 C). After further centrifugation (2200 X g for 10 min at 22 C) of the PRP, a pellet was obtained. This pellet was resuspended and washed twice with HEPES-Tyrode’s buffered saline (NaCI 129 mmol/L, NaHC03 8.9 mmol/L, KHIPOl 0.8 mmol/L, MgCb 0.8 mmol/L, dextrose 5.6 mmol/L, HEPES 10 mmol/L, pH 7.40). It was then resuspended in HEPES-Tyrode’s buffered saline to a final platelet concentration of 4 X lO’/~l in aliquots of 0.2 mL. Platelet aggregation was calculated from the turbidity of the solution as measured by a four-channel aggregometer (Nikko Biosciense Co. Tokyo, Japan). Washed platelets were preincubated in the aggregometer for 5 min at 37 C and then incubated with rT,, Tq, Ta, Triac, or thyronine at concentrations ranging from 5-50 bmol/L for 2 min at 37 C before adding collagen, thrombin, or TPA. Control aggregations were induced by each agonist after preincubation by adding 0.02 mol/L NaOH alone. Aggregation was expressed either as the change in light transmission or as the maximum percentage increase in light transmission, with a 100% increase in transmission being defined as the maximum increase induced by an agonist of platelet aggregation (collagen, thrombin, or TPA) in the absence of any antagonist.
T IS WELL known that T3 and T4 have various biological activities and play an essential role in maintaining homeostasis in mammals (1). However, there are few reports regarding the biological activities of rT3, to which as much as 60% of T4 is metabolized in peripheral tissues (2-4). Recently, Hidaka and co-workers (5-7) reported that T3 and Tq inhibit the functions of platelets and vascular smooth muscle by inhibiting the activity of myosin light chain kinase (MLCK). In their reports, however, the activity of rT3 was not mentioned. Described in the present report is our study of the effect of rTj on human platelet functions. This study was performed to clarify further the role of rT3 outside the nucleus and to elucidate the possible mechanism by which rTj inhibits platelet aggregation. Materials
and Methods Serotonin
Materials T,, Ts, thyronine, 3,5,3’-triiodothyroacetic acid (Triac), 12O-tetradecanoylphorbol 13-acetate (TPA), EGTA, and thrombin were obtained from the Sigma Chemical Co. (St. Louis, MO). rT) was obtained from the Calbiochem Behring Co. (La Jolla, CA). Collagen was obtained from Hormon Chemie (Munich, Germany). [‘“C]Serotonin and 32P, specific activity 1.85-2.2 gigabecquerel (GBq)/mmol and 370 MBq/mL, respectively, were obtained from the Amersham Japan Co. (Tokyo, Japan). A sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis system was purchased from the Daiichi Chemical Co. (Kyoto, Japan). All thyroid hormones were dissolved in 0.02 mol/L NaOH.
Platelet
aggregation
Venous blood samples treated with the anticoagulant dextrose were prepared from healthy volunteers who Received October 25, 1991. * To whom requests for reprints
should
release from platelets
Washed platelets were incubated with [‘4C]serotonin (92.5 kBq/mL PRP) for 60 min at 37 C and spun at 2,200 X g for 8 min at 22 C. The pellet was resuspended in HEPES-Tyrode’s buffered saline to a concentration of 5 X 10”/~1. The labeled platelets were preincubated for 5 min at 37 C and then incubated with rT3, T,, or T2 at concentrations ranging from 5-50 pmol/L for 2 min at 37 C before the stimulation with collagen (final concentration 8 pg/mL). Control serotonin release was induced by collagen (8 Kg/ml) after the preincubation by adding 0.02 mol/L NaOH alone. The reaction was terminated with ice-cold EGTA and spun at 12,800 X 8 for 10 min at 0 C. The supernatants were mixed with 4 mL aqueous counting scintillant II (ACS II) and counted in a liquid scintillation counter. The data were expressed as percentage total platelet [‘“Clserotonin measured after lysing the platelets with 10% Triton X-100.
10% acid citrate had not been on
Protein min
be addressed.
phosphorylation
in platelets
Washed platelets were incubated with 32P (9.25 MBq/mL PRP) for 90 at 37 C and spun at 2200 x g for 8 min, 22 C. The pellet was
721
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722
MASAKI
TABLE 1. The inhibitory potency of thyroid hormones on collagen-induced platelet aggregation Thyroid hormones
I.50~WOllL)” 30 + 4 r’I’3 21+ 3 T4 16 rt 4 l-3 Triac 19 + 3 Thyronine >50 ’ The values are expressed as the mean + SE.
n 6 6 6 4 3
resuspended, preincubated, and then incubated with rTB, Td, or T3 in a manner identical to that in the experiment in which serotonin was released before the stimulation with collagen (final concentration: 8 rg/ mL). Control platelet protein phosphorylations were induced by collagen or TPA after preincubation by adding 0.02 mol/L NaOH alone. The reaction was terminated by adding sampling buffer (49.5 mmol/L TrisHCI, 6.9% SDS, 30% glycerol, pH 6.8, including bromphenol blue) at various time points. The samples were subjected to 12.5% SDS polyacrylamide gel electrophoresis. The gel was dried by a gel dryer (BioRad Laboratories Co, Richmond, CA) and exposed to Fuji x-ray film. The relative intensity of each band was quantitated from a densitometric tracing of the autoradiogram.
