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EFFECTS OF CLOFIBRATE ON PLATELETS AND EVIDENCE OF THE INVOLVEMENT OF PLATELET LIPIDS IN PLATELET FUNCTION HUZOOR-AKBAR AND N.G. ARDLIE Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, A.C.T. 2601, Australia.
(Received 19.8.1976; in revised form 17.11.1976. Accepted by Editor B. Firkin) ABSTRACT The effects of clofibrate, a recognised plasma lipid lowering agent, on platelet function and lipid metabolism were examined to determine the role of platelet lipids in platelet function and the mechanism of the inhibitory action of clofibrate on platelets. Clofibrate inhibited aggregation of human platelets by thrombin and collagen but did not inhibit the initial platelet-collagen interaction. The drug also inhibited the second phase of aggregation induced by epinephrine and ADP and inhibited the release of [3H]5 hydroxytryptamine by collagen and thrombin, and release of f3-glucuronidaseby thrombin. These findings suggest that the effect of clofibrate on aggregation is attributable to inhibition of the release reaction. Further investigation revealed that the drug decreased platelet factor 3 activity but not factor Xa forming activity. Clofibrate also decreased the incorporation of 14C-acetate, but not 14C-U-glucose into platelet phospholipids, glycerides and free fatty acids. Both immediate and progressive inhibitory effects were observed. Since clofibrate inhibited the release of contents from washed platelets and also inhibited lipid synthesis by washed platelets, it is possible that the two may be linked. Decreased platelet factor 3 activity may also contribute to inhibition of release. INTRODUCTION Heightened platelet function may be important in thrombogenesis and has been described in a variety of diseases. Recently, Carvalho and associates (1) reported an increase in platelet responsiveness to ADP, collagen and epinephrine in patients with familial type IIb hyperlipoproteinemia. They also
CLOFIBRATE AND PLATELET LIPIDS
showed that clofibrate (ethyl-p-chlorophenoxy isobutyric acid, (CPIB) decreased platelet sensitivity to aggregating agents in both normal subjects and in patients with type IIb hyperlipoproteinemia (2). Clofibrate is a well known plasma lipid lowering agent (3,4,5) but its influence on platelet function has been found to be independent of its effects on plasma lipids. Concentrations of clofibrate which normalized platelet function in hyperlipidemic patients did not significantly alter the plasma total cholesterol, LDL-cholesterol or triglyceride concentration (2). Although clofibrate-induced alterations in platelet function may not be dependent on plasma lipids it is still possible that they are mediated through changes in platelet lipids. We therefore examined the effects of clofibrate on platelet aggregation and release, platelet coagulant activities and platelet lipid metabolism in order to better understand the role of platelet lipids in platelet function and to determine how clofibrate alters platelet function. METHODS AND MATERIALS Methods for blood collection,preparation of platelet-rich plasma (PIG) and platelet-poor plasma (PPP), control of plasma pH, platelet counting, glassware, plastic tubes and anticoagulant solutions have been described previously (6,7). Platelet aggregation was measured by the turbidometric method (6). Platelet coagulant activities. Platelet factor 3 activity and factor Xa forming activity of platelets were assayed by the method of Walsh (8). Platelet release reaction. This was studied by measuring the release of radioactivity from plateletslabelled with t3Hl 5 hydroxytryptamine creatinine sulphate (i3H15HT) as described previously (61, and also by measuring the release of B-glucuronidase (E.C.126.96.36.199). Plateletswere washed three times (as described below) and incubated with i3H15HT (0.6~Ci/10g cells) at 37OC for 30 minutes in the third wash. The platelets were then washed once more and finally suspended in modified Tyrode's buffer (NaCl 8.Og/l, KC1 0.2g/l, NaHC03 l.Og/l, NaH2P04H20 O.O5g/l, CaC12 0.306g/l, MgC126H20 0.203g/l, glucose l.Og/l, bovine albumin 3.5g/l), pH 7.4,without apyrase. Release of fi-glucuronidasefrom plateletswas determined with a kit supplied by Sigma Chemical Co., St. Louis, MO., U.S.A. Platelet washing. To study the effects of clofibrate on the incorporation of 14C-acetate and U-l4C-glucose into platelet lipids, the platelets were isolated from PRP obtained from blood anticoagulated with ACD and washed by a method described elsewhere (6). TO enable estimation of platelet proteins, albumin was omitted from both washing and suspending solutions. Platelets were finally resuspended in a modified Tyrode's buffer, pH 7.4 and the platelet count adjusted to lO"/l. Albumin was retained in the washing and suspending fluids when platelets were isolated for studies of aggregation and the release reaction.
