THROMBOSIS RESEARCH 67; 505-516,1992 00493848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
KINETICS
OF MERTHIOLATE-INDUCED PLATELETS
AGGREGATION
OF HUMAN
P.V. Vrzheshch, A-V. Tatarintsev, E.V. Orlova, D.E. Yershov, S.D. Varfolomeyev Bio-Rad Laboratories T-0. BioChemMack; A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Lenin Hills, 119899 Moscow, Russia
(Received
30.4.1992;
accepted in revised form 17.7.1992 by Editor I.P. Baskova)
ABSTRACT Incubation of human platelet-rich plasma (PRP) or washed platelets with merthiolate (MT; sodium ethylmercurithiosalicylate; an inhibitor of lysophosphatide: arachidonoyl transferase) leads to irreversible platelet aggregation which is parallelled by an increase in thromboxane A 2 s y nthesis. MT-induced aggregation is preceded by a pronounced lag-period (0.5-10 min). Duration of the latter is inversely related to the concentration of MT ( [MT I). Platelet responses to MT are similar to those triggered by arachidonate (AA) in that the relationships of the aggregation rates both to [MT ] and [AA] are threshold and exhibit characteristic super-high values of the apparent Hill coefficients (h > 30). A typical MT-induced response can be subdivided in two sequential pha$es: i) and ii) indomethacin-abrogated rapid cyclooxygenase-independent slow aggregation, aggregation. MT-induced responses are blocked by PGE 1 or ajoene (which inhibits binding of fibrinogen to its cell surface receptor, GPIIb/IIIa). The obtained data are interpreted both quantitatively and qualitatively in terms of a mode1 assuming the existence of: i) a relationship between the rate of MT-inhibitable AA incorporation into phospholipids and the concentration of intracellular free AA, [AA] i ; ii) a certain threshold value of [AA] i essential for triggering the second phase of the aggregation.
INTRODUCTION Biochemical reactions linked to arachidonate (AA) liberation and conversion are of considerable importance for platelet activation. Inhibition of AA cyclooxygenation prevents the aggregatory responses induced by A23187, exogenous AA, or collagen [l 1. Secondary aggregation, release of intracellular granule constituents and phospholipase C activation induced by ADP or L-epinephrine are all blocked by cyclooxygenase inhibitors or thromboxane A2 [2]. (TxA2 ) receptor antagonists In dormant platelets the level of free AA is maintained low [3 ] due to rapid phosphplipid [S ] and acyl-CoA: lysophosphatide acyltransferase reacylation [4 ] catalysed by acyl-CoA-synthase (lysophosphatide: acyl transferase, LAT) [6]. Merthiolate (MT, sodium ethylmercurithiosalicylate) has been reported to inhibit LAT in human platelets and murine peritoneal macrophages, and as a result to block reincorporation of AA liberated through the action of phospholipase A 2 ; intraceklular metabolism of free AA is thereby enhanced [7-91. It has also been shown that in response to MT
Key words: Kinetics,
platelet aggregation,
merthiolate.
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synthesized by macrophages [ 111. PGI2 is released from cultured endothelial cells [lo 1 and PGE2 With platelet-rich plasma (PRP) as an experimental system both MT-induced aggregation and serotonin release have been described [12,13 1. In washed human platelets MT has been reported to [ 14 1. In addition, MT has induce aggregation [9,14 1, Ca 2 + mobilization [9 ] and chemiluminescence been found to potentiate production of leukotrienes by human polymorphonuclear leukocytes [15]. As it follows from the results described in [7,8 1, MT is a selective modulator of AA metabolism capable of increasing the level of intracellular free AA ( [AA ] . ) . Here we present results of our experimental inbestigation and theoretical description of the kinetics of MT-induced platelet aggregation.
MATERIALS
AND METHODS
, and bovine serum albumin (BSA) Reagents and buffers. AA, merthiolate, indomethacin, PGE were purchased from Sigma (St. Louis, USA), PGE 2 , PG Ff 2a , glucose, sodium dodecylsulphate (SDS) and HEPES from Serva (Heidelberg, Germany), fibrinogen from Boehringer (Mannheim, Germany). [3 HI-AA (100 Ci/mM) was obtained from the Institute of Medical Genetics, Russian Academy of Sciences. Ajoene was produced by J.V. BioChemMack (MOSCOW,Russia). , 2.8 mM KCl, 0.35 mM Na HP0 Buffer A: 135 mM NaCl, 12 mM NaHCO , 5 mM HEPES, 5.5 mM glucose, 12 mM citric acid (pH 6. 3 1. Buffer B: 135 mM NaCl, 11 mM r4aHC03 , 2.8 mM KCl, 0.35 mM Na HP0 , 5 mM HEPES, 5.5 mM glucose, 1 mg/ml BSA (pH 7.35). Preparation of $ RP. 4 lood was drawn from antecubital veins of healthy male volunteers into 3.8% trisodium citrate (9:1, v/v), or acid-citrate-dextrose (pH 4.5; 6:1, v/v) if further processing was required, and centrifuged at 110 g (RT) for 15 min. Preparation of l3 H I-AA-labelled washed platelets. PRP was layered on Ficoll-Paque (PharmaciaLKB, Uppsala, Sweden) (l:l, v/v) and centrifuged at 250 g CRT) for 30 min. Cells from the interphase were collected, diluted twofold by buffer A and centrifuged at 860 g CRT) for 15 min. The for 30 min at pellet was resuspended in buffer A and incubated with [ 3 HI-AA (7.5 @X/ml) 37 0 C. To remove the excess of the label, cells were washed twice in the same buffer (630 g, 10 min, RT) and finally resuspended in buffer B at a concentration of 2 * 108 per ml. Aggregation assav. Platelet aggregation was measured turbidimetrically [ 16 ] using a double-beam aggregometer (Thromlite-1006, J.V. BioChemMack (Moscow, Russia)). Aliquots of PRP (250 1.11in glass cuvettes) were stirred (1000 rpm) in the cell of the device with inhibitors or their vehicle controls at 37 O C for 1 min followed by addition of the stimuli. Indomethacin and PGE 1 were dissolved in ethanol, ajoene in dimethylsulphoxide (DMSO), MT in buffer B, AA in autologous PPP. The final concentrations of the organic solvents never exceeded 0.5% v/v. In studying the responses of washed platelets, aliquots of cells (250 ~1) were pipetted into siliconized (Sigmacote R ) glass cuvettes, and stirred (1000 rpm, 37 0 Cl with fibrinogen (0.5 mg/ml), CaC12 (2 mM) and MgC12 (2 mM) for 1 min prior to addition of merthiolate. Analvsis of I 3 H l-AA metabolites. Washed platelets (250 ~1) were incubated in the aggregometer cuvette. Each sample was processed as follows. The reaction was terminated by addition of 0.1 ml 0.4% formic acid (the resultant pH being of about 3.0), the reaction mixture centrifuged at 1000 g for 5 min CRT) and the supernatant extracted thrice by ethylacetate (3x1.5 ml). The organic phase was collected, desiccated with sodium sulphate, evaporated under nitrogen, dissolved in 0.5 ml of ethylacetate and evaporated once again. The residue was redissolved in 50 1.11ethylacetate and the standards of prostaglandins were added (PGF 2a and PGE 2 , 2 pg each in 15 ~1 ethanol). The products were separated by TLC at RT on silica plates (Merck, Darmstadt, Germany) 1.5 cm width in the system of ethylacetate, 2,2,4_trimethylpentane, acetic acid and water 100:500:10:110 (v/v/v v, upper phase). The standards were visualized by exposing the plates to iodine vapour. To determine the distribution of radioactivity relative to R f , each plate was cut into 5 mm sections. The sections were transferred into scintillation vials containing 1 ml of 0.1 y0 sodium dodecylsulphate each and allowed to stand for 1 hr at RT prior to addition of the dioxane scintillant (10 ml per vial). Counts were performed on a liquid scintillation counter (Marck 111).