Statistics All values were presented as the mean + SE. Statistical significance was determined by two-way analysis of variance.
Results Effect of rT3 on platelet aggregation Platelet aggregation was induced by collagen at concentrations of 1-8 rg/mL in a dose-dependent manner. Control
ET AL.
JCE & M .1992 Vol75.No3
experiments revealed that collagen at a final concentration of 8 pg/mL and thrombin at a final concentration of 0.1 U/ mL after preincubation by adding 0.02 mol/L NaOH induced the maximum platelet aggregation. T, and TJ, at concentrations ranging from 5-50 pmol/L, inhibited collagen-induced platelet aggregation in a dosedependent fashion; the concentrations that inhibited platelet aggregation by 50% (Iso) were 21 + 3 and 16 + 4 pmol/L, respectively (Table 1). rTg at concentrations of 5-50 pmol/L also inhibited collagen-induced platelet aggregation in a dose-dependent manner (Fig. l), and for rTg the Isowas 30 + 4 pmol/L (Table 1). Triac also inhibited collagen-induced platelet aggregation, with its Isobeing 19 + 3 pmol/L. However, up to 50 pmol/L thyronine caused no inhibition of collagen-induced platelet aggregation (Table 1). Figure 2 shows the dose-dependent inhibitory potency of these hormones. Platelet aggregation was induced by TPA at a final concentration of 2 ng/mL and by thrombin at a final concentration of 0.1 U/mL. However, rT3 did not inhibit the platelet aggregation induced by TPA and by thrombin (Fig. 3). TJ and Tq also did not inhibit at all either TPA- or thrombininduced platelet aggregation (Fig. 3). Effect of rT3 on serotonin releasefrom platelets Collagen (final concentration: 8 pg/mL) after the preincubation of 0.02 mol/L NaOH released approximately 10% of total added [‘*C]serotonin from platelets. T4 and T3 at 5-50
FIG. 1. Effect of rT3 (5-50 rmol/L) on collagen (8 pg/mL)-induced platelet aggregation The extent of aggregation was expressed as the change in light transmission. Control indicates aggregation after preincubation by adding 0.02 mol/ L NaOH, without rT3. 5, 10, 20, 30, 40, and 50 amol/L indicate the concentrations of rT3.
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rTg AND
COLLAGEN-INDUCED
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IO 0
I 0
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30
40
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Hormones
50
(u moi/L)
ji: 104 "
FIG. 2. Effect of thyroid hormones on platelet aggregation induced by collagen (8 rg/mL). Aggregation was expressed as the maximum percentage increase in light transmission, with 100% increase in transmission being defined as the maximum collagen-induced increase. l -0, TB; A- - -A, Tq; o----O, rT3; U----U, thyronine; and I- - - -m, Triac.
0
;o
3b
Concentration
(pmol/L)
i0 of Thyroid
Hormones
FIG. 4. Effect of thyroid hormones on l*C serotonin release from platelets when they are induced by collagen to aggregate. The data are expressed as the percentage control release without rT3. l - - - -0, TB; A- l l -A, T,; and o----O, rT3.
(A) -2 z I 2 z
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90
80 70 60 56
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,
,
,
,
10
20
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20
30
40
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Concentration of Thyroid Hormones FIG. 3. Effect of T,, T3, and rTg at 10 and 50 pmol/L on platelet aggregation induced by TPA (2 ng/mL) (A) or thrombin (0.1 U/mL) (B). Aggregation was expressed as the maximum increase in light transmission, with 100% increase in transmission being defined as the maximal increase induced by TPA (2 ng/mL) or thrombin (0.1 U/mL) (B). l - - - -0, TB; A- - -A, T,; and o--O, rT3.
pmol/L and rTg inhibited in a dose-dependent manner [‘“Cl serotonin releasefrom platelets (Fig. 4). Effect of rT3 on protein
phosphorylation
in platelets
Control phosphorylation of 20 kilodalton (kDa) and 40kDa proteins in platelets was induced by collagen (8 &mL) (left three lanes,Fig. 5). rTg (30 pmol/L) inhibited the 20-kDa
Colla en (8vg Bml)
Colla
en
(8l.y Bml) rT3 3011mol/L
0
30sec
60sec
60sec
120sec
FIG. 5. Effect of rTQ (30 rmol/L) on collagen (8 pg/mL)-induced platelet protein phosphorylation. The left-most three lanes show protein phosphorylation induced in platelets by collagen (8 pg/mL) by adding O.OZmol/L NaOH in the absence of rT3. The right-most two lanes show protein phosphorylation in platelets induced by collagen in the presence of rTg (30 rmol/L).
