THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1959 hy The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 15, Ieaue of May 25, pp. 8575-6579.1959 Printed in U.S.A.

Thyroid Hormones Inhibit Platelet Function and Myosin Light Chain Kinase*

(Received for publication, December 21, 1987)

Shigeo Mamiya, Masatoshi HagiwaraS, Shigeo Inoue, and Hiroyoshi HidakaSQ From the Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Edobashi, Tsu, Mie 514 and the $Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan

We examined the extranuclear effects of thyroid hor- mones on human platelets. Pretreatment with DL-thy- roxine or DL-triiodothyronine inhibited collagen-in- duced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no signifi- cant effects. In contrast to collagen, 12-0-tetradeca- noylphorbol- 13-acetate-induced aggregation was not affected by thyroid hormones at the same concentra- tion range. Thyroxine also inhibited the release of [‘“CI serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000- dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/ phospholipid-dependent enzyme) and CAMP-depend- ent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 PM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also de- signed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine- affinity chromatography. A markedly increased puri- fication was thus achieved, and DEAE-cellulose and L- thyroxine-affinity chromatography were made feasi- ble. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated re- actions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin.

Thyroid hormones show a wide variety of biological activi- ties, and .they exert profound effects on various metabolic processes in almost all types of cells. The major cellular activities of these hormones have been described in terms of nucleus-mediated events (l), and nuclear-binding sites are found in various tissues (2). Other effects of the hormones may be expressed by extranuclear mechanisms (3). Using

* This work was supported in part by a grant-in-aid for the scien- tific research from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom all correspondence should be addressed Dept. of Phar- macology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.

platelets from healthy humans, we examined the extranuclear effects of thyroid hormones on these tissues.

In the presence of activation, platelets respond by shape change, aggregation, release reaction, and clot retraction. Here, contractile proteins seem to have important roles (4). Other workers (5-7) suggested that protein phosphorylation is involved in shape change and the release reaction of plate- lets. The Ca2+-calmodulin-dependent phosphorylation of my- osin light chain (MLC)’ catalyzed by MLC kinase may be the major regulatory system of contractile proteins in platelets (5-9).

We report here that thyroid hormones, in particular thy- roxine, inhibit platelet function and MLC phosphorylation via suppression of MLC kinase, at the micromolar level of the hormone, in vitro, therefore, thyroxine can serve as a useful ligand to purify MLC kinase from human platelets.

EXPERIMENTAL PROCEDURES

Chemicals-DL-Thyroxine, DL-triiodothyronine, and DL-tyrOSine were purchased from Sigma. Other derivatives of thyroid hormones were generous gifts of Dr. A. Nagasaka (Fujita Gakuen University). All other chemicals used were of analytical grade or better.

Platelet Aggregation and Release Reaction-Washed platelets were prepared from the blood of healthy adult volunteers who had not been on any drug for at least 1 week before venipuncture. The platelet- rich plasma was obtained by centrifugation (164 X g, for 10 rnin), at room temperature. After two washes, the pellet was suspended in Hepes-buffered saline (135 mM NaCl, 2.7 mM KCl, 1 mM MgCL, 20 mM Hepes, 5 mM glucose, pH 7.35). Platelet aggregation was studied turbidimetrically, using a Rikadenki 4 channel aggregometer (RAM41), the final volume of the platelets being 0.3 ml. The platelets were preincubated with thyroid hormones in the aggregometer for 2 min at 37 “C, before stimulation with agonists.

Serotonin release was measured as described by Costa and Murphy (10). Washed platelets were incubated for 60 min at 25 “C with 0.1 pCi/ml of [“C]serotonin (Amersham Corp.), spun at 600 X g for 5 min and the supernatant discarded. The pellet was resuspended in the same buffer to give a count of 5-10 X 10s/ml. The reaction was terminated with 1.5% formalin. Samples of the supernatant were mixed with 10 ml of ACS I1 (Amersham Corp.) and counted in a LKB liquid scintillation counter. The data were expressed as a percentage of the total platelet [“Clserotonin, measured after lysis of the plate- lets with 1% Triton X-100.

