HDL₃-Mediated Inhibition of Thrombin-Induced Platelet Aggregation and Fibrinogen Binding Occurs via Decreased Production of Phosphoinositide-Derived Second Messengers 1,2-Diacylglycerol and Inositol 1,4,5-tris-Phosphate

From the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität, Münster (J.-R.N., M.W., G.A.); the Institut für Arterioskleroseforschung an der Universität Münster (M.W., U.S., G.A.); Experimentelle Hämostaseforschung, Medizinische Klinik und Poliklinik, Innere Medizin A, Münster (B.K., S.W.); and Universitätklinik Marienhospital, Ruhr-Universität Bochum, Herne (M.T.), Germany.

Correspondence to Michael Walter, MD, Institut für Klinische Chemie und Laboratoriumsmedizin, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany.

	Abstract

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Abstract—We demonstrate that physiological concentrations of HDL₃ inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL₃-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL₃ on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL₃ with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL₃ were not blocked by H89, a protein kinase A inhibitor. In addition, HDL₃ failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL₃-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL₃-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.

Key Words: high-density lipoprotein • protein kinase C • signal transduction • platelet aggregation • fibrinogen

	Introduction

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Both HDL and platelets are intimately involved in the pathogenesis of atherosclerosis, thrombosis, and coronary heart disease.^{1 2 3} Numerous observations demonstrated a direct effect of HDL on platelet functions.⁴ Platelet hyperreactivity was noted in subjects with hypoalphalipoproteinemia.⁵ Decreased activation of platelets by strong agonists such as thrombin and collagen has been reported in the presence of physiological concentrations of HDL.^{6 7 8} Furthermore, HDL inhibited thromboxane A₂ liberation, and addition of HDL to clotting blood diminished platelet thromboxane A₂ formation capacity.^{9 10}

The mechanism underlying the effects of HDL on platelet function is little understood. HDL₃, the major HDL subfraction in blood, is known to interact with specific binding sites on platelets that appear to be identical with GP IIb/IIIa.^{11 12 13 14} In human platelets, HDL₃ was also shown to activate cellular phospholipases, resulting in the formation of DAG.^{15 16 17 18} The involvement of both phosphatidylcholine-specific phospholipase C and phosphatidylcholine-specific phospholipase D was postulated.^{16 17 18} Recently, the PKC-dependent enhancement of the Na⁺/H⁺ antiport in the presence of HDL₃ has been reported.¹⁹ Both HDL₃-induced DAG formation and Na⁺/H⁺ exchange stimulation were markedly decreased after covalent modification of HDL₃ with tetranitromethane or DMS, otherwise known to abolish HDL₃ binding. The relevance of HDL₃ binding and HDL₃-induced cell signaling to the effects of these lipoproteins on platelet functions is not yet entirely clear.

The aim of the present study was to gain further insight into the HDL-platelet interactions. We demonstrate that HDL₃ at physiologically relevant concentrations inhibits thrombin-induced platelet fibrinogen binding and aggregation. The inhibitory effect of HDL₃ is associated with reduced formation of the phosphoinositide-derived second messengers DAG and Ins(1,4,5)P₃, and occurs parallel to the activation of PKC.

	Methods

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Materials
Thrombin, fura 2-AM, tri-n-octylamine, apyrase (grade VIII), DMS, and the authentic lipid standards were from Sigma Chemical Company, Deisenhofen, Germany. Ins(1,4,5)P₃ radioreceptor kit, [³²P]orthophosphoric acid and [³³P]orthophosphoric acid were purchased from NEN-DuPont, Dreieich, Germany. Biotrack cAMP detection kit and [1-¹⁴C]arachidonic acid were from Amersham, Braunschweig, Germany. Silica gel 60 plates and solvents for TLC were obtained from Merck, Darmstadt, Germany. Calphostin C, bis-indolylmaleimide (GF109203X), IBMX, and forskolin were obtained from Calbiochem, Bad Soden, Germany. Highly purified, plasminogen-free fibrinogen was supplied by Enzyme Research Labs. GLY-PRO-ARG-PRO (GPRP) was from Novabiochem, Bad Soden, Germany.