protein phosphorylation induced in platelets by collagen to 5.5 f 3.2% (n = 4) of control phosphorylation as shown in the right-most two lanes in Fig. 5. To a lesserextent, however, rTg (30 pmol/L) also inhibited collagen-induced 40-kDa protein phosphorylation to 43 + 2.6%-(n = 4) (Fig. 5).
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MASAKI
724
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ET
JCE & M * 1992 Vo175*No3
AL.
+ 9% (n = 4) of control (right-most two lanes,Fig. 6). Thus, the inhibition by rTg of TPA-induced protein phosphorylation was much lessthan that induced by collagen. As shown in Fig. 7, it is remarkable that the inhibitory effect of rTg on collagen-induced 20-kDa protein phosphorylation was much greater than that on TPA- and collagen-induced 40-kDa protein phosphorylation. This difference is significant as determined by two-way analysis of variance (P < 0.001) (Fig. 7). Discussion
TPA(2 rig/ml)
TPA(2 rig/ml) rTJf30 ii mol/L
0
3Osec
6Osec
120sec
60sec
120sec
FIG. 6. Effect of rTg on TPA (2 ng/mL)-induced platelet protein phosphorylation. The left-most four lanes show protein phosphorylation induced by TPA (2 ng/mL) by adding O.OZmol/L NaOH in the absence of rT3. The right two lanes show protein phosphorylation induced by TPA (2 ng/mL) in the presence of rT3 (30 pmol/L).
rTB
7. Effect of rTg on collagen- and TPA-induced platelet protein phosphorylation. The data are expressed as the percentage control protein phosphorylation induced by TPA (2 ng/mL) or collagen (8 rg/ mL). n , LO-kDa; 0, 40-kDa protein phosphorylation induced by collagen (8 rg/mL) in the presence of rT3; and Cl, 40-kDa protein phosphorylation induced by TPA (2 ng/mL) in the presence of rT!+ rTs inhibited collagen-induced 20-kDa protein phosphorylation. rTS also inhibited collagen-induced 40-kDa protein phosphorylation, but to a lesser extent. In contrast, the inhibitory potency of rTs on TPA-induced 40-kDa protein phosphorylation was much weaker than that of collagen-induced 20-kDa protein phosphorylation. FIG.
Similarly, TPA (2 ng/mL) after preincubation by adding 0.02 mol/L NaOH induced phosphorylation of the 40-kDa protein (left four lanes, Fig. 6). However, rT3 (30 pmol/L) inhibited phosphorylation of the 40-kDa protein to only 81.5
Thyroid hormones can be detected in plasma and virtually every organ and they have a variety of physiological actions. However, it is generally accepted that rTg is merely an inactive metabolite of Tq and that it has few biological actions (8). In the present study, we show clearly that rTg inhibits collagen-induced human platelet activation at concentrations of 5-50 pmol/L in a dose-dependent manner, as do T4 and TJ. rTJ, Tl, and T3 did not differ significantly in their ability to inhibit platelet activation. Although thyroid hormones are reported to have some extranuclear effects (9, lo), there is a general agreement that most of the characteristic biological effects of TJ and Tq are mediated by the interaction of TBwith specific nuclear receptors (11, 12). It is very interesting that the inhibition by rT, of collagen-induced platelet aggregation is mediated not by interaction with a nuclear receptor but by an extranuclear action. We know this to be true because platelets have no nuclei. Tq and T3 are reported to inhibit collagen-induced platelet aggregation by inhibiting the activity of MLCK (6). In the present study, rTg similarly inhibited phosphorylation of the 20-kDa protein identified as myosin light chain (13). Moreover, rT3 also inhibited the following phosphorylation of a 40-kDa protein (target protein of protein kinase C), albeit to a lesserextent. These data suggest that rTg inhibits collagen-induced platelet aggregation partly by inhibiting protein kinase C. However, rTg does not inhibit thrombin- and TPA-induced platelet aggregation. Thrombin activates both phosphatidylinositol turnover and the Ca-calmodulin-MLCK pathway. TPA is a direct activator of protein kinase C. The inhibition by rT3 of TPA-induced 40-kDa protein phosphorylation was significantly less intense than its inhibition of collagen-induced 40-kDa protein phosphorylation. It was reported previously that ML-9, which is an MLCK-specific inhibitor, inhibited to a lesserextent both the 20-kDa protein and 40kDa protein in platelets (14). Therefore, we hypothesize that rT3 inhibits collagen-induced platelet aggregation mainly by inhibiting MLCK activity. rT3, Th, T3, and Triac are all potential inhibitors of platelet aggregation, but thyronine doesnot inhibit collagen-induced platelet aggregation. This suggeststhat iodination is critical to the inhibitory effect of rT3. In this study, the effective concentration of rTJ was about 30 pmol/L, which was much-greater than the physiological
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 05:20 For personal use only. No other uses without permission. . All rights reserved.