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RESULTS
Platelet aggregation studies. Incubation of human platelets (both PRP and washed platelet suspension) with MT results in their irreversible aggregation. Tracings of MT-induced aggregation obtained using the turbidimetric technique are biphasic. The following parameters may be used for (the retention time, i.e. time interval their quantitative characterization (Fig. IA, insert): i) T preceding the onset of the response); ii) V (initial rat: of aggregation); iii) V (maximal rate of aggregation numerically equal to the slope oPthe curve at the point of its inflection); iv) 7 (the lagperiod, i.e. time interval numerically equal to the intercept cut on the time axis by the tangent drawn through this point); v) 6T (the extent of aggregation, i.e. maximal light transmission increment). Platelet responses to exogenous AA are similar to those triggered by MT in that in either case irreversible aggregation is preceded by a lag-period, and the relationships of V to [MT] or [AA] are threshold. In contrast with MT-elicited responses, tracings of AA-induced light transmission increments are monophasic and their quantitation according to the procedure described above (Fig. IA, insert) gives only 3 measurable parameters, viz 7, V, and 6T (note that in this case the following equalities are formally correct for 7 andV :T =T, v =V). Figure 1 shows the relationshrps of V”andor to [MT] (panel A) and [AA J (panel B). The relationships of V to the concentration of either of the agonists ([A]) may be described by the Hill equation: hA V (1) ([Al/KA )
-
=
VCO
hA 1 + ([A]/KA ) where KP equals to EC50, h A is the Hill coefficient and V, is the value of V at [A J ->a). As 11ustrated, the characteristic threshold-like appearance of these relationships is markedly pronounced. The Hill equation gives their satisfactory approximation (Eq. 1) at h>30. The values of K A for MT-induced aggregation of PRP are in the range of lOO-200pM; in the system of washed platelets K amounts to 7.5 PM. The relationships of 6T to [MT ] or [AA ] are also highly cooperative (data not s Rown). As shown in Fig. 1, 7 is inversely related to [MT I (panel A). With AA as an inducer of aggeragation the relationship is similar, but much less pronounced, the values of 7 being reliably lower (Fig. 1, panel B). MT-induced
TxA2
svnthesis. Kinetic studies of MT-induced aggregation and accumulation of clearly demonstrate that the aggregatory response (Fig.2, curve 3) is parallelled by concurrent TxA synthesis (assessed by [ 3 H ]-TxB 2 release into the medium; Fig. 2, curve 4 xA2 is not formed throughout the lag-period. 1). As illustrated, It is also noteworthy that MTinduced TxA 2 s y nthesis may proceed in the absence of stirring (Fig.2, curve 2), when aggregate formation cannot be detected turbidimetrically (Fig.2, curve 4).
[ 3 H J-AA-metabolites
Inhibition of MT-induced aggregation. Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,1 I-triene-9-oxide, has previously been reported to inactivate the platelet fibrinogen receptor and thereby interfere with formation of intercellular receptor-fibrinogen-receptor bonds [ 17 1. Hence, ajoene is thought to be a versatile inhibitor of platelet aggregation [ 18 1. Preincubation of platelets with ajoene completely inhibits both primary and secondary phases of MT-induced aggregation (data not shown). Indomethacin, the well-known cyclooxygenase inhibitor, does not affect the first phase of the aggregatory response (characterized by 7 ), but blocks its second phase (characterized by and V 7 and V). Data from these experiments aYe summar?zed in Fig.3, which shows the relationships of 7, to the concentration of the inhibitor. Increasing indomethacin input progressively ‘S tbtts and O tn the vsecond phase of the aggregatory response. Quantitative manifestation of this tendency appears as a decrease in 7 and V which finally reach their minimal values equal to 7 and V respectively (Fig. 3). Hence, the first slow wave of the biphasic MT-induced response”is independent of the cyclooxygenase pathway of AA metabolism, while the second rapid wave of aggregation strongly depends on the ability of platelets for AA cyclooxygenation. It should be noted that inhibition by indomethacin of both MT- and M-induced aggregatory responses exhibits the same degree of cooperativity, since in either case the relationships of U to to the concentration of indomethacin are characterized by equally high values of the apparent Hill coefficients [ 19 1.
’
MERTHIOLATE-INDUCED
508
A
ho
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AGGREGATION
Z,min
1000
2000 [ Merthiolate] , ,+IM
14
+ 095
i 0
LArachidonic
acidJ , pM
Figure 1. The relationships of the maximal rate of aggregation W; closed circles, curves 1 and 3) and of the lag-period preceding the onset of the response (T; open circles, curves 2 and 4) to the concentrations of merthiolate (MT) (panel A) and arachidonic acid (AA) (panel B). All numerical data are derived from the aggregation tracings obtained in a single experimental setting with plateletrich plasma (PRP) of the same donor. Replicate experiments with samples of PRP from 6 donors gave similar results. Insert: a typical tracing obtained by the turbidimetric technique and the method proposed for its quantitation (scheme; for details see the text). Addition of the stimulus is marked with an arrow.
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MERTHIOLATE-INDUCED
I
0
/
I
2
I
AGGREGATION
I
I
509
1
4 Time , min
6
Figure 2. Synthesis of thromboxane associated with MT-induced aggregation. Addition of MT (50 PM, arrow) to washed platelets (2 10 * per ml in buffer B supplemented with 0.5 mg per ml fibrinogen, 2 mM CaCl and 2 mM MgC12 ) stirred at 1000 rpm induces their aggregation (curve 3) and accumulation of [ 3iI ]-TxB 2 in the medium (curve 1, open circles). In the absence of stirring aggregates are not formed (curve 4), while the increase in the level of [ 3 H ]-TxB2 is still detectedkurve 2, closed circles).
v and V, , %min-’ 100
t and Z,,min 10 /
[Indomethacin],yM of MT-induced aggregation by indomethacin. Aliquots of PRP were Fieure 3. Inhibition preincubated with varying concentrations of indomethacin (37 o C, 1000 rpml for 1 min, followed by addition of MT (400 PM) and the resultant light transmission increments were monitored continuously. The following parameters were derived from each of the obtained tracings: maximal W, . open ctrcles) and mtttal ‘Vo , closed circles) rates of aggregation, lag-period (7, open circled), retention time (7 o , closed circled) (curves 1 through 4,respectiveiy).