Lipoprotein Isolation
HDL₃ (d=1.125 to 1.210 g/mL) was isolated from human plasma by discontinuous KBr gradients as described by Havel et al.²⁰ The HDL₃ was dialyzed against 0.3 mmol/L Tris-HCl/0.14 mol/L NaCl, pH 7.2, and when necessary concentrated using the Minicon B15 concentrator (Amicon). The apoE content did not exceed 0.2% of the total HDL₃ protein. For oxidative modification, HDL₃ was incubated for 20 hours with 5 µmol/L CuSO₄ as described by Ardlie et al.²¹ In some experiments, HDL₃ was modified with DMS as described by Chaco et al.²²

Platelet Isolation
Venous blood was drawn from healthy volunteers aged 25 to 55 years who had not received any medication during the previous 10 days. The blood was collected into 5-mL tubes (Sarstedt) containing 0.5 mL of 0.1 mol/L trisodium citrate solution and centrifuged at 120g for 10 minutes. The platelet rich plasma was then mixed with ACD (6 mmol/L citric acid, 12 mmol/L trisodium citrate, and 19.4 mmol/L glucose [2:9 vol/vol]). Iloprost (a stable prostacylin analogon) and apyrase (grade VIII) (an adenosine 5'-triphosphatase and an adenosine 5' diphosphatase) were added to final concentrations of 0.01 µmol/L and 0.5 U/mL, respectively. Platelet-rich plasma was centrifuged at 750g for 20 minutes. The platelets were resuspended in a buffer containing (mmol/L) 5 HEPES, 145 NaCl, 5 KCl, 5 glucose, 1.1 NaHCO₃, and 0.35% albumin (wt/vol). For fibrinogen binding experiments, platelets were gel filtered on a Sepharose 2B column (Pharmacia, Uppsala, Sweden) and resuspended in HEPES-Tyrode buffer ([mmol/L]3.5 HEPES, 3 NaH₂PO₄, 5.5 glucose, 1 CaCl₂, 1 MgCl₂, 2.7 KCl, 137 NaCl, and 0.35% albumin [wt/vol], pH 7.4).

Platelet Fibrinogen Binding
Fibrinogen was conjugated with FITC according to the method of Xia et al.²³ The protein/FITC ratio was 1:5. Fibrinogen-FITC was used within 1 week after conjugation. Gel-filtered platelets (5x10⁸/mL) were preincubated for 10 minutes at 37°C with 0.15 g/L fibrinogen-FITC, and with either HDL₃ or with the same volume of buffer. Platelets were then activated with varying concentrations of human -thrombin in the presence of 1.25 mmol/L GPRP (an inhibitor of fibrin polymerization), and the reaction was stopped after 3 minutes by fixation with formaldehyde (0.5% vol/vol, final concentration). After 30 minutes' fixation, the platelets were carefully washed with Isoton II solution (Becton Dickinson, Heidelberg, Germany) and the FITC fluorescence of 5000 platelets was measured using a FACScan instrument (Becton Dickinson, Heidelberg, Germany). Unspecific binding was determined by competition experiments with nonlabeled fibrinogen and with fibrinogen-FITC binding to platelets from patients with Glanzmann thrombasthenia type I. Quantum 26 Premixed (Flow Cytometry Standards Corp) was used for the calibration of the fluorescence signal according to the manufacturer's instructions.