rTg AND level. However, it would kinds of activity of rT3.
be interesting
COLLAGEN-INDUCED to investigate
some
References 1. Shambaugh III GE. 1986 Biologic and cellular effects. In: Ingbar SH, Braverman LH, eds. Werner’s the thyroid. 5th ed. Philadelphia: JB Lippincott Company; 201-18. 2. Papavasiliou SS, Martial JA, Latham KR, Baxter JD, 1977 Thyroid hormone like actions of 3,3’,5’-L-triiodothyronine and 3,3’-diiodothyronine. J Clin Invest. 60:1230-9. 3. Fishman N, Huang YP, Tergis DC, Rivlin RS. 1977 Relation of triiodothyronine and reverse triiodothyronine administration in rats to hepatic L-triiodothyronine aminotransferase activity. Endocrinology. 100:1055-9. 4. Chopra IJ. 1977 A study of extrathyroidal conversion of thyroxine(TI) to 3,3’,5-triiodothyronine(T3) in vitro. Endocrinology. 101:453-63. 5. Hagiwara M, Mamiya S, Ochiai M, Hidaka H. 1988 Thyroid hormones inhibit the Ca” calmodulin-induced activation of myosine light chain kinase. Biochem Biophys Res Commun. 152:270-6. 6. Mamiya S, Hagiwara M, Inoue S, Hidaka H. 1989 Thyroid hormones inhibit platelet function and myosine light chain kinase. J
PLATELET
AGGREGATION
Biol Chem. 264:8575-9. 7. Ishikawa T, Chijima T, Hagiwara M, Mamiya S, Hidaka H. 1989 Thyroid hormones directly interact with vascular smooth muscle strips. Mol Pharmacol. 35:760-5. 8. Chopra IJ. 1986 Nature, sources and relative biological significance of thyroid circulating hormones. In: Ingbar SH, Braverman LE, eds. Werner’s the thyroid. 5th ed. Philadelphia: JB Lippincott Company, 136-53. 9. Segal J, Hubert H. 1989 A rapid extranuclear effect of 3,5,3’-triiodo thyronine on sugar uptake by several tissues in the rat in vivo. Evidence for a physiological role for the thyroid hormone action at the level of the plasma membrane. Endocrinology. 124:2755-64. 10. Siegrist-Kaiser CA, Aubry CJ, Tranter MP, Ekenbarger DM, Leonard JL. 1990 Thyroxine dependent modulation of actin polymerization in cultured astrocytes. J Biol Chem. 265:5296-302. 11. Oppenheimer JH. 1985 Thyroid hormone action at the nuclear level. Ann Intern Med. 102:374-84. 12. Samuels HH, Forman BM, Horowitz ZD, Ye ZS. 1988 Regulation of gene expression by thyroid hormone. J Clin Invest. 81:957-67. 13. Adelstein RS, Conti MA, Anderson Jr W. 1973 Phosphorylation of human platelet myosin. Proc Nat1 Acad Sci USA. 70:3115-9. 14. Saitoh M, Naka M, Hidaka H. 1986 The modulatory role of myosin light chain phosphorylation in human platelet activation. Biochem Biophys Res Commun. 140:280-7.
