THROMBOSIS RESEARCH 66; 537-547,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

CELL

RESPONSE

KINETICS: THE PHENOMENON AGGREGATION OF HUMAN

Peter V. Vrzheshch, A.N. Belozersky

Institute

OF SUPERCOOPERATIVITY PLATELETS

IN

Alexander V. Tatarintsev, Dimitry E. Yershov, Sergey D. Varfolomeyev of Physico-Chemical Biology , Moscow State University, 119899, Moscow, Russia.

(Received 8.10.1992; accepted in revised form 9.3.1992 by Editor I.P. Baskova)

ABSTRACT Concentration - response relationships of human platelet aggregation rates were analyzed for a variety of agonists and inhibitors. Their approximation by the Hill equation showed that the values of the Hill coefficient (h) were agonist-dependent and increased as follows: h AJ)P = h L-EPINEPHRINE = hpAF = hpGH2 = hU46619 < results were . The hPMA < hA23187 < hMERTHIOLATE = h ARACHIDONATE interpreted in terms of a model assuming varying degrees of cooperativity for each step of signal transduction involved in platelet aggregation. Super-high values of h (>30) obtained with arachidonate and merthiolate, as well as in the case of inhibition of an arachidonate-induced response by indomethacin and PTA 2 , suggested that at least exhibited one region of signal transduction pathway leading to aggregation supercooperative properties.

INTRODUCTION Platelet aggregation has long been recognized as a typical cell response. It is suggested that platelet aggregation may provide an adequate model for studies of cell response kinetics and stimulusresponse coupling (1). The high sensitivity of platelets to various physiological stimuli (2)) the possibility of suicidal (irreversible) aggregation and spontaneous clumping imply that the kinetics of platelet responses ought to be stringently regulated. Platelet activation may be triggered off by a variety of agonists, such as ADP (3). L-epinephrine (9), which all (EPI) (4)) thrombin (51, collagen (61, PAF (71, vasopressin (81, PGH 2 and TxA 2 operate via specific surface receptors (2). As a rule, a signal from the occupied receptor is transduced through the plasma membrane by GTP-binding proteins (lo), thereby activating effector systems, e.g. phospholipases C (11) and A (12), adenylate cyclase (13), ionic channels (14,1.5), etc. The effector syste s modulate the concentrations of intracellular messengers such as IP3 (16), DAG (16), Y+ (15), CAMP (171, AA and its metabolites (12). Platelet aggregation is associated with Ca cytoskeleton rearrangements (18), protein phosphorylation (19), clusterization of surface receptors (20) and secretion of intracellular granule constituents (2). The terminal intracellular step of signal

Key words: Kinetics,

supercooperativity,

platelet aggregation,

537

arachidonic

acid.

PLATELET AGGREGATION

538

Vol. 66, No. 5

KINETICS

transduction is the exposure of fibrinogen receptors and the binding of fibrinogen to the platelet surface (21); cell-cell collisions ultimately enable aggregate formation (221 .Hence platelet aggregation may be viewed as a result of successive activation of intracellular regulatory systems forming the stimulus-response apparatus (2,23). We show that the dose-response relationships obtained for some platalet aggregation inducers and inhibitors are characterized by ‘threshold’ effects. The results obtained are interpreted in terms of a model assuming varying degrees of kinetic cooperativity for each step of signal transduction.

MATERIALS

AND

METHODS

Preparation of PRP: Blood was drawn from cubital veins of normal volunteers into l/10 vol. of 3.8% trisodium citrate and centrifuged at 150 g CRT) for 15 min. Aggregation of platelets: Aggregation was measured by monitoring light transmission in 0.25 ml aliquots of PRP at 1000 rpm. We used an improved aggregometer model providing precise temperature control (37 + 0.0.5O Cl. The aggregation rate (F) was calculated from the slope of each aggregation curve. The inhibitors (if used) were introduced into PRP 1 min before the addition of the stimuli. , arachidonic acid and RGD Reagents: PAF, A23187, ADP, PMA, U46619, indomethacin, PGEl were purchased from Sigma (USA), sodium merthiolate (thymerosa - from Loba Chemie Fischamend (Austria), PTA2 - from Calbiochem-Behring (USA), epinephrine - from Serva (FRG). at Moscow State University (courtesy of Dr. A.A. Shchogolev Ajoene and PGH2 were synthesized and S.I. Shram). Final concentrations of solvents (ethanol, DMSO) did not exceed 0.2% (v/v) and were maintained constant throughout each experiment. Arachidonic acid was dissolved in autologous PPP.