Platelet Aggregation
Platelet aggregation was monitored in the Elvi 611 aggregometer adjusted to its maximal sensitivity as described by Born.²⁴

Expression of CD 62 on the Platelet Surface
Platelets were activated with -thrombin in the presence of 1.25 mmol/L GPRP and fixed as described above. Aliquots of 0.2 mL were incubated with monoclonal antibodies against CD 62 (CLB-thromb/6) or isotype-matched mouse IgG (Coulter) at a final concentration of 5 mg/L. For fluorescence labeling, 0.1 mL sheep anti-mouse F(ab)₂-FITC fragments (62.5 mg/L) was added for a further 30 minutes. Finally, the platelets were washed in PBS and resuspended in Isoton II solution. Fluorescence was measured using a FACScan instrument (Becton Dickinson, Mountain View, Calif).

Determination of Phospholipid Turnover
Inositol phospholipid turnover was determined as described previously.²⁵ Briefly, platelets (1x10⁹/mL) were incubated with [³²P]orthophosphoric acid (0.1 mCi/mL) for 90 minutes at 37°C, washed, and adjusted to 1x10⁹/mL. After adding -thrombin, reactions were stopped at various time points and lipids were extracted according to Bligh and Dyer.²⁶ PtdInsP₂, phosphatidylinositol 4-monophosphate, and PtdOH were separated by TLC on silica gel 60 plates impregnated with 1% (wt/vol) potassium oxalate and 1 mmol/L EDTA using chloroform/acetone/methanol/glacial acetic acid/water (40:15:13:12:7, vol/vol/vol/vol/vol) as a solvent system. The TLC plates were then subjected to autoradiography.

Determination of DAG Formation
Determination of DAG formation was accomplished as described previously.²⁵ Briefly, platelets (1x10⁹/mL) were incubated for 60 minutes with 0.1 µCi/mL [¹⁴C]arachidonic acid at 30°C. After stimulation with -thrombin, the reactions were terminated and radiolabeled lipids were extracted as described above. TLC was performed essentially as described by Gruchalla et al.²⁷ Autoradiography was performed thereafter.

Ins(1,4,5)P₃ Release
After stimulating platelets (1x10⁹/mL) with -thrombin, the reaction was terminated by addition of 1 g/L TCA 1:5 (vol/vol). The precipitate was removed by centrifugation and the water soluble-phase was neutralized using tri-n-octylamine/freon (1:3, vol/vol). Aliquots of 0.1 mL were used for the Ins(1,4,5)P₃ determination. A commercially available Ins(1,4,5)P₃ radioreceptor assay kit (NEN DuPont, Dreieich, Germany) was applied according to the manufacturer's instructions.

Determination of the Intracellular Ca²⁺ Concentration
Intracellular calcium measurements were performed using the Ca²⁺-sensitive fluorescence probe fura 2–AM according to established methods.^{28 29} At concentrations of HDL₃ up to 1.5 g/L, no major quenching effects on the fura 2 fluorescence signal were observed.

Determination of cAMP Formation
For cAMP determination, platelets (10⁹/mL) were incubated for 5 minutes with 10 µmol/L IBMX to prevent cAMP degradation and then stimulated with HDL₃. The reaction was stopped by extraction with ice-cold ethanol (1:2 vol/vol). After pelleting the cellular debris, the extracts were evaporated and redissolved in 0.25 mL distilled water. cAMP was determined in 50-µL aliquots using a commercially available kit (Amersham, Braunschweig, Germany) according to the supplier's instructions.

Determination of the Protein Phosphorylation
Platelets were labeled with 0.1 mCi/mL [³²P]orthophosphoric acid or [³³P]orthophosphoric acid for 90 minutes at 37°C, washed, and adjusted to 1x10⁹/mL. The reactions were terminated by adding 10% TCA (1:1 vol/vol). The precipitate was washed twice and dissolved in 0.2 mL of 0.2 mol/L NaOH. An equal volume of double-concentrated SDS reducing gel sample buffer was added, and the samples were boiled for 5 minutes and electrophoresed by 14% SDS–polyacrylamide gel electrophoresis according to Laemmli.³⁰ Phosphorylated bands were detected by autoradiography. The autoradiograms were then analyzed by densitometry. Alternatively, dried gels were analyzed with a BAS 1500 phosphoimager (Fuji Film) equipped with the Tina 2.0 evaluation program (Raytest).