MERTHIOLATE-INDUCED
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AGGREGATION
Z, min
to /min
60
[PGE,], nM Fipure 4, Inhibition of MT-induced aggregation by PGEl . The experimental protocol is described under Fig. 3, exept that [MT ] = 470 PM; the relationships of V (closed circles) and T (open circles) [PGE 1 I are presented.
Preincubation of platelets with PGEl leads to a prolongation of the lag-period and a decrease in the maximal rate of aggregation (increase in 7, reduction of V; Fig. 4); the parameters of the first phase are affected similarly (data not shown). As illustrated, the relationship of V to [PG ] is not threshold, the the observed values of Hill coefficient bein in the range of 0.7-1.3. I!!h ibition by PGE 1 of AA-induced aggregation is characterized by equally low values of the Hill coefficient [19]. Kinetic model of MT-induced azzrezation. The obtained data are described both quantitatively and qualitatively in terms of a model assuming that: i) [AAJ is increased as a consequence of MTinduced inhibition of lysophospholipid acylation; ii) a certain threshold level of [AA 1. is required for initiation of irreversible aggregation. This model corresponds to the scheme depicted in Fig. 5 (insert). The kinetics of variation of [AA]i is described by the following differential equation:
d[fil
i = v,
- v_
(2)
dt Here V + is the overall rate of reactions leading to AA release (a parameter considered herein to be constant) and V_ is the rate of AA reincorporation, which is in turn related to the bulk [MT ] as given by equation (3): ko [uli (3) v_ = 1 + WI/K1 where k o is the rate constant of the pseudo-first order reaction, and KI equals to ICSo for the relationship of the rate of AA reincorporation to the bulk [MT] introduced into the system. The first order with respect to [AA]. (Eq. 3) reflects the well-known fact that the value of in platelets is extremely low $1. Assuming MT to be added at zero time (t = 0) and its I”li concentration to vary from zero to the final value ([MT]) we obtain the solution of Eq. 2 in the following form:
to
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MERTHIOLATE-INDUCED
AGGREGATION
ko v, (1 + [MT]/Kf
[AAli
)
V+
) e ko
ko
t
1 + [MTl/KI
[MT]
-(
=
511
(4)
KI
Expression (4) describes the kinetics of MT-induced variation of [AA] . ~ Time interval required is determined from to attain the critical (i.e. inducing irreversible aggregation) value of ]A], : expression (5); it is assumed that this critical value is attained at the bulk [MT 1 = KA
P=Ym
(p+
KI KA
[MT 1
WT 1 ) ln(
KA
) [MT]-
(5)
KA
(the value reached by T at [MT] ->oO the rate of MT-induced platelet aggregation to the into the system. When the relationship of T to [MT] is represented graphically by plotting T /Too it becomes evident that the obtained function depends exclusively on the against [MT I/K calculated from exprestiion (5) fo ratio. T RL e relationships of 7 /7 oo to WI/K* K /KI di @erent values of this ratio are compared to those obtained experimentally in Fig. 5. It is apparent = 0.1. that expression (5) gives a satisfactory description of our experimental data at Q /KI
10
calculated from Equation (5) for Figure 5. The relationships of T/T o. to WTl/K A equal to 0.001, 0.03, 0.1, 1, 10, 100 and 1000 (curves 1 through 6, respectively K /ICI TRLe experimental = 0.32 min values of 7 (closed circles) are the same as in Fig. 1 (panel A); 7 Qo and K A = 75pM. Insert: Schematic illustration of the kinetic model of MT-induced aggregation.
MERTHIOLATE-INDUCED
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AGGREGATION
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MERTHIOLATE ,a” LYSO-PL 4 (LYSO-PAF)
\-
PAF +‘*
* * -+ 6
FIBRINOGEN RECEPTOR ACTIVATION 4
SLOW __,
AGGRE6ATlON (PHASE I)
AACoA PL
AdENE I T AA
Q ,+
PGU,
4
TxA,+
ST- -+FIBRIN&EN RECEPTOR ACTIVATION
b
RAPID AGGREGATION (PHASE 1 )
A6 intracellular
Figure
6.
of second
The proposed mechanism of the aggregatory effects of AA and MT. Generation messengers and active intermediates formed through hydrolysis of PL is depicted schematically. Sites of inhibition are marked with - *
DISCUSSION The proposed mechanism of MT-induced aggregation is depicted in Fig. 6. Stimulation of platelets with various agonists results in the release of AA from membrane phospholipids 120 1. Two pathways of AA liberation have been described: 1) hydrolysis by phospholipase A2 [3,21-23 I and phospholipase C / 1,2-diacylglycerol-lipase-dependent cleavage [21,23-25 I. Cyclooxygenase converts AA to PGH2 the latter being further transformed into TxA2 by thromboxane synthase [26,27]. These highly labile AA metabolites are potent platelet agonists; their binding to specific receptors induces G-proteindependent phospholipase C activation with the resultant initiation of the phosphoinositide turnover, [Ca 2 + Ii mobilization and other events terminating in aggregate formation. active Other products formed through phospholipase A 2 activation are the physiologically lysophospholipids [30-331 and the platelet-activating factor (PAF) [34]. Arachidonate-specific acyl-CoA synthase [35] and polyunsaturated fatty acid-specific acyl-CoAlysophosphatide acyltransferase 1361 which have been identified in platelets selectively decrease the levels of free AA and, consequently, PGH and TxA M?-induced i&bition of AA reincorporation into phospholipids should elevate the level of [AA] i and increase the proportion of lysophospholipids, thereby leading to enhanced synthesis of cyclooxygenase metabolites, platelet activation and aggregation. Indeed, MT has been reported to induce aggregation and serotonin release in PRP [12,13 1, synthesis of cyclooxygenase products 17-9 ] aggregation [9,14], [Ca 2 + ] i mobilization 19 ] and chemilumenescence [ 14 I in washed platelets Incubation of platelets with MT leads to irreversible aggregation which is preceded by a prolonged lag-period and parallelled by TxA2 synthesis (Fig. 2). Reincorporation of AA into membrane lysophospholipids of murine peritoneal macrophages is completely inhibited by 50 /JM MT 181. Moreover, at 40 PM MT significantly potentiates synthesis of leukotrienes induced by FMLP or C3a in human polymorphonuclear leukocytes [15 1. It is noteworthy that in our experiments the same concentrations of MT have been demonstrated to induce TxR2 accumulation (Fig. 2).
