The interaction between estradiol and human platelets

Cl~nzca Chikca Acta, 49 (1973) z87-2g3 (‘1 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

287

CCA 6129

THE

INTERACTION

I:. D. PLO’IKA,

BETWEEN

T. F. NIKOLAI

Jlavshfi~,ld Clizic Foundation (U.S..4 .) (I
ESTRADIOL

AND

HUMAN

PLATELETS”

A;VD S. S. HAGUE

for Medical

h’csijavch and Education,

Inc., Marshfield,

Wk.,

54449

3. 1973)

Human platelets have been incubated in vitro with 3H-estradiol. After incubation approximately 2% of the estradiol was associated with IO* platelets. Varying time or temperature had little effect on the association. Repeated washing of the platelets removed most of the associated estradiol. A highly positive correlation was found between the amount incubated and the amount bound to the platelet in the range of IO to 518 x 10-l~ M incubated. After incubation, about 10% of the estradiol was converted to estrone. A synthetic estrogen, ethinyl estradiol, had the highest binding to platelets followed by estradiol, estrone and progesterone. Estrone sulfate, corticosterone, and cortisol demonstrated almost no binding. These data suggest a loose binding of estradiol to human platelets.

INTRODUCTIOK

An association between high levels of circulating estrogens and thrombotic disease has been implicated in several reports. In a review of coagulation studies in women using hormonal contraceptives, Dugdale and Masil suggest that estrogen increases platelet function and accelerates clotting whereas progestin increases fibrinolytic activity. Poller et al.2 found a significant increase in platelet aggregation in women taking combined estrogen-progestin oral contraceptives. However, this increase was less than that which occurs during the third trimester of pregnancy. Elkeles et aL3 described an increased response to adenosine diphosphate (ADP) in men taking estrogen. These worker9 suggested that the response differed with the structure of the estrogen involved. They found that a synthetic estrogen altered the electrophoretic response to ADP more than natural estrogens. However, Bennet et aL4 found that estriol slightly increased platelet adhesiveness in men and Caspary and Peberdys reported increased platelet adhesiveness when an estrogen-progestin contraceptive was taken. Farbiszewski and KurowskaG noted significantly accelerated platelet aggregation after 6 and 12 months on oral contraceptives but only a slight and non-significant * ;1 preliminary report on this work was presented at the 56th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, 1972 (see Fed. Proc. 31 : 245, 1972).

288

PLOTKA

d tr/.

increase in platelet adhesiveness. Mettler and Selchow’ recently confirmed the ohservation of increased platelet aggregation during oral contraceptive therapy ant1 noted no change in platelet adhesiveness. Because of the confusing data on estrogenic effects on platelets and thrombotic phenomena, we determined to test the hypothesis that estrogens have a direct cffcct on platelets and this effect is direct in the terms which we currently understantl estrogen

action.

Tritiated steroids were obtained from New England Nuclear, Boston, Massachusetts. These included estradiol-6,7-:‘H, 46 Ci/mM ; estrone-6,7-“H, 42.4 Ci/mhl ; estriol-6,7-%, 45 Ci/mM; estrone-6,7-3H sulfate, 40 Ci/mlM; ethinyl 1.2 Ci~rn~I; progesterone-~,z-~H, 30 Ci/mM; corticosterone-1,2-W,

estradinL3H(G), 50 Ci/m%; and cortisol-1 ,z-“H, 44 CijmX. Radiochemical purity was checked by thin-layer chromatography and found to be greater than 97% in all cases. linlabeled steroids were purchased from Steraloids, Inc., Pauling, N.Y. Blood was drawn from the cubital vein of healthy volunteers into IO ml vacuum tubes containing 12 mg sodium EDTA and aliquots taken for hematocrit and platelet counting. Platelet-rich plasma (PRP) was obtained bv centrifuging the blood ftlr 15 min at 250 xg and 4”. Platelets were isolated by centrifuging the PRP for 15 min at 2500 x g and 4O. The supernatant was discarded and the tubes allowed to drain on filter paper for I min. The platelet pellets were then suspended in 0.05 XI phosphate buffer, pH 7.3. The steroid, also suspended in phosphate buffer, was added to the platelet suspension and I ml portions were transferred to Falcon Plastic tubes. Xn aliquot was taken for platelet counting. Unless otherwise stated, incubation was performed at 37’ in a metabolic shaker for 30 min and was stopped by isolating the platelets by centrifugation for IO min at 2500 g and 4”. The platelet pellets were washed with I ml of cold phosphate buffer, reisolated, and counted for radioactivity in a liquid scintillation counter (Packard Instrument Co., Downers Grove, Ill.). The supernatant and wash were also countetl for radioactivity. Platelets were counted on a hemocytometer after dilution with I 7; ammonium oxalate. For identification of the labeled compounds bound, the incubated platelets were isolated as above. The platelet pellet was suspended in 1.0 ml of hypotonic buffer and sonicated. The lysate was then extracted three times with 2.0 ml of diethyl ether and the extract chrolnatographed on thin-layer plates in 8.5% methanol in methylene chloride or on Sephadex LH-zo columns in benzene-methanol (85 : IS). l%e areas corresponding to authentic estradiol and estrone were counted for radioactivity. RESULTS