	Results

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Effect of HDL₃ on Platelet Aggregation
Incubation of platelets with thrombin (0.005 to 0.1 U/mL) caused platelet aggregation in a concentration-dependent manner (not shown). Maximal responses were observed by thrombin concentrations of 0.08 to 0.1 U/mL. As shown in Figure 1, the preincubation of the platelet suspension with HDL₃ for 2 minutes decreased both the rate and the extent of aggregation in a concentration-dependent fashion. In 3 independent experiments, we evaluated the effect of different concentrations of HDL₃ on the extent of platelet aggregation. HDL₃ inhibited the aggregation by 25.9±7.1% (mean±SD), 44.5±5.8% (P<0.05), and 82.3±6.0% (P<0.01) at 0.6, 0.9, and 1.2 g/L, respectively. In the presence of 1.0 g/L oxidized HDL₃ (ox-HDL), thrombin-induced aggregation was inhibited by 62.0±9.9% (P<0.05).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of HDL3 on thrombin-induced platelet aggregation. Washed platelets (5x108/mL) were stimulated with 0.1 U/mL thrombin in the presence of various concentrations of HDL3. Platelet aggregation was monitored in a Born aggregometer. Results are representative of 3 independent experiments.

Effect of HDL₃ on Platelet Fibrinogen Binding
Resting platelets bound 400±100 (n=7) fibrinogen molecules per cell. HDL₃ alone in concentrations up to 2 g/L did not induce fibrinogen binding to the platelet surface. At an HDL₃ concentration of 1.2 g/L, 400±100 (n=3; NS) fibrinogen molecules per platelet were bound. As shown in Figure 2, activation of platelets with thrombin resulted in fibrinogen-FITC binding in a concentration-dependent manner. The binding found at a thrombin concentration of 0.1 U/mL averaged 90 500±9800 fibrinogen molecules (n=7) and was not substantially changed at higher thrombin concentrations. When isolated platelets were preincubated with 1.2 g/L HDL₃ for 5 minutes, the fibrinogen binding to platelets activated with 0.1 U/mL thrombin averaged 17 200±4600 (n=3, P<0.001). Preincubation of platelets with 1.0 g/L ox-HDL and subsequent stimulation with 0.1 U/mL thrombin led to a binding of 15 900±600 fibrinogen molecules per platelet (n=3, NS versus native HDL₃). In contrast to native and oxidized HDL₃, DMS-HDL₃ at a concentration of 1.0 g/L failed to inhibit the thrombin-induced fibrinogen binding in human platelets. Under this experimental condition, a binding of 89 000±7300 fibrinogen molecules per platelet was found (n=3, NS versus thrombin alone).

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of HDL3 on thrombin-induced platelet fibrinogen binding. Gel-filtered platelets (5x108/mL) were activated with 0.02 to 0.2 U/mL thrombin in the presence of 0.15 g/L fibrinogen-FITC and 1.0 g/L HDL3 () or an equal volume of buffer (). The fluorescence of platelet-bound fibrinogen-FITC was measured from 5x103 platelets by using flow cytometry. Results represent mean±SD from 3 independent experiments.

Effect of HDL₃ on the Thrombin-Induced CD 62 Expression on the Platelet Surface
To assess the effect of HDL₃ on granule secretion, expression of CD 62 on the platelet surface was measured using flow cytometry. HDL₃ alone at concentrations up to 2.0 g/L failed to induce the expression of CD 62. The effects of 1.0 g/L HDL₃ on thrombin-induced expression of CD 62 are demonstrated in Figure 3. Preincubation of platelets with HDL₃ for 2 minutes moderately inhibited expression of CD 62 in thrombin-activated human platelets.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of HDL3 on thrombin-induced platelet granule secretion. Gel-filtered platelets (5x108/mL) were activated with 0.02 to 0.1 U/mL thrombin in the presence of 1.0 g/L HDL3 () or an equal volume of buffer (). Activation was stopped after 5 minutes by addition of formaldehyde. After fixation, platelets were incubated with antibodies against CD 62 followed by sheep F(ab)2 anti-mouse IgG-FITC. Expression of CD 62 on the platelet surface was taken as a measure of granule secretion. The results are representative of 2 independent experiments.