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Vol. 75, No. 3 Printed m U S.A
3,3’,5’-Triiodothyronine Inhibits Human Platelet Aggregation HIROYA MASAKI*, MITSUSHIGE NISHIKAWA, MASAYOSHI YOSHIMURA, NAGAOKI TOYODA, NOR10 YOSHIKAWA, AND MITSUO INADA Second Department
of
Internal
Medicine,
Kansai
Medical
Collagen-Induced
MASAYA URAKAMI, YASUKIYO MORI,
University,
Moriguchi
City, Osaka 570, Japan
ABSTRACT To clarify further the activity of rTcl, we examined the effect of rT:? on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rTa, T,, Ta, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rTij inhibited by 50% collagen-induced platelet aggregation was 30 f 4 (mean ? SE) Fmol/L. rT:$, T,, and T:] did not
differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT:, inhibited collagen-induced phosphorylation of the 20. kilodalton protein (myosin light chain) in platelets. In contrast, rTrl did not inhibit 12-0-tetradecanoylphorbol13-acetate (TPA)or thrombin-induced platelet aggregation and inhibited only minimally TPAinduced 40-kilodalton protein phosphorylation. These results suggest that rTn inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT:+ (J Clin Endocrinol Metab 75: 721-725,1992)
I
any drug for at least 2 weeks before venipuncture. The platelet rich plasma (PRP) was obtained from the supernatant after centrifugation (250 X g for 10 min at 22 C). After further centrifugation (2200 X g for 10 min at 22 C) of the PRP, a pellet was obtained. This pellet was resuspended and washed twice with HEPES-Tyrode’s buffered saline (NaCI 129 mmol/L, NaHC03 8.9 mmol/L, KHIPOl 0.8 mmol/L, MgCb 0.8 mmol/L, dextrose 5.6 mmol/L, HEPES 10 mmol/L, pH 7.40). It was then resuspended in HEPES-Tyrode’s buffered saline to a final platelet concentration of 4 X lO’/~l in aliquots of 0.2 mL. Platelet aggregation was calculated from the turbidity of the solution as measured by a four-channel aggregometer (Nikko Biosciense Co. Tokyo, Japan). Washed platelets were preincubated in the aggregometer for 5 min at 37 C and then incubated with rT,, Tq, Ta, Triac, or thyronine at concentrations ranging from 5-50 bmol/L for 2 min at 37 C before adding collagen, thrombin, or TPA. Control aggregations were induced by each agonist after preincubation by adding 0.02 mol/L NaOH alone. Aggregation was expressed either as the change in light transmission or as the maximum percentage increase in light transmission, with a 100% increase in transmission being defined as the maximum increase induced by an agonist of platelet aggregation (collagen, thrombin, or TPA) in the absence of any antagonist.
T IS WELL known that T3 and T4 have various biological activities and play an essential role in maintaining homeostasis in mammals (1). However, there are few reports regarding the biological activities of rT3, to which as much as 60% of T4 is metabolized in peripheral tissues (2-4). Recently, Hidaka and co-workers (5-7) reported that T3 and Tq inhibit the functions of platelets and vascular smooth muscle by inhibiting the activity of myosin light chain kinase (MLCK). In their reports, however, the activity of rT3 was not mentioned. Described in the present report is our study of the effect of rTj on human platelet functions. This study was performed to clarify further the role of rT3 outside the nucleus and to elucidate the possible mechanism by which rTj inhibits platelet aggregation. Materials
and Methods Serotonin
Materials T,, Ts, thyronine, 3,5,3’-triiodothyroacetic acid (Triac), 12O-tetradecanoylphorbol 13-acetate (TPA), EGTA, and thrombin were obtained from the Sigma Chemical Co. (St. Louis, MO). rT) was obtained from the Calbiochem Behring Co. (La Jolla, CA). Collagen was obtained from Hormon Chemie (Munich, Germany). [‘“C]Serotonin and 32P, specific activity 1.85-2.2 gigabecquerel (GBq)/mmol and 370 MBq/mL, respectively, were obtained from the Amersham Japan Co. (Tokyo, Japan). A sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis system was purchased from the Daiichi Chemical Co. (Kyoto, Japan). All thyroid hormones were dissolved in 0.02 mol/L NaOH.
Platelet
aggregation
Venous blood samples treated with the anticoagulant dextrose were prepared from healthy volunteers who Received October 25, 1991. * To whom requests for reprints
should
release from platelets
Washed platelets were incubated with [‘4C]serotonin (92.5 kBq/mL PRP) for 60 min at 37 C and spun at 2,200 X g for 8 min at 22 C. The pellet was resuspended in HEPES-Tyrode’s buffered saline to a concentration of 5 X 10”/~1. The labeled platelets were preincubated for 5 min at 37 C and then incubated with rT3, T,, or T2 at concentrations ranging from 5-50 pmol/L for 2 min at 37 C before the stimulation with collagen (final concentration 8 pg/mL). Control serotonin release was induced by collagen (8 Kg/ml) after the preincubation by adding 0.02 mol/L NaOH alone. The reaction was terminated with ice-cold EGTA and spun at 12,800 X 8 for 10 min at 0 C. The supernatants were mixed with 4 mL aqueous counting scintillant II (ACS II) and counted in a liquid scintillation counter. The data were expressed as percentage total platelet [‘“Clserotonin measured after lysing the platelets with 10% Triton X-100.
10% acid citrate had not been on
Protein min
be addressed.
phosphorylation
in platelets
Washed platelets were incubated with 32P (9.25 MBq/mL PRP) for 90 at 37 C and spun at 2200 x g for 8 min, 22 C. The pellet was
721
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 05:20 For personal use only. No other uses without permission. . All rights reserved.