RESULTS As it is seen from Fig.1, the relationships of aggregation may be approximated by the Hill equation curves (Fig.11: F

rate (Fl to the agonist

concentration

[A I

(lAl/QhA

-

=

F MAX

(1) 1 +

(W/KA)hA

where KA is the EC50, F - the estimated The values of hA calcu “1”ated for different

maximum value of F, h - the Hill coefficient. A reliably increase as 9 0110~s: h DP = h EPI

=

h P&F = h PG/l2 = hU4p619 < h PYA < hA23187, ’ h MERTHIOL&TE =%“Rz$CHADON$TE (Ta e 1). Simt arly the re ationships o F to the mhtbitor concentration [I I may e escn ed by equation 2: -=

(2) F

where

1

F1 1 +

t[tI/K1)ht

FI and F are the aggregation rates in the presence and in the absence of I, respectively, h I - the Hill coefficient (Fig. 2, Table 11. K or PTA 2 (but not PGE 1 ) are used for inhibiting AA-induced aggregation, the obtained dose-response relationships are characterized by super-high values of hI (Fig. 2, Table 1). Studies of AA- and merthiolate-induced aggregation produce similar results for h A (Fig. I, Table 1). Super-high va,lues of 3, are also obtained for the so-called secondary aggregatory responses (Fig. 3). (When studied turbo imetrically, EPI-induced aggregation of human platelets appeares as a biphasic response in which the first phase is conventionally termed primary and the second one secondary aggregation; Fig.3, insert). The values of h estimated for the relationships of rates of primary aggregation (V1 ) to 1EPI I are low (Fig.1, Ta %le 11. Conversely, the relationships of rates of

PLATELET AGGREGATION

Vol. 66, No. 5

KINETICS

539

1.0

Wmax

1

0 FIG 1 The relationship ~;-%l;;l;;$-~A;

2

PI /ha

of F to [A 1. 1 - ADP, K = 0.5 uM* 5 - A%,,;, pi”’ 2=- :%+6 - A,6:r’el;a;;T5 K lfi uM. Theoreiical curves are plotted by using eq.1 with hA 1.6 (a), 3.0 (b), 10 Cc) and 30 Cd).

.

1.0

0. . FI

F

I b-

(curve c), of F to the concentration of PGE (curves a,b), PTA FIG. 2. The relationship indomethacin (curve d). Aggregation was induced by 20 uM A b P (curve b), 500 u L AA (curves CUM) and hI a,c,d). Theoretical curves are plotted by using eq. 2. The respective values of K for curves (a) through (d): 0.0568 and 1.3; 0.162 and 1.3; 1.0 and lob; 1.62 and 100.

540

PLATELET AGGREGATION

TABLE NUMERICAL

No

PARAMETERS

CHARACTERIZING

Vol. 66, No. 5

KINETICS

1: THE RELs\TIONSHIP

Agonist

OF P TO [A] OR [I]

hI

hA

1.

ADP (n=6)

3.0 (2.0 - 5.0)

1.6 (1.3 - 2.0)

2.

EPI (n=3)

6.0 ( 4.0- 7.0)

1.7 (1.5

3.

PAF (n=3)

0.2 (0.1 - 0.3)

1.6 (1.4

4.

PMA (n=S)

0.45 (0.3 - 0.5)

2.9 (1.7

5.

A23187 (n= 11)

1.1 (0.4 - 4.0)

10 (5 - 20)

6.

AA (n=S)

27s (200 - 400)

> 30

7.

Merthiolate

175 (150 - 200)

> 30

- 1.8) - 1.7) - 3.3)

(n=5)

8.

PGH2 (n=3)

0.5 (0.3 - 0.8)

1.5 (1.4 - 1.7)

9.

U46619 (n = 3)

1.2 (0.7 - 1.8)

2.3 (1.S - 2.4)

10.

AA, 500 urn

indomethacin

2.1 (1.6 - 3.0)

PGEl

0.13 (0.06 - 0.17)

2.0 (1.3 - 2.0)

PGEl

0.10 (0.06 - 0.15)

2.0 (1.3 - 2.4)

PGEl

1.0 (0.5 - 1.0)

0.9 (0.7 - 1.3)

PTA2

0.9(0.5-1.0)

>30

RGD

4800(4500-5000)

2.5 (2.0 - 2.7)

ajoene

50(35-75)

2.7 (2.4 - 3.5)

>30

(n=3) 11

AA, 500 urn (n = 3)

12.

ADP, 20 urn (n=ll)

13.

Merthiolate, 500 uM (n=3)

14.

AA. 500 uM (n=3)

15.

ADP. 20 uM (n=3)

16.

ADP, 20 uM (n=7)

17.

A23181,

indomethacin

7(55-&O)

3.9 (3.2 - 4.4)

2.5 uM (n=3)

* Numerical

data are medians;

maximal and minimal valuaes are given in brackets.

secondary aggregation, V (and of the resultant light transmission increments, d Tl to [EPI ] are characterized by super-htg.?t values of hA (Fig.31. To obtain theoretical interpretation of our data, we have analysed a model of cell response generation (in preparation) in which the response of cell is considered as a manifestation of successive series of biochemical events (28-32) (Fig.41. This view is in accordance with the current concept of a ‘platelet activation cascade’ resulting in aggregate formation (3, 16, 17, 21, 22, 26, 27, 33). Under the steady state conditions, the relationship of F to [A ] or [I ] for this model may be approximated by equations 1 and 2, respectively. The parameters of these equations depend on the pathway of signal transduction involved and on the site affected by A or I. It should be noted that either of the parameters characterizing the functional activity of a cell (concentration of intracellular metabolites or activated receptors, enzyme activity, rate of secretion, ets. ) may by used as a measure of the cell response, F. If the Hill equation is used for describing the relationship of the accumulation rate of a certain second messenger (e.g., [Ca2+ ] ) to the concentration of the previously generated messenger (e.g., IP 3 1, 3 extreme modes of signal amplification are revealed (Table 2). When signal amplification is maximal , hA (or h ) reaches its highest value. If the signal is not amplified, hA approximates to - 1 (or h I = - 1). IF one of the signal transduction steps (s) exhibits a high degree of cooperativity (Ng ---> 00) the relationship of F to [A ] is threshold (h A ---> 00) A similar result (h I ---> 00 ) is obtained when I affects the stimulus - response apparatus at a site preceding the high-cooperativity step k). This mode of signal amplification is called bifurcational,

