Polyamines and platelet aggregation

Life Sciences Vol . 19, pp . 257-264, 1976 . Printed in the U.S .A .

Pergamon Pries

POLYAMINES AND PLATELET AGGREGATION Owen Rennert, Bruce Buehler, Thomas Miale, and Daniel Lawson Division of Genetics, Endocrinology and Metabolism Departments of Pediatrics, Biochemistry and Neuroscience University of Florida, Gainesville-Sunland Research Laboratory Gainesville, Florida 32610, U. S. A. (Received

in

final form June 7, 1976)

High blood concentrations of the naturally occurring polyamines have been reported in leukemia, psoriasis, cystic fibrosis and polySpermidine and spermine inhibit in vitro platecythemia rubra vera . let aggregation of platelet rich plasma preparations in which ADP and Ristocetin are the agglutinating agents . The proposal is made that these organic cations may modulate in vivo platelet agglutinability. An absolute requirement for platelet aggregation independent of the inducThe addition of competitive ining agent is adenosine diphosphate (ADP) (1) . hibitors of ADP to platelet-rich plasma (PRP), such as adenosine or 2-chloro adenosine, inhibits aggregation induced by serotonin, collagen, epinephrine or thrombin (2) . Experimental data (3) supports the postulation of specific membrane receptors for 5-hydroxytryptamine, ADP and epinephrine. The aggregation process involves the binding of ADP to membrane receptors, the formation of calcium bridges between ADP and platelet surfaces with a resultant decrease in platelet surface charge . However, the molecular mechanisms whereby ADP induces This investigation identifies aggregation require experimental elucidation (4) . the effect of the naturally-occurring polyamines, spermidine and spermine, on At in vitro platelet aggregation induced by ADP, epinephrine and ristocetin . physiologic concentrations spermidiue and spermine inhibit components of the aggregation process. Methode Platelet rich plasma (PRP) was prepared from blood anticoagulated with 3 .8% sodium citrate (1 .0 ml to 9 ml blood) . The specimens were centrifuged at 150 x The supernatant was examined to exclude the pre g for 5 minutes at 20-25° C. sence of erythrocytes, subsequently decanted into a polyethylene test tube and Platelet aggregation studies incubated for 30-60 minutes at room temperature . were performed within 2 hours of collection of the specimen . Aggregation was monitored in a Bio/DATA Aggregation Profiler, model PAP2A which employs an optical scanner, continuous amplifier and automatic recorder Turbidimetric changes were measured as calibration and standardization system . originally described by Born (5) . Blood was obtained from Caucasian, male, subjects ranging in age from 10-38 years . All had taken no medications for at least 36 hours and none had taken aspirin for at least 1 week . The volunteers were in good health ; 2 of the 257

Polyamines and Platelet Aggregation

258

Vol . 19, No . 2

subjeçts had psoriasis . Inducers of aggregation employed were : (a) ADP, 1 and 2 pM, (b) Ristocetin (Abbot Laboratories, Chicago, Ill .), 1 and 1 .2 mg/ml, (c) Epinephrine, 2 and 5 pM . Platelet aggregation was assessed in the presence of spermidine or spermine at a concentration of 1 PM . Spermidine " HC1 (SpD) and spermine " HC1 (SpM) were purchased from Sigma Chemical Corporation (St . Louis, Mo .), as were ornithine and putrescine which were assayed in equimolar concentrations to the polyamines . Results After a constant optical density was achieved, addition of ADP to control PRP (Fig . 1) induced a 45% decrease in turbidity and 2 UM ADP reduced it 70% (Table 1) . Prior addition of 1 yM SpD reduced the efficacy of ADP-induced ag gregation (1 pM) from 45% to 19% (Table 1, Fig. 1) . The inhibitory effect of 1 IN SpD was overcome by 2 vM ADP .