CLOFIBRATE AND PLATELET LIPIDS
Incorporation of "C-acetate or U-14C-glucose. lml aliquots of washed platelets were incubated with "'C-acetate (3pCi), clofibrate (2.3mM) and acetate (2uM) for 90 minutes at 37OC in a shaking water bath. Incorporation of U- "C-glucose into platelets was also studied by incubating lml aliquots of platelet suspension with U-14C-glucose (5pCi) and clofibrate (2.3mM) for 180 minutes at 37°C in a shaking water bath. Linearity of incorporation of 14C-acetate and U-14C-glucose was checked by incubating the mixtures for 1, 2 and 3 hours. Extraction of lipids. The platelets were immediately centrifuged at 1100s for 15 minutes at the conclusion of each incubation. The supernatant was discarded and the platelets were washed twice with 2ml of modified Tyrode's buffer. Finally, the platelets were resuspended in lml of modified Tyrode's buffer and transferred to conical flasks. Em1 of methanol was added and the mixture was heated on a hot plate. 16ml of chloroform and 0.5 ml of normal plasma (as lipid carrier) were added and after vigorous shaking the mixture was kept at room temperature. The extract was filtered and washed three times with 4ml of Folch reagent (9) and then 0.05M NaCl (1 vo1/5 vol extract) was added to separate the phases. The upper phase was discarded and the lower phase of the extract was washed twice with the upper phase of Folch reagent. The solvent was evaporated on a hot plate under nitrogen. Separation of lipid classes. Platelet lipids were separated into ohosoholinids (PL). mono and dicrlvcerides (GLY), free fatty acids -(FFAj, triglycerides (TG) and sterol esters (SE) on thin layer chromatographic plates (40~ thickness) using petroleum ether: ether:acetic acid (82:lE:l) as solvent. Platelet lipids corresponding to lipid standards were visualized with either iodine vapour or Rhodamine 6G. After localization, the silica gel bands were scraped and the lipids were extracted from the gel with appropriate solvents. Phospholipids were extracted with chloroform:methanol:acetic acid:water (200:156:20:4) while other lipids were extracted with diethyl ether. After the solvent was evaporated, samples were counted in a liquid scintillation counter. Saponification of lipids. Extracts of total platelet lipids were saponified as described by Rodbell (10). The saponifiable lipids were extracted by shaking the samples with 6ml of heptane. 4ml of the heptane phase was evaporated and counted in a liquid scintillation counter. Reagents. Epinephrine bitartrate (Calbiochem., San Diego, California, U.S.A.) and adenosine diphosphate (ADP, Sigma Chemical co., St. Louis, MO., U.S.A.) were stored in aliquots at -20°C in modified Tyrode's solution at a concentration of 10B2M and diluted before use. Bovine thrombin (Parke Davis & Co., Detroit, Michigan, U.S.A.) was stored in aliquots at -2O'C in modified Tyrode's solution at a concentration of lOOOU/ml (NIH) and diluted before use. Collagen (Sigma Chemical Co., St. Louis,
CLOFIBRATE AND PLATELET LIPIDS
MO., U.S.A.) was an acid soluble preparation of achilles tendon. Clofibrate, sodium salt of CPIB was obtained from Imperial Chemical Industries Ltd., Macclesfield, Cheshire, England and diluted in modified Tyrode's solution. Russell's Viper Venom (RW) (Burroughs Wellcome Research Laboratories, Beckenham, England) was dissolved in modified Tyrode's solution and stored A rase (E.C.188.8.131.52) (Sigma Chemical Co., St. Louis, ::.:"::s.A#- was stored in aliquots of 3.4mg/mlat -2O'C. m 5 hydroxytryptamine creatinine sulphate (['HISHT), specific activity 27mCi/mg, 1-"C-acetate, specific activity 6OmCi/mmol, and D[U-'4Cl glucose, specific activity 3mCi/mmol were obtained from the Radiochemical Centre, Amersham, England. Arachidonic acid was obtained from Sigma Chemical Co., St. Louis, MO., U.S.A. 8-glucuronidase kits were obtained from Sigma Chemical Co., St. Louis, MO., U.S.A. RESULTS Clofibrate and platelet aggregation The in vitro inhibitory effect of clofibrate on platelet aggregation caused by epinephrine is shown in Fig. la. .0.4mM clofibrate was ineffective whereas 0.8mM produced variable inhibition in different individuals, the inhibitory effect being as little as 14% in one individual and up to 100% in other individuals after 200 minutes incubation. A variable effect of 1.5mM clofibrate was also seen in different individuals. Maximal inhibition was achieved after 120 to 240 minutes incubation and ranged from 22-100%. The same variability was also observed with 2.3mM clofibrate. Complete inhibition of aggregation caused by 4UM epinephrine was observed after one to 80 minutes with 2.3mM clofibrate in different individuals; complete inhibition of aggregation by 8l~Mepinephrine was only observed after much longer incubation times, eg. 215 minutes. These results show