Vol. 66, No. 5

PLATELET AGGREGATION

541

KINETICS

since it formally describes the system’s behaviour in case a true bifurcation (leap) of M concentration occurs during the s step. Thus, each region of signal transduction pathway ( F’$+4 lg. ) IS characterized by a certain degree of kinetic cooperativity, amounting to the product of the respective Hill coefficients calculated for all steps constituting the region. The contribution of cooperativity of the region to hA (or h f ) depends on the properties of the signal transduction pathway (Table 2). According to our model, the diversity of regulatory systems in a cell appears as a multitude of interacting ‘stimulus-response coupling units’ 6RCU.s). Depending on the response, the kinetics of cell behaviour may be described by analysing a single SRCU (Fig. 4) or a superposition of several SRCUs.

“rnax ’ 2

6T

LJ “1

lmin

I

2

[EPll

t P”M

FIG 3 Epinephrine-induced aggregation of human PRP. The relationship of the rate of secondary A aggregation (V2, open circles) to [EPI] (solid line) is described by the equation :

-=

“2 ” “lax 2

(Wl/KA)hA 1 + ([EPI]/KA)hA

- 0.925 uM, h where K = 30. Closed c&s are the valu?s of the aggregation amplitude (dT, percent light transmission measured 5 min after the onset of the response). The right part of the equation may also be used for the description of the relationship of dT to [EPI 1. Insert: A typical tracing of EPI-induced aggregation. Both primary and secondary phases of the respectively) are clearly noticeable. As illustrated, response (characterized by V 1 and V the preincubation with indomethacin (20 uM, 3 i? o C, I min prior to addition of EPI) abrogates secondary aggregation.

542

v

PLATELET AGGREGATION

KINETICS

Vol. 66, No. 5

ACTIVATOR A RECEPTOR R

. II

Step 1 Step i

I \ \

4’ k St1 .-M /

step

s

Step n-l FIG h

4 The model of cell response generation. A - activator, R - receptor for the parameter measured to characterize the cell response; Ei (i = 0,1,2... n-l) regulating second messengers M i+l , k . - constants of the expenditure messengers (i = 1,2, . ..n). The relationship of the M 1 accumulation rate described by eq. 3: cue PI

A; I - inhibitor:

F -

- the effector systems rate of the second ( v 1 ) to [A ] is

(3)

“1 = If0 +

where R, stands for the activator complex, RA ; c by the occupied receptor [M i ] is described by eq.

Step n (cell response)

VI

total R concentratton; K, is the constant of dissociation of receptor- the proportionality factor characterizing the efficacy of E o activation (RA). The relationship of the M i+l accumulation rate (v i+l ) to 4: Ni

“,IMil v. It1 =

The relationship

of F to [M,

, Ni Ki +

I is described

i=1,2,...n-1 (4)

Ni

[Mil N,

by eq. 5: nnlMnl

F=

(5)

N”

If I interacts

with Ei at step j, the relationship

Kn + IM,l

N”

of vitl

“j+~(l’l) =

to [I ] is described

by eq.6:

*vj+l(O)

’ + 1’1 where j = 0,1,2,3, ...n-1. and v .+1 (0) is the accumulation Note that eqs. 3 and 4 are valid fo! vj+., (0).

(6) rate (v

j+l

) in the absence

of I.

PLATELET AGGREGATION

Vol. 66, No. 5

KINETICS

543

TABLE 2

A PROPOSED MODEL OF CELL RESPONSE GENERATION: MODES OF SIGNAL AMPLIFICATION

Conditions f&owing the mode of signal amplification

Mode

11

B n-l -

Maximal amplification

L

r

Kn

B n-2 --

K n-l

1

1 B.

I

I

‘I + 1 “’ Nn-l

I

Bi Ki+l

1

l/Wi

Nn

-N

.”

j+lNj+2

‘n

Nn_l)

>>

1

i = 0.1.2

-

.N1N2

%

J

Ki+l -1

Absence of amphfication

hA

Nn-l

1

n-l

I 1 I-l N. ’

Bi.1

-

Ki

Ni

K,

1.0 - 1.4

BO

1.0 - 1.6

oo

hA

--- > 00,

h I

Cell response kinetics: the phenomenon of supercooperativity in aggregation of human platelets.

Concentration-response relationships of human platelet aggregation rates were analyzed for a variety of agonists and inhibitors. Their approximation b...
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