Protein Kinase Assay-MLC kinase was prepared from fresh hu- man platelets, using the method of Hathaway and Adelstein (8). Calmodulin was purified from frozen bovine brain by the procedures described by Endo et al. (11). MLC was prepared from chicken gizzard by the method of Hathaway and Haeberle (12). MLC kinase activities were measured by quantification of [32P]phosphate into isolated MLC, as described (13), in the presence and absence of thyroid hormones. Kinase assay was performed at 30 “C in a final volume of 0.2 ml containing 50 mM Tris-HC1, pH 7.0,lO mM magnesium acetate,

The abbreviations used are: MLC, myosin light chain; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

8575

8576 Thyroid Hormones Inhibit MLC Kinase 0.1 mM calcium chloride, or 1 mM EGTA, 100 ng of calmodulin, 5- 100 p M [Y-~*P]ATP (4 X lo5 cpm), 20 p M smooth muscle 20-kDa MLC, and the enzyme (specific activity 1.3 pmol/min/mg). Protein kinase C and CAMP-dependent kinase were also prepared from fresh human platelets, according to Hidaka and Tanaka (14), and Beavo et al. (15), respectively. Protein kinase C activity was assayed in a reaction mixture containing 50 mM Tris-HC1, pH 7.0, 10 mM mag- nesium acetate, 0.5 mM calcium chloride or 1 mM EGTA, 10 pg of phosphatidylserine, 3.3-20 p~ [Y-~'P]ATP, 20 p~ 20-kDa gizzard MLC, and the enzyme (specific activity 0.1 pmol/min/mg). CAMP- dependent kinase activity was assayed in a reaction mixture contain- ing 50 mM Tris-HC1, pH 7.0, 10 mM magnesium acetate, 2 mM EGTA, 1 p M CAMP, 3.3-20 p~ [Y-~'P]ATP, 20 p~ 20-kDa gizzard MLC, and the enzyme (specific activity 2.0 pmol/min/mg).

Phosphoproteins in Platelet-For analysis of phosphoproteins, washed platelets were incubated with 0.1 mCi/ml of [32P]orthophos- phate (Japan Radioisotope Association). At various periods, the re- action was terminated by addition of a sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% SDS, 8 M urea, 1 mM dithiothreitol, 0.1 M Tris-HC1, pH 6.8). The sample was subjected to SDS-PAGE, under the conditions de- scribed by Laemmli (16). The separating and stacking gels contained 15 and 5% acrylamide, respectively. The gel was stained with Coo- massie Brilliant Blue, destained, and exposed to a Kodak X-Omat R P x-ray film. The relative intensity of each band was quantitated by densitometric tracing on the autoradiogram, using a GS3000 Transmittance/Refractance scanning densitometer.

Preparation of L-Thyroxine-coupled Sepharose-The gel used for affinity chromatography was prepared by coupling L-thyroxine to cyanogen bromide-activated Sepharose 4B (Pharmacia LKB Biotech- nology Inc.) as follows. L-Thyroxine (10 mg) was dissolved in 10 ml of 50 mM borate buffer, pH 8.0, containing 50% dimethyl sulfoxide and 0.1 M NaC1, and added to 10 ml of settled cyanogen bromide- activated Sepharose 4B. After an overnight incubation at 4 "C, the resin was incubated in 1.0 M ethanolamine, pH 8.0, for 2 h at room temperature and washed with 100 ml of distilled water, alternating 20 mM Tris-HC1, pH 7.5, containing 5 mM MgC12,O.l mM CaC12, and 100 mM NaCl. The amount of L-thyroxine coupled to cyanogen bromide-activated Sepharose 4B was determined to be 320 pg/ml gel, according to decrease in the amounts of L-thyroxine, measured by absorbance a t 240 nm in the supernatant of reaction mixture after the coupling reaction.