Effect of HDL₃ on Thrombin-Induced Inositol Phospholipid Turnover and PtdOH Formation
The physiological concentration of HDL₃ in blood varies between 0.8 and 1.2 g/L, and the inhibitory effects of HDL₃ on platelet aggregation and fibrinogen binding were observed in a similar concentration range. Therefore, 1.0 g/L HDL₃ was chosen as a standard concentration for the study of second-messenger formation. HDL₃ did not influence resting levels of PtdInsP₂. PtdOH resting levels were increased after 2 minutes' incubation with HDL₃ by 38±6% above the resting level (not shown).

Figure 4 shows the time course of the thrombin-induced changes in the ³²P radioactivity associated with PtdOH or PtdInsP₂ in ³²P-labeled platelets in the absence and presence of HDL₃. Addition of thrombin alone (0.1 U/mL) caused a rapid breakdown of endogenous PtdInsP₂. [³²P]PtdInsP₂ decreased by 47% 15 seconds after stimulation. Rapid resynthesis was then initiated, raising PtdInsP₂ levels up to 53% above the resting level at 120 seconds. Preincubation of the platelet suspension with HDL₃ for 2 minutes markedly inhibited PtdInsP₂ turnover. The initial decrease by 28% occurred at 30 seconds and was followed by a slow increase up to 10% above the resting level 120 seconds after stimulation with thrombin. Thrombin alone (0.1 U/mL) induced a progressive formation of [³²P]PtdOH up to 396% above the resting level at 120 seconds. In the presence of HDL₃ the thrombin-induced production of PtdOH was decreased both with respect to the initial velocity and the final extent (up to 202% above the resting level at 120 seconds).

View larger version (19K): [in this window] [in a new window]   Figure 4. Time course of [32P]PtdInsP2 turnover and [32P]PtdOH formation in platelets exposed to thrombin in the presence or absence of HDL3. 32P-labeled platelets (1x109/mL) were preincubated for 2 minutes in the presence () or absence () of 1.0 g/L HDL3 and then stimulated with 0.1 U/mL thrombin. At the indicated times, inositol phospholipids and PtdOH were extracted as described in "Methods." A, [32P]PtdInsP2 turnover. B, [32P]PtdOH formation. Changes in 32P radioactivity are expressed as percentage of controls representing the radioactivity of [32P]phosphatidylinositol 4-monophosphate and [32P]PtdOH in unstimulated platelets. The phospholipid radioactivity in unstimulated platelets did not change over the time course of the experiments. The data are representative of 3 independent experiments.