Ia

a

l ur

SPD -luM ADP

1uMSPD- 2YMAOP

2 4A ADP

tkne, minubbs

FIG . 1 ADP-induced aggregation of PRP plot of decrease in turbidity (ordinate) against time (abscissa) . The Nocardia antibiotic Ristocetin induced rapid platelet aggregation at concentrations of 1.0 and 1 .2 mg/ml (Fig . 2) . At both these concentrations turSpermidine totally blocked the response bidity decreased 75% or more (Table 1) . to Ristocetin . Epinephrine-induced aggregation in control PRP was not inhibited by polyamines and perhaps was increased (Table 1, Fig. 3) .

Vol . 19, No . 2

Polyamines and Platelet Aggregation

ô

1

I

1

I

a +I O

259

c~

N N

1

I

.1 %O

C% N

~t

1

N

O

N .C

l+1

V1

~O

d u1

e~1 *

Oo V

~O

cm 1~

N

e+1

N

.-1

It LM

00 rl

~O Co

O> Go

m N . P W O

al

m

e

19

d

b

41

W

ID ü n

4

IÂ

If

N

1

O R! Q ly M

rl

rl

Ô Y

N

91

en

I

I

n n

9

w 0 w u:3

00 d

N

u1

19

N

?

-Y -H ~p

OH -H ^

Ch %O

O% v1

CO +1

V1 -H

ID

0~0

n

O

Ir

-*

. O t

~O

'Y

V1

N

O;

IO

1~

N

rl v

CV v

I-1 v

N v

rl

o~ r4 r -1

A~ a 1-1 O V3

4j

+

M

m 14

it

V4 Ca W 14 001,

V

Polyamines and Platelet Aggregation

260

Vol . 19, No . 2

F

ö

tkne, mimtes

FIG. 2 ADP-induced aggregation of PRP from a patient with psoriasis .

time, minutes FIG. 3 Ristocetin-induced platelet aggregation.

Vol . 19, No . 2

Polyamines and Platelet Aggregation

26 1

Addition of spermidine or spermine to PRP in the absence of an aggregation inducer at a 100-fold excess of that used with aggregation-inducers (Table 1) had no effect ; addition of ornithine or arginiue to PRP in combination with ADP, epinephrine or Ristocetin had no effect . A previous report by our group indicated that blood levels of spermidine and spermine were 1.5-2 times elevated in psoriasis . The data depicted in Fig. 4 and Table 1 suggest that a significant reduction of ADP-induced aggregation, 17% of control aggregation, occurs with psoriatic PRP in the absence of added polyamines . The addition of exogenous spermidine results in an additive effect, further decreasing platelet aggregation. Though poly-fines did not influence epinephrine-induced aggregation, in control PRP diminished aggregation was observed in the two patients with psoriasis, and Ristocetin-induced aggregation was indistinguishable from controls .

1 um SPD-1um ADPP 1uM ADPp

s

4

tkne,

minubsS FIG . 4

Epinephrine-induced aggregation. Epin - PRP from control Epinp - PRP from a patient with psoriasis. Discussion Elevations in the circulating blood levels of polyamines have been reported in leukemia (6,7), psoriasis (8), polycythemia rubra vera (9), other neoplasms In leukemia, increased bleeding times (7) and cystic fibrosis (CF) (10,11) . are clinically observed which are disproportionate to the degree of thrombocytopenia present . In the dermal hyperproliferative condition psoriasis, the diagnostically useful sign, the Auspitz phenomenon, is said to result from abnormality of vascularity in the psoriatic lesion . However, the capillary bleeding that occurs following removal of a scaly lesion (Auspitz) may also reflect an