Preparation of Platelet Myosin Light Chain Kinase by L-Thyroxine- coupled Sepharose-Human platelets were prepared from 600 ml of fresh blood by differential centrifugation. The pellet was resuspended in 150 mM NaC1, 0.5 mM EDTA, 1 mM dithiothreitol, centrifuged a t 1,500 X g for 15 min and the procedure repeated twice. After the final wash the pellet was resuspended in 25 ml of 20 mM Tris-HC1, pH 7.5, 2 mM dithiothreitol, 10 mM EDTA, 1 mM EGTA, 0.1 mM phenyl- methylsulfonyl fluoride, soybean trypsin inhibitor a t 0.1 mg/ml and the platelets then disrupted by sonication (30 s a t frequency 7, Branson Sonifier model W-225R). The resulting lysate was sedi- mented at 28,000 X g for 30 min, and the supernatant was collected for subsequent purification as described helow. The extract was applied to a DEAE-cellulose column (1.5 X 14 cm) that had been equilibrated with 20 mM Tris-HC1, pH 7.5, 10 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 20 mM KC1. The column was washed until no further protein was eluted. The enzyme was then eluted with a 300-ml linear KC1 gradient (20-500 mM KC1) a t a flow rate of 15 ml/h and 1.5-ml fractions were collected. Kinase activity was deter- mined in the presence of either 0.1 mM CaC12 and 0.1 p M calmodulin or 1 mM EGTA. The active fractions obtained by ion exchange chromatography were pooled. This pool was then further purified by L-thyroxine-affinity chromatography. Twenty ml of the enzyme so- lution were applied to a 2 x 3-cm L-thyroxine-affinity Sepharose column and the column then washed with 2 bed volumes of buffer A (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) containing 0.5 M NaC1, then, Buffer A, Buffer A containing 20% glycerol, and Buffer A containing 0.5 M NaCl and 20% glycerol, respectively. Kinase was eluted from the column with a 100-ml linear arginine gradient (0.3-0.6 M arginine).

Protein was quantified by the method of Bradford (17).

RESULTS

Effect of Thyroid Hormones on Platelet Aggregation and Release Reaction-DL-Thyroxine and DL-triiodothyronine in- hibited collagen-induced platelet aggregation, in a dose-de-

S A .s L 100

L

C Q) 0 Q) L

n o

B O

1 oa

., 3 5 1 0 30

Concentration of Antagonist ( L I R A )

"

4 L 100 A231 87

30

10

I 3

conlrol

100 1 TPA

FIG. 1. A, Effect of thyroid hormones and their derivatives on platelet aggregations induced by collagen. Aggregation was quanti- tated as the maximal percentage increase in light transmission with 100% increase in transmission defined as the maximal increase in- duced by 2 pg/ml of collagen without antagonist. Each point repre- sents the mean of three experiments. L-thyroxine (W), D-thy- roxine (W), L-triiodothyronine (A-A), D-triiodothyronine (A-A). * L-Diiodothyronine, L-diiodotyrosine, L-tyrosine, D-tyro- sine, and L-thyronine had no significant effect up to 30 pM

collagen (2 pg/ml), TPA (20 ng/ml), and A23187 (0.2 pM). Platelets (X- - -X). B, effect of L-thyroxine on platelet aggregation induced by

were preincubated a t 37 "C for 2 min without (control) or with L- thyroxine at concentrations ranging from 3 to 30 p ~ . The extent of aggregation was expressed as the change in light transmission.

Thyroid Hormones Inhibit MLC Kinase 8577

* O t 1 CO"l,Ol

- 3..M

5 M

- 30 M

10 M

0 30 60 90 Ttme (sec)

FIG. 2. Effect of L-thyroxine on ["C]serotonin release from platelets in response to collagen. The labeled platelets were prein- cubated without (control) or with L-thyroxine a t concentrations rang- ing 3-30 p~ before stimulation with collagen (3 pg/ml). The reaction was terminated with 1.5% formalin, and the supernatant was counted in a liquid scintillation counter. The data were expressed as a per- centage of the total platelet ["Clserotonin. Each point represents the mean of four experiments.