AXD

DISCUSSIOS

EDTA as a chelator of both Ca and Mg ions reportedly causes platelets to swell with inflow of fluid and loss of ions. I~Jorder to assure ourselves that the &radio1 binding was not just due to an influx of material with extracellular fluid, we compared the binding of estradiol to platelets isolated using acid-citrate-dextrose (ACD) as the

ESTRADIOL

AND HUMAN

PLATELETS

289

anticoagulant with platelets isolated using EDTA as anticoagulant. As can be seen in Table I, the anticoagulant used had no effect on the amount of estradiol bound to the platelets. The yield of platelets, however, was greatly increased with use of EDTA. Since ACD apparently does not cause an influx of fluids into platelets, the estradiol binding cannot be attributed to that phenomenon. In the initial experiments we tested the effect of time of incubation on the binding of estradiol to platelets. Platelets were isolated and incubated for varying times from o to 90 min at 37’ (Table II). The amount of estradiol associated with platelets was relatively constant throughout the times tested. These data are similar to those found by Brinkmann et aL8, who found that uptake of steroids by intact erythrocytes in vitro was independent of time, temperature, and steroid concentration. The effect of incubation temperature on the binding of estradiol to platelets was also investigated, and as can be seen in Table III, the binding appears to be temperature independent. Brinkmann et al.* found that temperature variations between 4 and 45” had little effect on the binding of steroids by erythrocytes. Simonsson9, however, found that the in vitro uptake of cortisol by rabbit polymorphonuclear leukocytes was temperature sensitive. This may indicate that a different mechanism is involved in polymorphonuclear leukocytes as compared to platelets and erythrocytes. TABLE

I

EFFECT OF AxTICoAGUL.ANT AXI2 TEMPERATURE OF IsoLATIoN ox PLATELET YIELD 4N” ESTRADIOL HINI)ING

._

Na-EDTA

Temperature I’C)

Acid-Citrate-Dextrose Yield x 10~

Molecules bound

4 23

2.21 2.13

129 & 6* 131 xt 5

-Yield x 10~

Molecules bound

5.52 6.86

129 + 5 131 & 6

* lLlean 5 standard error. TABLE

II

1SCUBATIoN

Time (min) 0

15 30 4.5 60 90

TIME

VERSUS

ASSOCIATION

OF

ESTRADIOL*

IN

Number of incubations

Amount* * associated with 10~ platelets (“/b)

4 8 x 8 8 8

I.38 * 0.07 1.38 & 0.07 1.39 & 0.06 I.55 + 0.13

HUMAN

PLATELETS

1.37 xt 0.07 1.54 * 0.15

* 133 x 10-14 moles were incubated. ** Mean f standard error. Td1BLE

III

INCUBATION

TEMPERATURE

VERSUS

ASSOCIATION

OF

Temperature PC)

Number of incubations

Amount* assoczated with 10~ platelets (76)

4 23 37

11 8 I1

2.76 f 0.35 2.82 l 0.07 3.01 + 0.19

* Mean f

standard

error.

ESTRADIOL

TO

HUMAS

PLATELETS

PLOTIiA

290

c’t d.