that there is an immediate inhibitory effect of clofibrate and that inhibition increases with prolonged incubation. They also show that there is a variable response to clofibrate in different individuals. The second phase of ADP-induced aggregation was inhibited by clofibrate (2.3mM) (Fig. lb). Sometimes there was an immediate effect, whereas inhibition was only observed after 80-140 minutes in other individuals. Clofibrate did not inhibit the first phase of ADP-induced aggregation. The inhibitory effect of clofibrate (2.3mM) on thrombininduced aggregation varied between individuals. For example, after 1 minute clofibrate inhibited aggregation by 0.25U of thrombin by 58% in one individual and 89% in another individual. The inhibitory effect increased with longer incubation. The extent of inhibition was also determined by the concentration of thrombin used to cause aggregation (Fig. lc). Aggregation by collagen was inhibited by 1.5mM clofibrate but not 0.8mM. Again the effect of clofibrate varied between individuals. Maximal inhibition by 2.3mM clofibrate was sometimes observed after one minute when a low concentration of
CLOFIBRATE AND PLATELET LIPIDS
FIG. 1 (a) Effect of clofibrate on platelet aggregation caused by epinephrine. Modified Tyrode's solution (MT) or different concentrations of clofibrate were added to PRP 1 minute before epinephrine (4uM). The different concentrations (mM) of clofibrate used are shown beside each tracing. (b) Effect of clofibrate on ADP-induced aggregation. Clofibrate (2.3mM) or MT was added to PRP 1 minute before ADP (8k1M). (c) Effect of clofibrate on aggregation caused by different concentrations of thrombin. PRP was incubated with clofibrate (2.3mM) or MT for 100 minutes before the addition of thrombin. The concentrations of thrombin are shown beside each tracing. (d) Progressive inhibition by clofibrate of aggregation caused by collagen. Prior to the addition of collagen (lOUg/ml), PRP was incubated with clofibrate (2.3mM) for different times (mins) (shown beside each tracing). (e) Effect of aspirin or clofibrate on aggregation by arachidonic acid. Clofibrate (2.3mM) or aspirin (5mM) was added to PRP 1 minute before arachidonic acid (0.3mM). (f) Effect of clofibrate on aggregation and clotting caused by epinephrine. Clotting is indicated by the change in light transmission at the end of each tracing. PPP (5O)Jlto 5OOpl platelet suspension) and Ca++ (5mM) were added immediately before epinephrine (8pM). The time (mins) of incubation of platelet suspension with clofibrate (2.3mM) is shown beside each tracing.
CLOFIBRATE AND PLATELET LIPIDS
collagen was used to cause aggregation. A progressive inhibitory effect with time was also observed (Fig. Id). Arachidonic acid is thought to release platelet constituents and thus cause aggregation by being converted to a labile cyclic endoperoxide (11). Aspirin inhibits the synthesis of this endoperoxide and consequently inhibits aggregation by arachidonic acid (12). Inhibition by aspirin of platelet aggregation induced by arachidonic acid is shown in Fig. le. In the same figure it can be seen that clofibrate failed to inhibit aggregation by arachidonic acid. In order to determine whether the inhibitory effect of clofibrate on platelet function involves plasma factors or reflects a direct effect of the agent on platelets, experiments were carried out with washed platelets. The results obtained with washed platelets were similar to those observed with PRP. Both immediate and progressive inhibitory effects were seen, and moreover, variable effects of clofibrate on platelets from different subjects were still obtained. Washed platelets did not respond to epinephrine unless plasma and calcium were present. In this test system epinephrine aggregated platelets, although no primary phase was observed, and the samples eventually clotted. Clofibrate prolonged the lag phase between the addition of epinephrine and the onset of platelet aggregation and this inhibitory effect increased with time (Fig. If). It is also noteworthy that clofibrate progressively increased the clotting time (Fig. If). Clofibrate and the platelet release reaction The effect of clofibrate on the platelet release reaction was examined, because the observed inhibitory effects of this agent on aggregation could be due toinhibitionof the release of platelet contents. To assess the release from dense and ugranules, [3H]5HT and O-glucuronidase respectively were measured in the suspending medium. The results are shown in Table 1. Clofibrate inhibited the release of both L3H]5HT and O-glucuronidase and the inhibitory effect on release of [3H]5HT increased with time. Clofibrate and platelet coagulant activities After 90 minutes incubation with clofibrate platelet factor 3 activity had decreased by 23% (P