I

5 I I n I L '

Control T h y r o x l n e -

N I I

0 20 4 0 60 Time (sec)

FIG. 3. Effect of L-thyroxine on protein phosphorylation of 20-kDa protein in platelets prelabeled with [32P]orthophos- phate (0.1 mCi/ml) in response to collagen (2 pglml). Platelets were preincubated a t 37 "C without or with L-thyroxine before stim- ulation. At various periods, the reaction was terminated, the sample subjected to 15% SDS-PAGE, stained with Coomassie Blue, de- stained, and exposed to x-ray film. The relative intensity of the 20- kDa band was quantitated by densitometric tracing on the autoradi- ogram. Inset, autoradiograph of SDS-PAGE showing the effect of L- thyroxine (30 p ~ ) on platelet protein phosphorylation.

pendent fashion (Fig. IA), but L-diiodothyronine, L-diiodo- tyrosine, DL-tyrosine, and L-thyronine, up to 30 PM, had no significant effect on the collagen-induced platelet aggregation. Fig. 1B shows inhibitory effects of L-thyroxine on collagen, calcium ionophore A23187, and TPA-induced platelet aggre- gation. L-Thyroxine was also an effective inhibitor of A23187- induced platelet aggregation but did not significantly affect the aggregation induced by TPA, a direct activator of protein kinase C.

In the next experiments, the data of which are shown in Fig. 2, we examined the effect of thyroxine on ['4C]serotonin

TABLE I Inhibition of platelet protein kinase actioities b.v DL-thvroxine (TI)

and DL-triiodothyronine (Td The values are means f 1 S.D. of two or three determinations. The

assays of protein kinases are described under "Experimental Proce- dures." Gizzard MLC was used as a substrate of all protein kinases.

Protein kinases Conc. of Im"

L-TI D-T, L-Ts D-Ta

P M

MLC kinase 11.4 f 2.2 14.4 f 0.2 49 f 21 52 k 11 Protein kinase C 72.5 f 7.5 205 k 75 630 f 50 550 f 50 CAMP-dependent 140 f 20 185 f 25 550 ? 50 575 f 125

protein kinase Ii Im, concentration which caused a 50% inhibition of enzyme

activity.

5 I O I5 2 0

FIG. 4. Dixon plots of inhibition of platelet MLC kinase by L-thyroxine. The assay was performed in the presence of 0.4 pg/ml (O), 1.0 pg/ml (O), and 5.0 pg/ml (A) of calmodulin.

release from collagen-stimulated platelets. The results of these studies indicated that L-thyroxine dose-dependently inhibited the collagen-induced release of ['4C]serotonin. Only a weak release reaction was observed at the concentration of 30 PM. Thus, thyroxine seems to inhibit the stimulus-coupled release process in platelets.

Effect of L-Thyroxine on Protein Phosphorylation in Plate- let-In attempt to relate platelet function to protein phospho- rylation, we measured changes in phosphorylation of proteins in platelets that had been incubated with L-thyroxine, before the addition of collagen. In the control platelets, collagen induced a rapid phosphorylation of the 20-kDa protein. On the other hand, L-thyroxine inhibited this phosphorylation, dose-dependently. A marked inhibition was observed with 30 PM of L-thyroxine (Fig. 3). The 20-kDa protein has been identified as the light chain of platelet myosin (18) and L- thyroxine inhibited the 20-kDa light chain phosphorylation. These findings are compatible with the results of platelet aggregation.