722
MASAKI
TABLE 1. The inhibitory potency of thyroid hormones on collagen-induced platelet aggregation Thyroid hormones
I.50~WOllL)” 30 + 4 r’I’3 21+ 3 T4 16 rt 4 l-3 Triac 19 + 3 Thyronine >50 ’ The values are expressed as the mean + SE.
n 6 6 6 4 3
resuspended, preincubated, and then incubated with rTB, Td, or T3 in a manner identical to that in the experiment in which serotonin was released before the stimulation with collagen (final concentration: 8 rg/ mL). Control platelet protein phosphorylations were induced by collagen or TPA after preincubation by adding 0.02 mol/L NaOH alone. The reaction was terminated by adding sampling buffer (49.5 mmol/L TrisHCI, 6.9% SDS, 30% glycerol, pH 6.8, including bromphenol blue) at various time points. The samples were subjected to 12.5% SDS polyacrylamide gel electrophoresis. The gel was dried by a gel dryer (BioRad Laboratories Co, Richmond, CA) and exposed to Fuji x-ray film. The relative intensity of each band was quantitated from a densitometric tracing of the autoradiogram.
Statistics All values were presented as the mean + SE. Statistical significance was determined by two-way analysis of variance.
Results Effect of rT3 on platelet aggregation Platelet aggregation was induced by collagen at concentrations of 1-8 rg/mL in a dose-dependent manner. Control
ET AL.
JCE & M .1992 Vol75.No3
experiments revealed that collagen at a final concentration of 8 pg/mL and thrombin at a final concentration of 0.1 U/ mL after preincubation by adding 0.02 mol/L NaOH induced the maximum platelet aggregation. T, and TJ, at concentrations ranging from 5-50 pmol/L, inhibited collagen-induced platelet aggregation in a dosedependent fashion; the concentrations that inhibited platelet aggregation by 50% (Iso) were 21 + 3 and 16 + 4 pmol/L, respectively (Table 1). rTg at concentrations of 5-50 pmol/L also inhibited collagen-induced platelet aggregation in a dose-dependent manner (Fig. l), and for rTg the Isowas 30 + 4 pmol/L (Table 1). Triac also inhibited collagen-induced platelet aggregation, with its Isobeing 19 + 3 pmol/L. However, up to 50 pmol/L thyronine caused no inhibition of collagen-induced platelet aggregation (Table 1). Figure 2 shows the dose-dependent inhibitory potency of these hormones. Platelet aggregation was induced by TPA at a final concentration of 2 ng/mL and by thrombin at a final concentration of 0.1 U/mL. However, rT3 did not inhibit the platelet aggregation induced by TPA and by thrombin (Fig. 3). TJ and Tq also did not inhibit at all either TPA- or thrombininduced platelet aggregation (Fig. 3). Effect of rT3 on serotonin releasefrom platelets Collagen (final concentration: 8 pg/mL) after the preincubation of 0.02 mol/L NaOH released approximately 10% of total added [‘*C]serotonin from platelets. T4 and T3 at 5-50
FIG. 1. Effect of rT3 (5-50 rmol/L) on collagen (8 pg/mL)-induced platelet aggregation The extent of aggregation was expressed as the change in light transmission. Control indicates aggregation after preincubation by adding 0.02 mol/ L NaOH, without rT3. 5, 10, 20, 30, 40, and 50 amol/L indicate the concentrations of rT3.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 05:20 For personal use only. No other uses without permission. . All rights reserved.
rTg AND
COLLAGEN-INDUCED
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3 E % B
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6 70s 60% 50;a, 40-
60-
lYx 30.c 5 20-
IO 0
I 0
10
20
Concentratmn
30
40
of Thyrad
Hormones
50
(u moi/L)
ji: 104 "
FIG. 2. Effect of thyroid hormones on platelet aggregation induced by collagen (8 rg/mL). Aggregation was expressed as the maximum percentage increase in light transmission, with 100% increase in transmission being defined as the maximum collagen-induced increase. l -0, TB; A- - -A, Tq; o----O, rT3; U----U, thyronine; and I- - - -m, Triac.
0
;o
3b
Concentration
(pmol/L)
i0 of Thyroid
Hormones
FIG. 4. Effect of thyroid hormones on l*C serotonin release from platelets when they are induced by collagen to aggregate. The data are expressed as the percentage control release without rT3. l - - - -0, TB; A- l l -A, T,; and o----O, rT3.
(A) -2 z I 2 z
x :.g pm a p
(%) 100
v “‘.*
90
80 70 60 56
1,
0
0
c.-.-. . . . . . . . . . . . . . . . . . .__. . . . . . . . . ...::::*Q
,
,
,
,
10
20
30
40
,
+40K
50 (~,mol/L)
Concentration of Thyroid Hormones
W z &g s 2 z
(96) 100 ~:,*“.-.-
.--.....