262

Polyamines and Platelet Aggregation

Vol. 19, No . 2

abnormality of platelet aggregability. Recent reports (12,13) postulated that the prostaglandin effect on ADP-induced aggregation was abnormal in CF patients . This observation and the reports of aberrant polyamine blood levels in CF patients (10,11) are consistent with a potential physiologic role of spermine and spermidine in platelet aggregation. The concentration of polyamines used in these aggregation studies is within physiologic mammalian tissue levels ; in human blood the concentration of spermidine and spermine is 1-4 vM . The blood concentration of these organic cations in CF, psoriasis and leukemia (6,8,10) are 1-5 times higher than used in these platelet studies . Induction of platelet aggregation by ADP involves changes in platelet morphology, an energy-dependent contractile phenomenon, as well as changes in the adhesive properties of the platelet and the subsequent secretory activity characterized by ADP, ATP, serotonin catecholamine, K+, Ca++ and platelet-factor release . Epinephrine-induction of aggregation occurs in the absence of shape and volume changes in the platelet (13) . The mechanism of Ristocetininduced aggregation is still unclear, however, an abnormality in this type of aggregation has been reported only in Von Willebrand's disease and relates to an unknown defect in the adhesive properties of the platelet (14) . The in vitro effects of polyamines on aggregation may allow a further identification of these phenomena and may be functionally significant in some of the disease states associated with abnormal polyamine levels . Calciumand magnesium are divalent cations required for the in vitro induction of platelet aggregation. Though the molecular mechanism of ADP's action in aggregation is not well understood, it is clear that it interacts with specific Recent receptors on the platelet membrane and thereby induces agglutination. data give evidence to the involvement of cAMP in this process (16) and still another line of evidence (4) involves the energy producing hydrolysis of ATP to ADP in this phenomenon . Recent work in our laboratory (17) indicates that spermine, at 1 uM concentration, has the capacity to inhibit adenylate cyclase. Additionally the polyamines are organic cations and may displace calcium and magnesium from their sites without fulfilling their functional roles . Unpublished results in our laboratory indicate specific interactions of polyamines with erythrocyte membrane preparations . Each of these considerations in regard to the platelet system requires further elucidation. Acknowledgement The authors acknowledge with thanks the support of the National Institutes of Health Training Grant No . A1î05680-05, the National Cystic Fibrosis Research Foundation, and Boehringer-Mannheim . References 1. 2. 3. 4. 5. 6. 7. 8. 9.

G. V. R. BORN, Ann. NY Acad . Sci . 201 4-12 (1972) . F. MICHAL, M . MAGUIRE, and G. GOUGH, Nature 222 1073-1074 (1969) . E. LUSCHER, S. PFUELLER, and P. MASSINI, Ser. Haematol . 6 392-391 (1973) . H. KATTLOVE, Am . J. Physiol. 226 325-329 (1974) . G. V. R. BORN, and M. CROSS, J. Phvsiol. 168 178-195 (1963) . 0 . RENNERT, T. MIALE, J . SHUKLA, D . LAWSON, and J . FRIAS, Blood (in press) . D. H. RUSSELL, Polyamines and Neoplastic Growth , Raven Press, New York (1973) . M. PROCTOR, V. FLETCHER, J . SHUKLA, and 0. RENNERT, J. Invest . Dermatol . 65 409-411 (1975) . H. DESSER, P . HOCKER, M. WEISER, and J. BÖHNEL, Clin . Chim . Acta . 63 243-247 (1975) .

Vol . 19, No . 2 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .

Polyamines and Platelet Aggregation

0 . RENNERT, J . FBIAS, and D . LaPOINTE, Fundamental Problems of Cvstic Fibrosis and Related Diseases , R . TOLANO, and J . MANGOS (ads .), p . 41, Symposia Specialist, Miami (1973) . D . LUNDGREN, P . FARRELL, and P . DI SANT'AGNESE, Clin . Chim . Acts . 62 357-362 (1975) . C . SAMUELS, and R . ELLIOTT, Lancet II 607 (1975) . G . RAMS, H . EMT, and S .. DOWLING, Lancet II 918 (1975) . J . O'BRIEN, Ann . Rev . Med . 17 275-290 (1966) . H . WEISS, In : Hematoloay . W . WILLIAMS, E . BEUTLER, A . ERSLEY, and R . BUNDLES (ads .), P . 1217-1222, McGraw-Hill Co ., N .Y . (1972) . J . McDONALD, and R . STUART, J . Lab . Clin . Med . 8 4 111-121 (1974) . R . WRIGHT, B . BUEHLER, S . SCHOTT, and 0 . RENNERT, Biochem . Biouhus . Res . Commun . (in press) .

26 3