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AGGREGATION
Aggregation of washed human platelets induced by methylmercury chloride (a compound related to MT chemically) is Inhibited by indomethacin [37 1. Methylmercury chloride has recently been shown to inhibit specifically the activity of lysophosphatide: acyltransferase in platelet microsomes ]38 1. In terms of our kinetic model, the characteristic lag inherent to MT-induced responses (Fig. 1) ts vtewed as a result of accumulation of free AA. It is suggested that the onset of irreversible aggregation corresponds to the attainment of a certain critical value of ]AAli which is required for triggering off this phase of the response. The values of 7 calculated from Eq. 5 correspond to those obtained experimentally (Fig. 5). The second phase of MT-induced response (Fig. lA, insert) is similar to AA-elicited aggregation in that it is irreversible, susceptible to inhibition by indomethacin (Fig. 3) or ajoene [19], and characterized by highly cooperative relationships of V to [MT ] (Fig. 1; Eq. 1). Increasing AA input results in inhibition of aggregation [39-441; analogous results are obtained with MT as a stimulus (data not shown). The first phase of MT-induced response (Fig. lA, insert) is ‘resistant’ to indomethactn (Fig 31. To date mechanisms underlying this phenomenon remain obscure. In the majority of cells including platelets biosynthesis of PAF is linked to AA liberation via a common Intermediate, I-o-alkyl-2arachidonoyl-at-glycero-3-phosphorylcholine (AA-GPC) [45]. Hydrolysis of AA-GPC by phosphohpase A2 initiates both PAF and eicosanoid biosynthesis [46 I. Cyclooxygenase-independent slow aggregation may be underlain by PAF production and/or lysophospholipid accumulation (Fig. 6). For iInstance, PAF-induced aggregation is known to be independent of TxA synthesis I47 1, while lysophosphatidylcholine has recently been demonstrated to act as a second2 messenger modulating the activity of protein kinase C ]48 1. It should be noted that other explanations cannot be excluded. Preincubation of platelets with PGE 1 has previously been shown to result in a prolongation of the lag-period preceding the onset of aggregatory, secretory and luminescence responses to MT ]I4 J. Our observation that pretreatment with PGEl postpones the onset of MT-induced aggregation and inhibits its maximal rate (Fig. 4) points to the existence of at least 2 intracellular targets for PGl$ , one possibly controlling AA accumulation, and the other associated with some terminal steps of platelet activation. Derangement of the former mechanism leads to a prolongation of the lag, interference with the latter affects the rate of aggregate formation (Fig. 6) PGEl is known to elevate intracellular CAMP [49], which in turn induces Ca sequestration ]49,50]; depletion of [Ca2+ ] i may lead to an inhibition of the activity of the Ca-dependent phospholipase A 2 and thereby decrease the rate of intracellular AA accumulation. Indeed, CAMP has been reported to affect the release of AA mediated by phospholipase A 2 [51 1. In addition, CAMP has been found to decrease the proportion of proteins available for protein kinase C-dependent phosphorylation [20]. The observed decrease in V (Fig 4) might partially be accounted for by either of these effects. The inferred ability of PGEl for affecting a certain terminal step of signal transduction leading to aggregation is indirectly corroborated by our recent observation that the values of the apparent Hill coefficients characterizing the relationships of the aggregation rates to [PGE, 1 are independent of the agonists used for triggering off the response (i.e. of the signal transduction pathways involved) and reliably approximate unity [ 19 1. ACKNOWLEDGEMENTS We are grateful to A. Turgiev for fruitful discussions
and help in preparation
of the manuscript.
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metabolites
LEFKOWIT,
smooth
in
J.B.
- muscle
29. BRASS, L.F., SHALLER, C.C. and BELMONTE, E.J. Inositol 1,4,5,-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipase C mediated by thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanism distinct from that of thrombin J. Clin. Invest. 79, 1269-1275, 1987. 30. BROEKMAN, M.J., WARD, J.W. and MARCUS, A.J. Fatty acid composition of phosphatidylinositol and phosphatidic acid in stimulated platelets. Persistence of arachidonyl-stearyl structure. .I. Biol. Chem. 256, 8271-8274, 1981. 31. MCKEAN, M.L., SMITH, J.B. and SILVER, M.J. J. Formation of lysophosphatidylcholine by human platelets in response to thrombin: support for the phospholipase A 2 pathway for the liberation of arachidonic acid. J Biol. C/ieni. 256, 1522-1524, 1981. 32. WALENGA, R.W., OPAS, E.E. and FEINSTEIN, M.B. Differential effects of calmodulin platelets. J. Biol. Chern. 256, 12523antagonists on phospholipase A 2 and C in thrombin-stimulated 12528, 1981. 33. BILLAH, M.M. and LAPETINA, E.G. Formation of lysophosphatidylinositol stimulated with thrombin or ionophore A23187. J. Biol. C/zau. 257, 5196-5200, 1982. 34. ROTH,
in
platelets
G.J. Platelet arachidonate metabolism and platelet-activating factor. In: Biochenristv D.R. and Shyman, M.A. (Eds.) Orlando: Academic Press, Inc., 1986, pp.69-113.
of
Platelets Phillips,
3.5. WILSON, D.B., PRESCOTT, S.M. and MAJERUS, P.W. Discovery of an Coenzyme-A synthetase in human platelets. J. Biol. C/rem. 257, 3510-3515, 1982. 36. MCKEAN,
M.L., SMITH,
J.B. and SlLVER,
M.J. Phospholipid
biosynthesis
arachidonoyl
in human
platelets.
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Formation of phosphatidylcholine 3-phosphocholine acyltransferase. 37. MACFARLANE, release via activation
AGGREGATION
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from I-acyl lysophosphatidylcholine by acyl-CoA-l-acyl-sn-glyceroJ. Biol. Chem. 256, 1278-1283, 1981.
D.E. The effects of methyl mercury on platelets. Induction of aggregation and of the prostaglandin synthesis pathway. Mol. Phamiacol. 19, 470-476, 1981.