&.z:
. . . . . . e&
+20K
90
80
s 60 ‘E g 50 5E 7001, 8 =Jz
0
,
,
,
,
10
20
30
40
, 50 (v mol/L)
Concentration of Thyroid Hormones FIG. 3. Effect of T,, T3, and rTg at 10 and 50 pmol/L on platelet aggregation induced by TPA (2 ng/mL) (A) or thrombin (0.1 U/mL) (B). Aggregation was expressed as the maximum increase in light transmission, with 100% increase in transmission being defined as the maximal increase induced by TPA (2 ng/mL) or thrombin (0.1 U/mL) (B). l - - - -0, TB; A- - -A, T,; and o--O, rT3.
pmol/L and rTg inhibited in a dose-dependent manner [‘“Cl serotonin releasefrom platelets (Fig. 4). Effect of rT3 on protein
phosphorylation
in platelets
Control phosphorylation of 20 kilodalton (kDa) and 40kDa proteins in platelets was induced by collagen (8 &mL) (left three lanes,Fig. 5). rTg (30 pmol/L) inhibited the 20-kDa
Colla en (8vg Bml)
Colla
en
(8l.y Bml) rT3 3011mol/L
0
30sec
60sec
60sec
120sec
FIG. 5. Effect of rTQ (30 rmol/L) on collagen (8 pg/mL)-induced platelet protein phosphorylation. The left-most three lanes show protein phosphorylation induced in platelets by collagen (8 pg/mL) by adding O.OZmol/L NaOH in the absence of rT3. The right-most two lanes show protein phosphorylation in platelets induced by collagen in the presence of rTg (30 rmol/L).
protein phosphorylation induced in platelets by collagen to 5.5 f 3.2% (n = 4) of control phosphorylation as shown in the right-most two lanes in Fig. 5. To a lesserextent, however, rTg (30 pmol/L) also inhibited collagen-induced 40-kDa protein phosphorylation to 43 + 2.6%-(n = 4) (Fig. 5).
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MASAKI
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+ 9% (n = 4) of control (right-most two lanes,Fig. 6). Thus, the inhibition by rTg of TPA-induced protein phosphorylation was much lessthan that induced by collagen. As shown in Fig. 7, it is remarkable that the inhibitory effect of rTg on collagen-induced 20-kDa protein phosphorylation was much greater than that on TPA- and collagen-induced 40-kDa protein phosphorylation. This difference is significant as determined by two-way analysis of variance (P < 0.001) (Fig. 7). Discussion
TPA(2 rig/ml)
TPA(2 rig/ml) rTJf30 ii mol/L
0
3Osec
6Osec
120sec
60sec
120sec
FIG. 6. Effect of rTg on TPA (2 ng/mL)-induced platelet protein phosphorylation. The left-most four lanes show protein phosphorylation induced by TPA (2 ng/mL) by adding O.OZmol/L NaOH in the absence of rT3. The right two lanes show protein phosphorylation induced by TPA (2 ng/mL) in the presence of rT3 (30 pmol/L).
rTB
7. Effect of rTg on collagen- and TPA-induced platelet protein phosphorylation. The data are expressed as the percentage control protein phosphorylation induced by TPA (2 ng/mL) or collagen (8 rg/ mL). n , LO-kDa; 0, 40-kDa protein phosphorylation induced by collagen (8 rg/mL) in the presence of rT3; and Cl, 40-kDa protein phosphorylation induced by TPA (2 ng/mL) in the presence of rT!+ rTs inhibited collagen-induced 20-kDa protein phosphorylation. rTS also inhibited collagen-induced 40-kDa protein phosphorylation, but to a lesser extent. In contrast, the inhibitory potency of rTs on TPA-induced 40-kDa protein phosphorylation was much weaker than that of collagen-induced 20-kDa protein phosphorylation. FIG.
