THROMBOSIS RESEARCH 57; 795-801,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
BRIEF POTENTIATION COMPOUNDS
COMMUNICATION
OF fiDP-INDUCED
PLATELET
Barbara
AGGREGATION
BY MERCURY
Kostka
Department of Biochemistry, Institute of Environmental Research and Bioanalysis, Medical ficademy, Muszyfiskiego 1, 90-145+bdt, Poland (Received 30.8.1989; accepted in revised form 22.11 .1989 by Editor V. Sultan) INTRODUCTION There is growing evidence that mercury compounds induce disorders in blood platelets functions. c1s exemplified by some authors, mercurials may both inhibit and stimulate the activities of blood platelets (l-4). We have recently observed that the effect of various mercury compounds on ADP-induced aggregation of the blood platelets differs considerably and most probably depends on the concentration as well as on the chemical properties of these compounds (5). We have also noticed an increased blood platelet reactivity to ADP in the presence of methylmercuric chloride (61. In this study we investigated the influence of the mercurials with different chemical specificity; mercuric chloride (MC), methylmercuric chloride (MMC) and phenylmercuric acetate (PMEII on ADP-induced platelet aggregation in in vitro conditions. MEITERIFILS CIND METHODS Methylmercuric chloride (ICN Pharmaceuticals, Inc. US&l, phenylmercuric acetate and mercuric chloride (POCH, Poland) and adenosine diphosphate (Merck) were used in buffered saline containing 0.154 M NaCl and 0.154 ?I Tris-HCl, pH 7.4 (9:l). PtlCI at concentrations above 10 AIM was prepared with distilled water. CIddition of 2I3,ul of water did not affect platelet aggregation. Platelet rich plasma (PRP) was separated from human blood obtained from normal, drug-free donors. Domestic pig blood was collected at a slaughterhouse. Albino Wistar rat blood was collected from carotid artery of ether anesthetized animals.
Words: platelet aggregation, mercuric phenylmercuric acetate, chloride.
Key
795
adenosine chloride,
diphosphate, methylmercuric
796
MERCURY AND PLATELET AGGREGATION
Vol. 57, No. 5
The
blood was placed in plastic tubes with 3.13X sodium citrate (9:l) and centrifuged at 120 x g for 20 minutes at room temperature to obtain PRP (human, pig) and at 630 x g for 5 minute5 to obtain PRP. rat Platelet poor plasma (PPPlwas obtained by centrifuging the remaining blood at 2800 x g for 10 minutes. The PRP was kept in capped tubes at ambient temperatures no longer than 4 hours from the moment of preparation to completion of the aggregation studies. An aggregometer equipped with a stirring device and attached to a chart recorder was used (71. Total volume of the sample was JOOrul. PRP sample was stirred in a siliconized glass tube in the aggregometer at 800 rpm at 37% for 5 minutes before the addition of 5~1 aliquots of the aggregating agent to provide the nominal final concentrations. Various concentrations of mercury compounds in the volume of 5 - 25 /ul or saline in the control tests were added to the PRP sample for time before CIDP addition. The aggregations were a given recorded against .a PPP reference until maximum aggregation was reached. speed 1 Changes in light transmission were recorded at a cm/min and the rate of aggregation was given as the maximum aggregation Xl. deflection of the aggregation curve (maximum Aggregation tracings (Figure 11 are representative of at least Data were analyzed by means of a 3 separate experiments. Student's t - test. the kinetic parameters of platelet In order to measure Louis was used aggregation the method described by Rossi and ADP alone, induced by (8). In this paper platelet aggregation PRP was ADP and MMC added simultaneously to as well as curves utilizing five measured. Five different aggregation different ADP concentrations (1,2,5,10 and 20pM) were prepared for each of the examined plasma samples. Similarly, at least curves with these five five different aggregation ADP, in the presence of MMC at the concentrations of made. The nonaggregating concentration (10 and 40 &IN) were paper speed was set at 5 cm per minute. A line tangential to the initial rapid wave of platelet aggregation was drawn and light platelet aggregation velocity (v) expressed as units of transmission per minute (XT min-*1 each was recorded for concentration of the aggregant. The reciprocal of the aggregation velocity was plotted against the reciprocal of the ADP concentrations and a regression line was calculated with the aid of a computer programme (Amstrad CPC 61281. At least 4 points were used to calculate each regression line. The point on the ordinate intercepted by regression line represents the reciprocal of the maximum aggregation velocity. The point on the abscissa intercepted by the regression line is the negative reciprocal of the ADP concentration required to produce half of the maximum platelet aggregation (-l/K'). The K' value QJM) for ADP-induced aggregation and K' in the case of ADP and MMC added simultaneously were calculated for each sample of PRP. RESULTS Different effects of mercury platelets agqregation induced by ADP. The examined mercurials: MC, MMC,
compounds PMA,
on
incubated
the with
blood rat
MERCURY AND PLATELET AGGREGATION
Vol. 57, No. 5
797
PRP do not affect the platelet aggregation caused by 10 PM CIDP when their concentrations are lower than 5pM (Table 1). Clt the concentration5 5 - 100 PM, MC decreases, whereas MMC increases significantly the ADP-induced aggregation. PMA inhibits competitively the process of aggregation at the concentrations concentration above, a up to 1001_rM. At the 1-M and significant increase of the aggregation is observed (Table 1, Fig 1 small inset). The PM&induced changes decreased as a result of the increasing ADP concentration (Fig 1). We observed that a 20-minute incubation of the blood platelets with PMA