38. HORNBERGER, W. and PATSCHEKE, H. Primary stlmuli of icosanoid release inhibit arachidonoyl-Cob synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets. Eur. J. Biochent. 187, 175181, 1990. 39. LINDER, B.L., CHERNOFF, A., KAPLAN, K.L. and GOODMAN, D.S. Release of plateletderived growth-factor from human platelets by arachidonic acid. Proc. Natl. Acad. Sci. USA 76, 41074111,1979. 40. AHARONY, D., SMITH, J.B. and SILVER, M.J. Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid. Biochint. Biophys. Acta 718, 193-200, 1982. 41. LINDER, B.L. and GOODMAN, D.S. Studies on the mechanism of the inhibition of platelet aggregation and release induced by high levels of arachidonate. Blood 60, 436-445, 1982. 42. NISHIKAWA, M., HIDAKA, H. and SHERAKAWA, S. Possible involvement of direct stimulation of protein kinase-C by unsaturated fatty acids in platelet activation. Biochern. Pltarmacol. 37, 3079-3089,1988. of exogenous arachidonate 43. KOWALSKA, M.A., RAO, A.K. and DISA, J. High concentrations inhibit calcium mobilization in platelets by stimulation of adenylate cyclase. Biochern. J. 253, 255-262, 1988. 44. SATO, T., HASHIZUME, T., NAKAO, K., AKIBA, S. and FUJII, T. Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide thromboxane A2 binding to the membrane receptor. Biochim. Biochys. Acta 992, 168-173, 1989. 45. HANAHAN, D.J. Platelet-activating Biochent. 55, 483-509, 1986.
factor - a biologically
active phosphoglyceride.
Annu. Rev.
46. KRAMER, R.M., JAKUBOWSKI, J.A. and DEYKIN, D. Hydrolysis of 1-alkyl-2-arachidonoyl-srtglycero-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A 2. Biocluin. Biophys. Acta 959, 269-279, 1988. 47. MCCULLOCK, R.K. and VANDOGEN, aggregation and secretion in human platelets.
R. Mechanisms of platelet-activating ProstagZandim39, 13-21, 1990.
48. OISHI, K., RAYNER, R.L., CHARP, P.A. and KUO, J.F. Regulation lysophospholipids. Potential role in signal transduction. J. Biol. C/zem. 263, 6865-6871,
factor-induced
of PKC 1988.
by
49. FEINSTEIN, M.B., EGAN, J-J., SHA’AFI, R.I. and WHITE, J. The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PGD2 , PGE 1 , forskolin). Biochem. Biophys. Res. Cornnum. 113, 598-604, 1983. 50. ZAVOICO, G.B. and FEINSTEIN, AMP antagonism between stimulators Convnun. 120, 579-585, 1984.
M.B. Cytoplasmic Ca2 + in platelets is controlled by cyclicand inhibitors of adenylate cyclase. Biocltern. Biopllys. Res.
51. PANNOCCHIA, A. and HARDISTRY, R.M. Cyclic AMP inhibits platelet activation independently of its effect on cytosolic free calcium. Biochent. Biophys. Res. Commur~. 127, 339-345, 1985.
KINETICS
OF MERTHIOLATE-INDUCED PLATELETS
AGGREGATION
OF HUMAN
P.V. Vrzheshch, A-V. Tatarintsev, E.V. Orlova, D.E. Yershov, S.D. Varfolomeyev Bio-Rad Laboratories T-0. BioChemMack; A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Lenin Hills, 119899 Moscow, Russia
(Received
30.4.1992;
accepted in revised form 17.7.1992 by Editor I.P. Baskova)
ABSTRACT Incubation of human platelet-rich plasma (PRP) or washed platelets with merthiolate (MT; sodium ethylmercurithiosalicylate; an inhibitor of lysophosphatide: arachidonoyl transferase) leads to irreversible platelet aggregation which is parallelled by an increase in thromboxane A 2 s y nthesis. MT-induced aggregation is preceded by a pronounced lag-period (0.5-10 min). Duration of the latter is inversely related to the concentration of MT ( [MT I). Platelet responses to MT are similar to those triggered by arachidonate (AA) in that the relationships of the aggregation rates both to [MT ] and [AA] are threshold and exhibit characteristic super-high values of the apparent Hill coefficients (h > 30). A typical MT-induced response can be subdivided in two sequential pha$es: i) and ii) indomethacin-abrogated rapid cyclooxygenase-independent slow aggregation, aggregation. MT-induced responses are blocked by PGE 1 or ajoene (which inhibits binding of fibrinogen to its cell surface receptor, GPIIb/IIIa). The obtained data are interpreted both quantitatively and qualitatively in terms of a mode1 assuming the existence of: i) a relationship between the rate of MT-inhibitable AA incorporation into phospholipids and the concentration of intracellular free AA, [AA] i ; ii) a certain threshold value of [AA] i essential for triggering the second phase of the aggregation.
INTRODUCTION Biochemical reactions linked to arachidonate (AA) liberation and conversion are of considerable importance for platelet activation. Inhibition of AA cyclooxygenation prevents the aggregatory responses induced by A23187, exogenous AA, or collagen [l 1. Secondary aggregation, release of intracellular granule constituents and phospholipase C activation induced by ADP or L-epinephrine are all blocked by cyclooxygenase inhibitors or thromboxane A2 [2]. (TxA2 ) receptor antagonists In dormant platelets the level of free AA is maintained low [3 ] due to rapid phosphplipid [S ] and acyl-CoA: lysophosphatide acyltransferase reacylation [4 ] catalysed by acyl-CoA-synthase (lysophosphatide: acyl transferase, LAT) [6]. Merthiolate (MT, sodium ethylmercurithiosalicylate) has been reported to inhibit LAT in human platelets and murine peritoneal macrophages, and as a result to block reincorporation of AA liberated through the action of phospholipase A 2 ; intraceklular metabolism of free AA is thereby enhanced [7-91. It has also been shown that in response to MT
Key words: Kinetics,
platelet aggregation,
merthiolate.
505
506
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synthesized by macrophages [ 111. PGI2 is released from cultured endothelial cells [lo 1 and PGE2 With platelet-rich plasma (PRP) as an experimental system both MT-induced aggregation and serotonin release have been described [12,13 1. In washed human platelets MT has been reported to [ 14 1. In addition, MT has induce aggregation [9,14 1, Ca 2 + mobilization [9 ] and chemiluminescence been found to potentiate production of leukotrienes by human polymorphonuclear leukocytes [15]. As it follows from the results described in [7,8 1, MT is a selective modulator of AA metabolism capable of increasing the level of intracellular free AA ( [AA ] . ) . Here we present results of our experimental inbestigation and theoretical description of the kinetics of MT-induced platelet aggregation.