Similarly, TPA (2 ng/mL) after preincubation by adding 0.02 mol/L NaOH induced phosphorylation of the 40-kDa protein (left four lanes, Fig. 6). However, rT3 (30 pmol/L) inhibited phosphorylation of the 40-kDa protein to only 81.5
Thyroid hormones can be detected in plasma and virtually every organ and they have a variety of physiological actions. However, it is generally accepted that rTg is merely an inactive metabolite of Tq and that it has few biological actions (8). In the present study, we show clearly that rTg inhibits collagen-induced human platelet activation at concentrations of 5-50 pmol/L in a dose-dependent manner, as do T4 and TJ. rTJ, Tl, and T3 did not differ significantly in their ability to inhibit platelet activation. Although thyroid hormones are reported to have some extranuclear effects (9, lo), there is a general agreement that most of the characteristic biological effects of TJ and Tq are mediated by the interaction of TBwith specific nuclear receptors (11, 12). It is very interesting that the inhibition by rT, of collagen-induced platelet aggregation is mediated not by interaction with a nuclear receptor but by an extranuclear action. We know this to be true because platelets have no nuclei. Tq and T3 are reported to inhibit collagen-induced platelet aggregation by inhibiting the activity of MLCK (6). In the present study, rTg similarly inhibited phosphorylation of the 20-kDa protein identified as myosin light chain (13). Moreover, rT3 also inhibited the following phosphorylation of a 40-kDa protein (target protein of protein kinase C), albeit to a lesserextent. These data suggest that rTg inhibits collagen-induced platelet aggregation partly by inhibiting protein kinase C. However, rTg does not inhibit thrombin- and TPA-induced platelet aggregation. Thrombin activates both phosphatidylinositol turnover and the Ca-calmodulin-MLCK pathway. TPA is a direct activator of protein kinase C. The inhibition by rT3 of TPA-induced 40-kDa protein phosphorylation was significantly less intense than its inhibition of collagen-induced 40-kDa protein phosphorylation. It was reported previously that ML-9, which is an MLCK-specific inhibitor, inhibited to a lesserextent both the 20-kDa protein and 40kDa protein in platelets (14). Therefore, we hypothesize that rT3 inhibits collagen-induced platelet aggregation mainly by inhibiting MLCK activity. rT3, Th, T3, and Triac are all potential inhibitors of platelet aggregation, but thyronine doesnot inhibit collagen-induced platelet aggregation. This suggeststhat iodination is critical to the inhibitory effect of rT3. In this study, the effective concentration of rTJ was about 30 pmol/L, which was much-greater than the physiological
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rTg AND level. However, it would kinds of activity of rT3.
be interesting
COLLAGEN-INDUCED to investigate
some
References 1. Shambaugh III GE. 1986 Biologic and cellular effects. In: Ingbar SH, Braverman LH, eds. Werner’s the thyroid. 5th ed. Philadelphia: JB Lippincott Company; 201-18. 2. Papavasiliou SS, Martial JA, Latham KR, Baxter JD, 1977 Thyroid hormone like actions of 3,3’,5’-L-triiodothyronine and 3,3’-diiodothyronine. J Clin Invest. 60:1230-9. 3. Fishman N, Huang YP, Tergis DC, Rivlin RS. 1977 Relation of triiodothyronine and reverse triiodothyronine administration in rats to hepatic L-triiodothyronine aminotransferase activity. Endocrinology. 100:1055-9. 4. Chopra IJ. 1977 A study of extrathyroidal conversion of thyroxine(TI) to 3,3’,5-triiodothyronine(T3) in vitro. Endocrinology. 101:453-63. 5. Hagiwara M, Mamiya S, Ochiai M, Hidaka H. 1988 Thyroid hormones inhibit the Ca” calmodulin-induced activation of myosine light chain kinase. Biochem Biophys Res Commun. 152:270-6. 6. Mamiya S, Hagiwara M, Inoue S, Hidaka H. 1989 Thyroid hormones inhibit platelet function and myosine light chain kinase. J
PLATELET
AGGREGATION
Biol Chem. 264:8575-9. 7. Ishikawa T, Chijima T, Hagiwara M, Mamiya S, Hidaka H. 1989 Thyroid hormones directly interact with vascular smooth muscle strips. Mol Pharmacol. 35:760-5. 8. Chopra IJ. 1986 Nature, sources and relative biological significance of thyroid circulating hormones. In: Ingbar SH, Braverman LE, eds. Werner’s the thyroid. 5th ed. Philadelphia: JB Lippincott Company, 136-53. 9. Segal J, Hubert H. 1989 A rapid extranuclear effect of 3,5,3’-triiodo thyronine on sugar uptake by several tissues in the rat in vivo. Evidence for a physiological role for the thyroid hormone action at the level of the plasma membrane. Endocrinology. 124:2755-64. 10. Siegrist-Kaiser CA, Aubry CJ, Tranter MP, Ekenbarger DM, Leonard JL. 1990 Thyroxine dependent modulation of actin polymerization in cultured astrocytes. J Biol Chem. 265:5296-302. 11. Oppenheimer JH. 1985 Thyroid hormone action at the nuclear level. Ann Intern Med. 102:374-84. 12. Samuels HH, Forman BM, Horowitz ZD, Ye ZS. 1988 Regulation of gene expression by thyroid hormone. J Clin Invest. 81:957-67. 13. Adelstein RS, Conti MA, Anderson Jr W. 1973 Phosphorylation of human platelet myosin. Proc Nat1 Acad Sci USA. 70:3115-9. 14. Saitoh M, Naka M, Hidaka H. 1986 The modulatory role of myosin light chain phosphorylation in human platelet activation. Biochem Biophys Res Commun. 140:280-7.
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