In order to determine if the steroid could be washed off the platelets, platelet suspensions were incubated at 37O with estradioL3H. The platelets were isolated b! centrifugation and repeatedly washed with cold isotonic buffer. Kg. I represents a plot of the percentage of the original steroid bound to the platelets as a func-tion of tllf number of washings. These data show that it is possible to remove the bound steroid almost completely from the cells by repeated washings. Similar results have been drmonstrated bv Brinkmann ~:tal.*, who reported that they could wash androstenedione and testosterone from red cells. Our data may indicate that the estrogens are loosel!. bound in the membrane. However, we cannot eliminate the possibility that tlie estradiol is in the cell and is leached out by repeated washing. DeMoor and Steenol” suggest that erpthrocytes may contain an agent for the binding of corticosteroids. They called this agent intracortin. Their later studies I1, however, did not confirm their previous results. \‘ermeulen12 concluded that corticosteroids are adsorbed at the reel cell surface. DeVenuto’3 has demonstrated the binding of steroids by isolated soluble membrane proteins and, to a lesser extent, by nonhemoglobinintracellular protein of erythrocytes. In our initial experiments 1.5 $2 of tritiated cstradiol was mixed with varying amounts of unlabeled estradiol and incubated with platelets for 30 min. A positilrr correlation (r = 0.95) was found between amounts incubated and amount bound to the platelet in the range of I0 to jrS i< Io- I3 31 incubated (Fig. 2). This correlation increased to 0.99 if several levels of steroids were incubated wit11 one platelet preparation, thereby eliminating differences due to platelet counts. As much as I .h5 x IO-” b1 estradiol was incubated without saturating the platelets. Since platelets may be altered during isolation and studies with isolated platelets may not reflect what happens under physiological conditions, the binding experiments were repeated with platelets in platelet-rich plasma. PKP was prepared by centrifuging fresll whole blood for IO min at 250 xg. Tritiated estradiol was added to the plasma and incubated at 37” for 30 min. The platelets wete isolated by centrifugation at zgooxg for IO min washed once with phosphate buffer to remove any remaining plasma and counted

for radioactivity.

ESTRADIOL AND HUMAN PLATELETS

0

50

291

100 150 200 250 300 350 400 450 500 550 600 Moles Incubated(~lO-'~i

2.

The relationship

of estradiol incubated

with estradiol bound to blood platelets.

Although the percent of estradiol bound was reduced to 0.02 to o.~%/Io~ platelets, we could still demonstrate a direct correlation between amount incubated and amount bound (Table IV). The amount bound appears to increase from 0.02 O/o/108 platelets when 5.4 x 10-1~ moles is incubated to o.~~/~/Io~platelets when 17.5x 1o-l2 moles is incubated. With higher amounts incubated the percent bound stayed at the higher level. The reduced binding in PRP was expected since the platelets are competing with the serum proteins, albumin, and sex hormone binding globulin, which bind estrogens with relatively high affinities. Brinkmann et aZ.* found that when progesterone was incubated with erythrocytes in whole blood, the amount bound was reduced considerably as compared to incubation in Krebs-Ringer buffer. They8 also reported that when erythrocytes in 50/O(v/v) pl asma were incubated with increasing amounts of progesterone, an increase in the percentage steroid bound to erythrocytes was observed. This is in contrast to their incubation studies with erythrocytes in Krebs-Ringer solutions. We also noted an increase in percentage steroid bound when incubating in PRP up to o.5°/o/~os platelets. This rise in binding percentage may be explained if it is assumed that specific estradiol binding proteins in plasma are first saturated, followed by a competition for binding between albumin and platelets. This is also in agreement TABLE

of Farese

IV

1% VitYO ISCUBATION Moles incubated ( x 1c14)

5.4

with the observations

OF

FSTRADIOL

IN PLATl?LET

Moles bound/+ztel~~t (x 10@2)

0.01

705 ‘749 3672 6418

0.96

10587

13.40

1.66

3.59 6.60

RICH

PLASMA

and Plager14 who showed that

292

PLOTKA

c’t al.

the binding of cortisol by erythrocytes depends on the amount and nature of plasma proteins present in the cell suspension. Comparing various steroids (Table V), we found that ethinyl estradiol had the greatest affinity of the estrogens followed by estradiol and estrone. Progesterone had comparable binding to estradiol and estrone. Estriol was intermediate and the corticoids, corticosterone and cortisol and a conjugated estrogen, estrone sulfate, hat1 much lower affinity. These data suggest that steroid polarity may affect the ability to associate with platelets. Brinkmann c’t nl.” demonstrated differences in the binding affinity of steroids for erythrocytes but could not make a direct correlation between the polarity of the steroid and the amount bound to the erythrocyte. We also cannot directly compare binding with polarity. After incubating the platelets with estradiol, we tentatively identified the associated hormone by chromatography. The platelets were washed and sonicated followed by extraction with diethyl ether and chromatography on thin-layer plates or on LH-20 columns. In both systems, about 9c% of the radioactivity migrated in the area corresponding to authentic estradiol standards (Table \‘I). About IO?/” migrated in the area of authentic estrone, indicating that platelets may metabolize a small portion of the estradiol to estrone. This does not preclude breakdown of estradiol, but purification of the estradiol before incubation did not alter the pattern. Bischoff and Bryson15 observed conversion of estrone to etradiol by an erythrocyte hemolysate and suggested that estrone penetrated into the red blood cell. Vermeulenl2, however, concluded that corticosteroids are adsorbed at the red cell surface. From our data, we cannot determine where the conversion is taking place. Platelets have been reported to take up and metabolize