MATERIALS
AND METHODS
, and bovine serum albumin (BSA) Reagents and buffers. AA, merthiolate, indomethacin, PGE were purchased from Sigma (St. Louis, USA), PGE 2 , PG Ff 2a , glucose, sodium dodecylsulphate (SDS) and HEPES from Serva (Heidelberg, Germany), fibrinogen from Boehringer (Mannheim, Germany). [3 HI-AA (100 Ci/mM) was obtained from the Institute of Medical Genetics, Russian Academy of Sciences. Ajoene was produced by J.V. BioChemMack (MOSCOW,Russia). , 2.8 mM KCl, 0.35 mM Na HP0 Buffer A: 135 mM NaCl, 12 mM NaHCO , 5 mM HEPES, 5.5 mM glucose, 12 mM citric acid (pH 6. 3 1. Buffer B: 135 mM NaCl, 11 mM r4aHC03 , 2.8 mM KCl, 0.35 mM Na HP0 , 5 mM HEPES, 5.5 mM glucose, 1 mg/ml BSA (pH 7.35). Preparation of $ RP. 4 lood was drawn from antecubital veins of healthy male volunteers into 3.8% trisodium citrate (9:1, v/v), or acid-citrate-dextrose (pH 4.5; 6:1, v/v) if further processing was required, and centrifuged at 110 g (RT) for 15 min. Preparation of l3 H I-AA-labelled washed platelets. PRP was layered on Ficoll-Paque (PharmaciaLKB, Uppsala, Sweden) (l:l, v/v) and centrifuged at 250 g CRT) for 30 min. Cells from the interphase were collected, diluted twofold by buffer A and centrifuged at 860 g CRT) for 15 min. The for 30 min at pellet was resuspended in buffer A and incubated with [ 3 HI-AA (7.5 @X/ml) 37 0 C. To remove the excess of the label, cells were washed twice in the same buffer (630 g, 10 min, RT) and finally resuspended in buffer B at a concentration of 2 * 108 per ml. Aggregation assav. Platelet aggregation was measured turbidimetrically [ 16 ] using a double-beam aggregometer (Thromlite-1006, J.V. BioChemMack (Moscow, Russia)). Aliquots of PRP (250 1.11in glass cuvettes) were stirred (1000 rpm) in the cell of the device with inhibitors or their vehicle controls at 37 O C for 1 min followed by addition of the stimuli. Indomethacin and PGE 1 were dissolved in ethanol, ajoene in dimethylsulphoxide (DMSO), MT in buffer B, AA in autologous PPP. The final concentrations of the organic solvents never exceeded 0.5% v/v. In studying the responses of washed platelets, aliquots of cells (250 ~1) were pipetted into siliconized (Sigmacote R ) glass cuvettes, and stirred (1000 rpm, 37 0 Cl with fibrinogen (0.5 mg/ml), CaC12 (2 mM) and MgC12 (2 mM) for 1 min prior to addition of merthiolate. Analvsis of I 3 H l-AA metabolites. Washed platelets (250 ~1) were incubated in the aggregometer cuvette. Each sample was processed as follows. The reaction was terminated by addition of 0.1 ml 0.4% formic acid (the resultant pH being of about 3.0), the reaction mixture centrifuged at 1000 g for 5 min CRT) and the supernatant extracted thrice by ethylacetate (3x1.5 ml). The organic phase was collected, desiccated with sodium sulphate, evaporated under nitrogen, dissolved in 0.5 ml of ethylacetate and evaporated once again. The residue was redissolved in 50 1.11ethylacetate and the standards of prostaglandins were added (PGF 2a and PGE 2 , 2 pg each in 15 ~1 ethanol). The products were separated by TLC at RT on silica plates (Merck, Darmstadt, Germany) 1.5 cm width in the system of ethylacetate, 2,2,4_trimethylpentane, acetic acid and water 100:500:10:110 (v/v/v v, upper phase). The standards were visualized by exposing the plates to iodine vapour. To determine the distribution of radioactivity relative to R f , each plate was cut into 5 mm sections. The sections were transferred into scintillation vials containing 1 ml of 0.1 y0 sodium dodecylsulphate each and allowed to stand for 1 hr at RT prior to addition of the dioxane scintillant (10 ml per vial). Counts were performed on a liquid scintillation counter (Marck 111).
Vol. 67, No. 5
MERTHIOLATE-INDUCED
AGGREGATION
507
RESULTS
Platelet aggregation studies. Incubation of human platelets (both PRP and washed platelet suspension) with MT results in their irreversible aggregation. Tracings of MT-induced aggregation obtained using the turbidimetric technique are biphasic. The following parameters may be used for (the retention time, i.e. time interval their quantitative characterization (Fig. IA, insert): i) T preceding the onset of the response); ii) V (initial rat: of aggregation); iii) V (maximal rate of aggregation numerically equal to the slope oPthe curve at the point of its inflection); iv) 7 (the lagperiod, i.e. time interval numerically equal to the intercept cut on the time axis by the tangent drawn through this point); v) 6T (the extent of aggregation, i.e. maximal light transmission increment). Platelet responses to exogenous AA are similar to those triggered by MT in that in either case irreversible aggregation is preceded by a lag-period, and the relationships of V to [MT] or [AA] are threshold. In contrast with MT-elicited responses, tracings of AA-induced light transmission increments are monophasic and their quantitation according to the procedure described above (Fig. IA, insert) gives only 3 measurable parameters, viz 7, V, and 6T (note that in this case the following equalities are formally correct for 7 andV :T =T, v =V). Figure 1 shows the relationshrps of V”andor to [MT] (panel A) and [AA J (panel B). The relationships of V to the concentration of either of the agonists ([A]) may be described by the Hill equation: hA V (1) ([Al/KA )
-
=
VCO
hA 1 + ([A]/KA ) where KP equals to EC50, h A is the Hill coefficient and V, is the value of V at [A J ->a). As 11ustrated, the characteristic threshold-like appearance of these relationships is markedly pronounced. The Hill equation gives their satisfactory approximation (Eq. 1) at h>30. The values of K A for MT-induced aggregation of PRP are in the range of lOO-200pM; in the system of washed platelets K amounts to 7.5 PM. The relationships of 6T to [MT ] or [AA ] are also highly cooperative (data not s Rown). As shown in Fig. 1, 7 is inversely related to [MT I (panel A). With AA as an inducer of aggeragation the relationship is similar, but much less pronounced, the values of 7 being reliably lower (Fig. 1, panel B). MT-induced
TxA2
svnthesis. Kinetic studies of MT-induced aggregation and accumulation of clearly demonstrate that the aggregatory response (Fig.2, curve 3) is parallelled by concurrent TxA synthesis (assessed by [ 3 H ]-TxB 2 release into the medium; Fig. 2, curve 4 xA2 is not formed throughout the lag-period. 1). As illustrated, It is also noteworthy that MTinduced TxA 2 s y nthesis may proceed in the absence of stirring (Fig.2, curve 2), when aggregate formation cannot be detected turbidimetrically (Fig.2, curve 4).
[ 3 H J-AA-metabolites
Inhibition of MT-induced aggregation. Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,1 I-triene-9-oxide, has previously been reported to inactivate the platelet fibrinogen receptor and thereby interfere with formation of intercellular receptor-fibrinogen-receptor bonds [ 17 1. Hence, ajoene is thought to be a versatile inhibitor of platelet aggregation [ 18 1. Preincubation of platelets with ajoene completely inhibits both primary and secondary phases of MT-induced aggregation (data not shown). Indomethacin, the well-known cyclooxygenase inhibitor, does not affect the first phase of the aggregatory response (characterized by 7 ), but blocks its second phase (characterized by and V 7 and V). Data from these experiments aYe summar?zed in Fig.3, which shows the relationships of 7, to the concentration of the inhibitor. Increasing indomethacin input progressively ‘S tbtts and O tn the vsecond phase of the aggregatory response. Quantitative manifestation of this tendency appears as a decrease in 7 and V which finally reach their minimal values equal to 7 and V respectively (Fig. 3). Hence, the first slow wave of the biphasic MT-induced response”is independent of the cyclooxygenase pathway of AA metabolism, while the second rapid wave of aggregation strongly depends on the ability of platelets for AA cyclooxygenation. It should be noted that inhibition by indomethacin of both MT- and M-induced aggregatory responses exhibits the same degree of cooperativity, since in either case the relationships of U to to the concentration of indomethacin are characterized by equally high values of the apparent Hill coefficients [ 19 1.