Effect of Thyroid Hormones on Protein Kinases-To ex- amine the effect of thyroid hormones on platelet protein kinases, we prepared three protein kinases which phospho- rylate MLC from fresh human platelets and compared the effect of hormones on the kinase activities in vitro. The results of these studies (Table I) indicated that DL-thyroxine and DL- triiodothyronine inhibited MLC kinase more markedly than findings with protein kinase C and CAMP-dependent protein kinase. In addition, we found that thyroxine was a more potent inhibitor of these three kinases than was triiodothy- ronine. A kinetic analysis was made of the mode of interaction

8578 Thyroid Hormones Inhibit MLC Kinase

r-

A 2 O O K -

11 6K- 97u-

66U-

43K-

1 _p

II

2 -"

43K-

31K-

22K-

14U-

FIG. 5. Binding of MLC kinase or calmodulin to L-thyroxine affinity column. One ml of chicken gizzard MLC kinase (0.2 mg/ ml) ( A ) or bovine brain calmodulin (0.2 mg/ml) ( B ) was applied to the L-thyroxine-Sepharose column (0.5 X 2 cm), preequilibrated with Buffer A containing 0.5 M NaCI. The column was washed with 5 ml of the same buffer, 5 ml of Buffer A and 5 ml of the Buffer A containing 20% glycerol and 0.5 M NaCI. The buffer was changed to elution buffer containing Buffer A and 1.5 M arginine. The eluted products (1 ml/fraction) were analyzed by SDS-PAGE. A, 10% SDS- PAGE of chicken gizzard MLC kinase. B, 17.5% SDS-PAGE of bovine brain calmodulin. Lane I . starting material; lam 2, void fraction; lane 3, eluted fraction.

FIG. 6. L-Thyroxine-af f in i ty chromatography. The DEAE fraction was applied to L-thyroxine-affinity Sepharose column. The washing buffers were Buffer A containing 0.5 M NaCI, Buffer A, Buffer A containing 20% glyc- erol, and Buffer A containing 0.5 M NaCl and 20% glycerol. The elution buffer contained L-arginine with a linear gra- dient concentration (0.3-0.6 M). Protein kinase activity was measured in the pres- ence of either 0.1 mM CaC12 and 0.1 p M calmodulin (A-A) or 1 mM EGTA (A-A). SDS-PAGE of active kinase fraction is shown in the inset. Positions of molecular weight standards are indi- cated on the left.

of L-thyroxine with MLC kinase. As indicated in Fig. 4, L- thyroxine was a competitive inhibitor versus calmodulin and the Ki value was calculated to be 2.6 PM. On the other hand, other derivatives of thyroid hormone, L-diiodothyronine, I.- diiodotyrosine, DL-tyrosine, and L-thyronine, did not signifi- cantly affect the MLC kinase activity (data not shown).

Purification of Platelet Myosin Light Chain Kinase by L- Thyroxine-coupled Sepharose-Our data revealed that the inhibition of MLC kinase by L-thyroxine was competitive, with respect to calmodulin. However, the L-thyroxine-induced inhibition of the enzyme could not be overcome by high concentrations of ATP or MLC (data not shown). Other workers found that thyroid hormone was not capable of binding to calmodulin (19). To determine whether or not thyroid hormone directly binds to MLC kinase, we prepared an affinity column, using L-thyroxine as the ligand. When gizzard MLC kinase or bovine brain calmodulin was applied to the affinity column of L-thyroxine-Sepharose, MLC kinase was selectively retained while the calmodulin passed through (Fig. 5). All these results indicate that the inhibitory action of L-thyroxine seems to be the result of direct effects on the calmodulin-binding site of the enzyme. We, therefore, at- tempted to purify platelet MLC kinase using an L-thyroxine- coupled Sepharose gel. Fig. 6 shows typical elution profiles for chromatography on DEAE-cellulose and L-thyroxine-af- finity Sepharose. After ion exchange chromatography, the active fractions were applied to an affinity column. The platelet MLC kinase was eluted with buffer containing L- arginine, in a linear gradient concentration (0.3-0.6 M). The SDS-PAGE for the active kinase fraction is shown in Fig. 6. The peak of protein kinase activity correlated with the ap- proximately 100-kDa subunit which contains other protein bands (90, 52, 38, and 20-kDa). Table I1 summarizes the recovery and activity of the platelet MLC kinase at the various

TABLE I1 Recovew and actiuih of Dlatelet MLC kinase with Durification

pmollmin mg pmollminlmg % -fold Extract 85.4 113 0.0076 100 1 DEAE-cellulose 40 3.6 0.110 46 14 Thyroxine- 16 0.03 5.90 19 780 affinity

%K-

116K- 97u- - .