BRIEF POTENTIATION COMPOUNDS
COMMUNICATION
OF fiDP-INDUCED
PLATELET
Barbara
AGGREGATION
BY MERCURY
Kostka
Department of Biochemistry, Institute of Environmental Research and Bioanalysis, Medical ficademy, Muszyfiskiego 1, 90-145+bdt, Poland (Received 30.8.1989; accepted in revised form 22.11 .1989 by Editor V. Sultan) INTRODUCTION There is growing evidence that mercury compounds induce disorders in blood platelets functions. c1s exemplified by some authors, mercurials may both inhibit and stimulate the activities of blood platelets (l-4). We have recently observed that the effect of various mercury compounds on ADP-induced aggregation of the blood platelets differs considerably and most probably depends on the concentration as well as on the chemical properties of these compounds (5). We have also noticed an increased blood platelet reactivity to ADP in the presence of methylmercuric chloride (61. In this study we investigated the influence of the mercurials with different chemical specificity; mercuric chloride (MC), methylmercuric chloride (MMC) and phenylmercuric acetate (PMEII on ADP-induced platelet aggregation in in vitro conditions. MEITERIFILS CIND METHODS Methylmercuric chloride (ICN Pharmaceuticals, Inc. US&l, phenylmercuric acetate and mercuric chloride (POCH, Poland) and adenosine diphosphate (Merck) were used in buffered saline containing 0.154 M NaCl and 0.154 ?I Tris-HCl, pH 7.4 (9:l). PtlCI at concentrations above 10 AIM was prepared with distilled water. CIddition of 2I3,ul of water did not affect platelet aggregation. Platelet rich plasma (PRP) was separated from human blood obtained from normal, drug-free donors. Domestic pig blood was collected at a slaughterhouse. Albino Wistar rat blood was collected from carotid artery of ether anesthetized animals.
Words: platelet aggregation, mercuric phenylmercuric acetate, chloride.
Key
795
adenosine chloride,
diphosphate, methylmercuric
796
MERCURY AND PLATELET AGGREGATION
Vol. 57, No. 5
The
blood was placed in plastic tubes with 3.13X sodium citrate (9:l) and centrifuged at 120 x g for 20 minutes at room temperature to obtain PRP (human, pig) and at 630 x g for 5 minute5 to obtain PRP. rat Platelet poor plasma (PPPlwas obtained by centrifuging the remaining blood at 2800 x g for 10 minutes. The PRP was kept in capped tubes at ambient temperatures no longer than 4 hours from the moment of preparation to completion of the aggregation studies. An aggregometer equipped with a stirring device and attached to a chart recorder was used (71. Total volume of the sample was JOOrul. PRP sample was stirred in a siliconized glass tube in the aggregometer at 800 rpm at 37% for 5 minutes before the addition of 5~1 aliquots of the aggregating agent to provide the nominal final concentrations. Various concentrations of mercury compounds in the volume of 5 - 25 /ul or saline in the control tests were added to the PRP sample for time before CIDP addition. The aggregations were a given recorded against .a PPP reference until maximum aggregation was reached. speed 1 Changes in light transmission were recorded at a cm/min and the rate of aggregation was given as the maximum aggregation Xl. deflection of the aggregation curve (maximum Aggregation tracings (Figure 11 are representative of at least Data were analyzed by means of a 3 separate experiments. Student's t - test. the kinetic parameters of platelet In order to measure Louis was used aggregation the method described by Rossi and ADP alone, induced by (8). In this paper platelet aggregation PRP was ADP and MMC added simultaneously to as well as curves utilizing five measured. Five different aggregation different ADP concentrations (1,2,5,10 and 20pM) were prepared for each of the examined plasma samples. Similarly, at least curves with these five five different aggregation ADP, in the presence of MMC at the concentrations of made. The nonaggregating concentration (10 and 40 &IN) were paper speed was set at 5 cm per minute. A line tangential to the initial rapid wave of platelet aggregation was drawn and light platelet aggregation velocity (v) expressed as units of transmission per minute (XT min-*1 each was recorded for concentration of the aggregant. The reciprocal of the aggregation velocity was plotted against the reciprocal of the ADP concentrations and a regression line was calculated with the aid of a computer programme (Amstrad CPC 61281. At least 4 points were used to calculate each regression line. The point on the ordinate intercepted by regression line represents the reciprocal of the maximum aggregation velocity. The point on the abscissa intercepted by the regression line is the negative reciprocal of the ADP concentration required to produce half of the maximum platelet aggregation (-l/K'). The K' value QJM) for ADP-induced aggregation and K' in the case of ADP and MMC added simultaneously were calculated for each sample of PRP. RESULTS Different effects of mercury platelets agqregation induced by ADP. The examined mercurials: MC, MMC,
compounds PMA,
on
incubated
the with
blood rat
MERCURY AND PLATELET AGGREGATION
Vol. 57, No. 5
797
PRP do not affect the platelet aggregation caused by 10 PM CIDP when their concentrations are lower than 5pM (Table 1). Clt the concentration5 5 - 100 PM, MC decreases, whereas MMC increases significantly the ADP-induced aggregation. PMA inhibits competitively the process of aggregation at the concentrations concentration above, a up to 1001_rM. At the 1-M and significant increase of the aggregation is observed (Table 1, Fig 1 small inset). The PM&induced changes decreased as a result of the increasing ADP concentration (Fig 1). We observed that a 20-minute incubation of the blood platelets with PMA