Stcvoid

Estradiol-17/j Ethinyl estradiol Estrone Progesterone &trio1 Corticosteronc Estrone sulfate Cortisol

dehydroascorbic

acid IS, demonstrating

Number of incubations

Amount* incubated

26 s 10 H

1201 1oj f 99 t

7 I3 I2 2”

191 100 ‘43 25.5

(molts

97’

% 10-14)

1 16 71

that oxida-

Amount* associated with 10~ platelets (96)

._.

1.99 _!_ 0.12 3.-t7 -L 0.0-l

2

c I + 0 rt ‘7 * -t

I.81

x

0.12

1.47

*

0-q

0.‘)7 $- 0.20 0.47 +: 0.04 o.27 J_ 0.03 o.17 I- 0.01 ~~~~~~ ~~~~

~

_

* Mean F standard error. TABLE

VI

COKVERSION~FESTRADI~~_ TO ESTRONE* .4 mount incubated moles ( x 1r14) ~~~ ~~__~ -

Percent @pea&g as estvone ___-

60 I .Zj

1.3.40

210

I2.41

.$ro

630

BY

PLATELETS

in vi&o

9.99

IO.53 9.4.5

* Tentative identification based on chromatographic and 8.5:: methanol in mcthylene chloride.

mobility

in benzene/methanol-85:

r.5 (v/v\

ESTRADIOL

AND

tive enzymes

HUMAN

293

PLATELETS

are present

in the platelet.

It remains

to be elucidated

whether

this

action is simply a transport mechanism or is related to the abnormal platelet behavior demonstrated in women taking oral contraceptives. Jenson and Jacobson17 have aptly demonstrated that estradiol action on the uterus is preceded by “binding” of the estradiol to uterine receptors. ACKNOWLEDGEMENT

This work was supported

in part by a grant

from the Wisconsin

Heart

Asso-

ciation. REFERENCES I M. DUGDALE AND A. T. MASI, in “Repovt on the Oral Contracefitives”, Advisory Committee on Obstetrics and Gynecology, Food and Drug Administration, 1969. 2 L. POLLER, C. M. PRIEST AND J. M. THOMSEN, Brit. Med. J.. 4 (1969) 273. 3 R. S. ELKELES, J. R. HAMPTON AND J. R. MITCHELL, _&met, 2 (1968) 315. 4 N. B. BENNETT, P. N. BENNETT, H. W. FULLERTON, C. M. OGSTON AND D. OGSTOX, Lancet, 2 (1966) 881. 5 E. A. C.~SPARY AND M. PEBERDY, Lancct, I (1965) 1142. 6 R. FABISZEWSKI AND T. KUROWSKA, Thromb. Diath. Haemorrh., 24 (1970) 304. 7 L. METTLER AND B. M. SELCHOW, Thromb. D&h. Haemorrh., 28 (1972) 213. 8 A. D. BRINKMANN, E. MULDER AND H. J. VAN DER MOLES, Res. Steroids, 4 (1970) 91. 9 B. SIMONSSON, Acta Endow.. 67 (1971) 634. 10 P. DE MOOR AXD 0. STEENO, Ann. Endow., 23 (1962) 99. II P. DE MOOR AND 0. STEENO, J. Endow., 26 (1963) 301. 12 A. VERMEULEN, Acta Endow., 37 (1961) 348. 13 I;. DE VENUTO, Proc. Sot. Exper. Biol. Med., 124 (1967) 478. 14 R. J. FARESE AXD J. E. PLAGER, J. Clin. Invest., 41 (1962) 53. ‘5 F. BISCHOFP AND G. J. BRYSON, J. A#. Physiol., 15 (1960) 515. 16 D. HORNIG, F. WEBER AXD 0. WISS, Clin. Chim. Acta, 33 (1971) 187. 17 E. V. JENSON AND H. I. JACOBSON, Recent Progr. Hormone Res., 18 (1962) 387.