’
MERTHIOLATE-INDUCED
508
A
ho
Vol. 67, No. 5
AGGREGATION
Z,min
1000
2000 [ Merthiolate] , ,+IM
14
+ 095
i 0
LArachidonic
acidJ , pM
Figure 1. The relationships of the maximal rate of aggregation W; closed circles, curves 1 and 3) and of the lag-period preceding the onset of the response (T; open circles, curves 2 and 4) to the concentrations of merthiolate (MT) (panel A) and arachidonic acid (AA) (panel B). All numerical data are derived from the aggregation tracings obtained in a single experimental setting with plateletrich plasma (PRP) of the same donor. Replicate experiments with samples of PRP from 6 donors gave similar results. Insert: a typical tracing obtained by the turbidimetric technique and the method proposed for its quantitation (scheme; for details see the text). Addition of the stimulus is marked with an arrow.
Vol. 67, No. 5
MERTHIOLATE-INDUCED
I
0
/
I
2
I
AGGREGATION
I
I
509
1
4 Time , min
6
Figure 2. Synthesis of thromboxane associated with MT-induced aggregation. Addition of MT (50 PM, arrow) to washed platelets (2 10 * per ml in buffer B supplemented with 0.5 mg per ml fibrinogen, 2 mM CaCl and 2 mM MgC12 ) stirred at 1000 rpm induces their aggregation (curve 3) and accumulation of [ 3iI ]-TxB 2 in the medium (curve 1, open circles). In the absence of stirring aggregates are not formed (curve 4), while the increase in the level of [ 3 H ]-TxB2 is still detectedkurve 2, closed circles).
v and V, , %min-’ 100
t and Z,,min 10 /
[Indomethacin],yM of MT-induced aggregation by indomethacin. Aliquots of PRP were Fieure 3. Inhibition preincubated with varying concentrations of indomethacin (37 o C, 1000 rpml for 1 min, followed by addition of MT (400 PM) and the resultant light transmission increments were monitored continuously. The following parameters were derived from each of the obtained tracings: maximal W, . open ctrcles) and mtttal ‘Vo , closed circles) rates of aggregation, lag-period (7, open circled), retention time (7 o , closed circled) (curves 1 through 4,respectiveiy).
MERTHIOLATE-INDUCED
510
Vol. 67, No. 5
AGGREGATION
Z, min
to /min
60
[PGE,], nM Fipure 4, Inhibition of MT-induced aggregation by PGEl . The experimental protocol is described under Fig. 3, exept that [MT ] = 470 PM; the relationships of V (closed circles) and T (open circles) [PGE 1 I are presented.
Preincubation of platelets with PGEl leads to a prolongation of the lag-period and a decrease in the maximal rate of aggregation (increase in 7, reduction of V; Fig. 4); the parameters of the first phase are affected similarly (data not shown). As illustrated, the relationship of V to [PG ] is not threshold, the the observed values of Hill coefficient bein in the range of 0.7-1.3. I!!h ibition by PGE 1 of AA-induced aggregation is characterized by equally low values of the Hill coefficient [19]. Kinetic model of MT-induced azzrezation. The obtained data are described both quantitatively and qualitatively in terms of a model assuming that: i) [AAJ is increased as a consequence of MTinduced inhibition of lysophospholipid acylation; ii) a certain threshold level of [AA 1. is required for initiation of irreversible aggregation. This model corresponds to the scheme depicted in Fig. 5 (insert). The kinetics of variation of [AA]i is described by the following differential equation:
d[fil
i = v,
- v_
(2)
dt Here V + is the overall rate of reactions leading to AA release (a parameter considered herein to be constant) and V_ is the rate of AA reincorporation, which is in turn related to the bulk [MT ] as given by equation (3): ko [uli (3) v_ = 1 + WI/K1 where k o is the rate constant of the pseudo-first order reaction, and KI equals to ICSo for the relationship of the rate of AA reincorporation to the bulk [MT] introduced into the system. The first order with respect to [AA]. (Eq. 3) reflects the well-known fact that the value of in platelets is extremely low $1. Assuming MT to be added at zero time (t = 0) and its I”li concentration to vary from zero to the final value ([MT]) we obtain the solution of Eq. 2 in the following form:
to
Vol. 67, No. 5
MERTHIOLATE-INDUCED
AGGREGATION
ko v, (1 + [MT]/Kf
[AAli
)
V+
) e ko
ko
t
1 + [MTl/KI
[MT]
-(
=
511
(4)
KI
Expression (4) describes the kinetics of MT-induced variation of [AA] . ~ Time interval required is determined from to attain the critical (i.e. inducing irreversible aggregation) value of ]A], : expression (5); it is assumed that this critical value is attained at the bulk [MT 1 = KA
P=Ym
(p+
KI KA
[MT 1
WT 1 ) ln(
KA
) [MT]-
(5)
KA
(the value reached by T at [MT] ->oO the rate of MT-induced platelet aggregation to the into the system. When the relationship of T to [MT] is represented graphically by plotting T /Too it becomes evident that the obtained function depends exclusively on the against [MT I/K calculated from exprestiion (5) fo ratio. T RL e relationships of 7 /7 oo to WI/K* K /KI di @erent values of this ratio are compared to those obtained experimentally in Fig. 5. It is apparent = 0.1. that expression (5) gives a satisfactory description of our experimental data at Q /KI
10
calculated from Equation (5) for Figure 5. The relationships of T/T o. to WTl/K A equal to 0.001, 0.03, 0.1, 1, 10, 100 and 1000 (curves 1 through 6, respectively K /ICI TRLe experimental = 0.32 min values of 7 (closed circles) are the same as in Fig. 1 (panel A); 7 Qo and K A = 75pM. Insert: Schematic illustration of the kinetic model of MT-induced aggregation.