Effect of HDL₃ on Thrombin-Induced DAG Formation in Platelets
Figure 5 shows the time course of the thrombin-induced changes in the ¹⁴C radioactivity associated with DAG in [¹⁴C]arachidonic acid–labeled platelets after preincubation with or without HDL₃. HDL₃ itself is known to induce a biphasic DAG formation.^{16 17 18} After 2 minutes' incubation with HDL₃, [¹⁴C]DAG resting levels were increased by 36±5%. Addition of 0.1 U/mL thrombin in the absence of HDL₃ caused a rapid formation of DAG reaching 274±47% (n=5) of the resting level 10 seconds after stimulation. The initial increase was followed by a slow and sustained decay approaching 157±30% of the resting level 60 seconds after stimulation. In the presence of 1.0 g/L HDL₃, the thrombin-induced [¹⁴C]DAG formation was markedly inhibited both with respect to the initial extent and subsequent decay (Figure 5). In contrast to native HDL₃, no inhibition of thrombin-induced DAG formation was observed in the presence of 1.0 g/L DMS-HDL₃ (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 5. Time course of [14C]-DAG formation in platelets exposed to thrombin in the presence or absence of HDL3. [14C]Arachidonic acid–labeled platelets (1x109/mL) were preincubated for 2 minutes in the presence of 1.0 g/L HDL3 (), 1.0 g/L DMS-HDL (), or an equal amount of buffer (), and then stimulated with 0.1 U/mL thrombin. At the indicated time, aliquots of the platelet suspension were withdrawn, extracted, and assayed for DAG levels as described in "Methods." Changes in 14C radioactivity associated with DAG are presented as percent of controls (not stimulated with thrombin). DAG radioactivity in buffer-treated controls did not change significantly over the time course of the experiments. DAG radioactivity in HDL3-treated controls was 36±5% higher than in buffer-treated controls. The data represent mean±SD from 5 independent experiments, except in the case of DMS-HDL, in which the mean from 2 independent experiments is given. *P<0.05, **P<0.01, buffer vs HDL3, determined with the Student's t test.

Effect of HDL₃ on Thrombin-Induced Ins(1,4,5)P₃ Formation in Platelets
The effect of HDL₃ on the time-dependent Ins(1,4,5)P₃ formation in response to thrombin was investigated using a commercially available radioreceptor assay. Preincubation of the platelet suspension with 1.0 g/L HDL₃ did not change the resting level of Ins(1,4,5)P₃, which was 18.2±1.5 pmol/10⁹ cells in unstimulated platelets. On stimulation with 0.1 U/mL thrombin, the mass content of Ins(1,4,5)P₃ rapidly increased, peaking within 10 seconds (Figure 6A). The maximal Ins(1,4,5)P₃ content after stimulation was 55.1±8.2 pmol/10⁹ cells. Ins(1,4,5)P₃ returned to nearly basal levels within 30 seconds, and no increase was observed thereafter. In the presence of 1.0 g/L HDL₃, the Ins(1,4,5)P₃ formation was considerably inhibited and delayed. The maximal Ins(1,4,5)P₃ content was noted at 20 seconds, and amounted 21.0±3.1 pmol/10⁹ cells. As shown in Figure 6B, HDL₃ inhibited the thrombin-induced Ins(1,4,5)P₃ formation in a concentration-dependent manner. Ten seconds after activation with 0.1 U/mL thrombin, the Ins(1,4,5)P₃ formation was decreased by 18.9±1.9% (P<0.05), 41.4±1.6% (P<0.01), 48.5±6.3% (P<0.05), and 57.6±6.9% (P<0.01) in the presence of 0.1, 0.25, 0.5, and 1.0 g/L HDL₃, respectively. In contrast, in the presence of 1.0 g/L DMS-HDL, the thrombin-induced Ins(1,4,5)P₃ formation averaged 64.3±6.3 pmol/10⁹ cells (n=3) and was not significantly different from that observed in the absence of HDL₃. Furthermore, a very short preincubation of platelets with HDL₃ (5 seconds) failed to affect the thrombin-induced Ins(1,4,5)P₃ formation, which under this experimental condition amounted to 51.4±5.9 pmol/10⁹ cells (n=3; NS versus thrombin alone).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of HDL3 on thrombin-induced Ins(1,4,5)P3 formation in human platelets. Washed platelets (1x109/mL) were preincubated for 2 minutes in the presence or absence of the indicated concentrations of HDL3 and then stimulated with 0.1 U/mL thrombin. A, Time course of Ins(1,4,5)P3 formation with () or without () HDL3 (1.0 g/L). Values are mean±SD from 3 independent experiments. B, Concentration dependence of HDL3-induced inhibition of Ins(1,4,5)P3 formation. Individual treatments (n=3) were significantly different from respective controls in the absence of HDL3. *P<0.05 and **P<0.01, as determined with Student's t test.