66K-

43K- 31K-

- """" ""

""""* ""_"" -"""

Thyroid Hormones

steps of the purification. The DEAE-cellulose pool repre- sented a 14-fold purification over 28,000 x g supernatant with a 46% overall recovery of activity. In the next L-thyroxine- Sepharose affinity column step, a markedly increased purifi- cation was achieved (780-fold).

DISCUSSION

A number of studies have shown that thyroid hormones have several extranuclear effects on cells (3, 20). In the present work, we obtained evidence that thyroid hormones inhibit collagen-induced platelet aggregation, the release re- action, and the phosphorylation of the 20-kDa MLC, in a dose-dependent fashion. Thyroxine also inhibits more mark- edly the MLC kinase than protein kinase C and CAMP- dependent protein kinase from platelets.

Pharmacological antagonism of protein phosphorylation is likely to be a fruitful approach to the manipulation of cellular responses and elucidation of the biological role of protein phosphorylation. The results of our study suggest that thy- roxine can serve as a pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions, without affecting the protein kinase C-dependent process, in platelets. In an attempt to relate protein phosphorylation to platelet function, we investigated the role of 20-kDa MLC phosphorylation, using the calmodulin antagonist W-7 (21). As an inhibitor of protein kinase C, we used H-7 (22), and a newly synthesized inhibitor of MLC kinase, ML-9 was also used (23). Our studies revealed that ML-9 also inhibits the collagen-induced platelet aggregation, the release reaction, and 20-kDa MLC phosphorylation but did not affect the protein phosphorylation induced by TPA (23). Using two inhibitors of MLC kinase, ML-9 and thyroxine, we confirmed that the kinase has an important role in collagen-stimulated platelet aggregation, release reaction, and 20-kDa MLC phos- phorylation. We also observed that following phosphorylation of 40-kDa peptide was depressed by thyroxine (Fig. 3, inset), although thyroxine produced little inhibition of Ca2'/phos- pholipid-dependent phosphorylation of 40-kDa peptide in cell-free assay system in doses up to 30 p~ (data not shown) as reported by Saitoh et al. (23) using ML-9. In addition, we considered that MLC kinase also plays a critical role in the A23187-induced platelet aggregation because thyroxine inhib- ited these aggregations. However, the mechanisms of inhibi- tion by ML-9 and thyroxine differed. The inhibition of MLC kinase by thyroxine was competitive with calmodulin, whereas ML-9 inhibited, in a competitive fashion, with ATP (24). Use of inhibitors of various aspects of MLC kinase should aid in elucidating effects of the kinase on platelet function.

Affinity chromatography, in other words biospecific adsorp- tion, has received considerable attention. Dramatic enzyme purifications have been observed using this technique. In most cases and for each individual enzyme, a different specific ligand bound to the matrix. In the present work, we used L- thyroxine as the ligand for the separation of MLC kinase since L-thyroxine is a selective inhibitor of MLC kinase. Indeed, platelet MLC kinase bound directly to L-thyroxine-

Inhibit MLC Kinase 8579

Sepharose, and a markedly increased purification was thus achieved. For preparation of MLC kinase, calmodulin has been used as a ligand of affinity chromatography. In our study, the purification of platelet MLC kinase by L-thyroxine affinity chromatography was more satisfactory than that by the calmodulin affinity reported by other investigators (8). L- Thyroxine can presumably serve as a substitute for calmo- dulin since it is commercially available and cost effective. We propose that L-thyroxine-affinity chromatography can serve as a pertinent purification procedure for MLC kinase.

Finally, we wish to propose that thyroid hormones or related analogs can be used as antiplatelet agents, as they inhibit MLC kinase. Further studies are underway to clarify the structural and stereochemical natures of thyroxine, as related to the inhibitory effects on MLC kinase and platelet function.

Acknowledgment-We thank M. Ohara of Kyushu University for comments on the manuscript.

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