MERTHIOLATE-INDUCED
512
AGGREGATION
Vol. 67, No. 5
MERTHIOLATE ,a” LYSO-PL 4 (LYSO-PAF)
\-
PAF +‘*
* * -+ 6
FIBRINOGEN RECEPTOR ACTIVATION 4
SLOW __,
AGGRE6ATlON (PHASE I)
AACoA PL
AdENE I T AA
Q ,+
PGU,
4
TxA,+
ST- -+FIBRIN&EN RECEPTOR ACTIVATION
b
RAPID AGGREGATION (PHASE 1 )
A6 intracellular
Figure
6.
of second
The proposed mechanism of the aggregatory effects of AA and MT. Generation messengers and active intermediates formed through hydrolysis of PL is depicted schematically. Sites of inhibition are marked with - *
DISCUSSION The proposed mechanism of MT-induced aggregation is depicted in Fig. 6. Stimulation of platelets with various agonists results in the release of AA from membrane phospholipids 120 1. Two pathways of AA liberation have been described: 1) hydrolysis by phospholipase A2 [3,21-23 I and phospholipase C / 1,2-diacylglycerol-lipase-dependent cleavage [21,23-25 I. Cyclooxygenase converts AA to PGH2 the latter being further transformed into TxA2 by thromboxane synthase [26,27]. These highly labile AA metabolites are potent platelet agonists; their binding to specific receptors induces G-proteindependent phospholipase C activation with the resultant initiation of the phosphoinositide turnover, [Ca 2 + Ii mobilization and other events terminating in aggregate formation. active Other products formed through phospholipase A 2 activation are the physiologically lysophospholipids [30-331 and the platelet-activating factor (PAF) [34]. Arachidonate-specific acyl-CoA synthase [35] and polyunsaturated fatty acid-specific acyl-CoAlysophosphatide acyltransferase 1361 which have been identified in platelets selectively decrease the levels of free AA and, consequently, PGH and TxA M?-induced i&bition of AA reincorporation into phospholipids should elevate the level of [AA] i and increase the proportion of lysophospholipids, thereby leading to enhanced synthesis of cyclooxygenase metabolites, platelet activation and aggregation. Indeed, MT has been reported to induce aggregation and serotonin release in PRP [12,13 1, synthesis of cyclooxygenase products 17-9 ] aggregation [9,14], [Ca 2 + ] i mobilization 19 ] and chemilumenescence [ 14 I in washed platelets Incubation of platelets with MT leads to irreversible aggregation which is preceded by a prolonged lag-period and parallelled by TxA2 synthesis (Fig. 2). Reincorporation of AA into membrane lysophospholipids of murine peritoneal macrophages is completely inhibited by 50 /JM MT 181. Moreover, at 40 PM MT significantly potentiates synthesis of leukotrienes induced by FMLP or C3a in human polymorphonuclear leukocytes [15 1. It is noteworthy that in our experiments the same concentrations of MT have been demonstrated to induce TxR2 accumulation (Fig. 2).
Vol. 67, No. 5
MERTHIOLATE-INDUCED
513
AGGREGATION
Aggregation of washed human platelets induced by methylmercury chloride (a compound related to MT chemically) is Inhibited by indomethacin [37 1. Methylmercury chloride has recently been shown to inhibit specifically the activity of lysophosphatide: acyltransferase in platelet microsomes ]38 1. In terms of our kinetic model, the characteristic lag inherent to MT-induced responses (Fig. 1) ts vtewed as a result of accumulation of free AA. It is suggested that the onset of irreversible aggregation corresponds to the attainment of a certain critical value of ]AAli which is required for triggering off this phase of the response. The values of 7 calculated from Eq. 5 correspond to those obtained experimentally (Fig. 5). The second phase of MT-induced response (Fig. lA, insert) is similar to AA-elicited aggregation in that it is irreversible, susceptible to inhibition by indomethacin (Fig. 3) or ajoene [19], and characterized by highly cooperative relationships of V to [MT ] (Fig. 1; Eq. 1). Increasing AA input results in inhibition of aggregation [39-441; analogous results are obtained with MT as a stimulus (data not shown). The first phase of MT-induced response (Fig. lA, insert) is ‘resistant’ to indomethactn (Fig 31. To date mechanisms underlying this phenomenon remain obscure. In the majority of cells including platelets biosynthesis of PAF is linked to AA liberation via a common Intermediate, I-o-alkyl-2arachidonoyl-at-glycero-3-phosphorylcholine (AA-GPC) [45]. Hydrolysis of AA-GPC by phosphohpase A2 initiates both PAF and eicosanoid biosynthesis [46 I. Cyclooxygenase-independent slow aggregation may be underlain by PAF production and/or lysophospholipid accumulation (Fig. 6). For iInstance, PAF-induced aggregation is known to be independent of TxA synthesis I47 1, while lysophosphatidylcholine has recently been demonstrated to act as a second2 messenger modulating the activity of protein kinase C ]48 1. It should be noted that other explanations cannot be excluded. Preincubation of platelets with PGE 1 has previously been shown to result in a prolongation of the lag-period preceding the onset of aggregatory, secretory and luminescence responses to MT ]I4 J. Our observation that pretreatment with PGEl postpones the onset of MT-induced aggregation and inhibits its maximal rate (Fig. 4) points to the existence of at least 2 intracellular targets for PGl$ , one possibly controlling AA accumulation, and the other associated with some terminal steps of platelet activation. Derangement of the former mechanism leads to a prolongation of the lag, interference with the latter affects the rate of aggregate formation (Fig. 6) PGEl is known to elevate intracellular CAMP [49], which in turn induces Ca sequestration ]49,50]; depletion of [Ca2+ ] i may lead to an inhibition of the activity of the Ca-dependent phospholipase A 2 and thereby decrease the rate of intracellular AA accumulation. Indeed, CAMP has been reported to affect the release of AA mediated by phospholipase A 2 [51 1. In addition, CAMP has been found to decrease the proportion of proteins available for protein kinase C-dependent phosphorylation [20]. The observed decrease in V (Fig 4) might partially be accounted for by either of these effects. The inferred ability of PGEl for affecting a certain terminal step of signal transduction leading to aggregation is indirectly corroborated by our recent observation that the values of the apparent Hill coefficients characterizing the relationships of the aggregation rates to [PGE, 1 are independent of the agonists used for triggering off the response (i.e. of the signal transduction pathways involved) and reliably approximate unity [ 19 1. ACKNOWLEDGEMENTS We are grateful to A. Turgiev for fruitful discussions
and help in preparation
of the manuscript.
REFERENCES I. SIESS, W. Molecular mechanisms
of platelet activation.
P/tysio/o@cal Rev. 69, 58-l 78, 1989.
2. SWEAT, J.D., BLAIR, I.A., CRAGOE, E.J. and LIMBIRD, L.E. Inhibitors of Na + /H + exchange block epinephrine-induced and ADP-induced stimulation of human platelet phospholipase C by blockade of arachidonic acid release at a prior step. J. Biol. Cherv. 261, 8660-8666, 1986. 3. BILLS, T.K., SMITH, J.B. and SIVER, M.J. Selective release of arachidonic phospholipids of human platelets in response to thrombin. J. CZin. Invest. 60, l-6, 1977.
acid
from
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