Effect of HDL₃ on Thrombin-Induced Intracellular Calcium Mobilization
The intracellular calcium concentration was measured in fura 2–loaded platelets (1x10⁸/mL). The resting [Ca²⁺]_i level in unstimulated platelets was 49±15.5 nmol/L (n=69) and was not affected by preincubation with 1.0 g/L HDL₃. The effect of HDL₃ on the time-dependent changes in the thrombin-induced release of [Ca²⁺]_i is presented in Figure 7A. The [Ca²⁺]_i level was elevated by 350±85 nmol/L in response to 0.1 U/mL thrombin. Preincubation of platelets for 2 minutes with 0.25, 0.5, and 1.0 g/L HDL₃ reduced the thrombin-induced response to 305±19 nmol/L (n=6), 226±21 nmol/L (n=7, P<0.05), and 118±45 nmol/L (n=23, P<0.001), respectively (Figure 7B). In the presence of DMS-HDL at a concentrations of 1.0 g/L, [Ca²⁺]_i increase averaged 290±21 nmol/L (n=4, NS versus thrombin) (Figure 7A). A short preincubation of platelets with 1.0 g/L HDL₃ (5 seconds) also failed to influence thrombin-induced [Ca²⁺]_i elevation, which under this experimental condition averaged 322±46 nmol/L (n=3). In the absence of extracellular calcium, the inhibitory effect of HDL₃ on thrombin-induced aggregation was also observed. Under this condition, 1.0 g/L HDL₃ reduced the thrombin-induced response to 125±19 nmol/L (n=5).

View larger version (20K): [in this window] [in a new window]   Figure 7. Effect of HDL3 on the thrombin-induced [Ca2+]i increases in human platelets. A, Fura 2–AM loaded platelets (1x108/mL) were preincubated in the presence of 1.0 g/L HDL3, 1.0 g/L DMS-HDL, or an equal amount of buffer and then stimulated with 0.1 U/mL thrombin. Tracings are representative of 3 series of independent experiments. B, Effects of different HDL3 concentrations on [Ca2+]i increases (Ca2+) induced by 0.1 U/mL thrombin. The results are expressed as mean±SD of 6 to 8 determinations. *P<0.05, ***P<0.01 as determined with Student's t test.

Effect of PKA and PKC Inhibition on the HDL₃-Mediated Inhibitory Effects on Platelet Activation
To examine the possible role of PKA and PKC in the inhibitory activity of HDL₃ on platelet activation, the effect on thrombin-induced Ins(1,4,5)P₃ formation and [Ca²⁺]_i release was evaluated in the presence of the PKA inhibitor H89 and 2 highly selective and structurally unrelated PKC inhibitors, calphostin C and bis-indolylmaleimide. All inhibitors failed to affect the increases of Ins(1,4,5)P₃ and [Ca²⁺]_i brought about by 0.1 U/mL thrombin (Table). Moreover, H89 at a concentration of 5 µmol/L failed to affect the inhibitory effect of HDL₃ on Ins(1,4,5)P₃ formation and [Ca²⁺]_i release (Table). By contrast, when platelets were stimulated with thrombin in the presence of 1.0 g/L HDL₃, both 0.2 µmol/L calphostin C and 5 µmol/L bis-indolylmaleimide partially reverted the inhibitory effect of HDL₃ on Ins(1,4,5)P₃ formation and [Ca²⁺]_i release (Table). Furthermore, the reverting effects of bis-indolylmaleimide were concentration dependent and were not seen at the inhibitor concentration of 0.1 µmol/L (Table). These data suggest that the inhibitory effect of HDL₃ on the thrombin-induced platelet activation is associated with the activation of PKC.

Effect of HDL₃ on cAMP Formation
In the next step, we investigated the effect of HDL₃ on the formation of cAMP, which is the physiological activator of PKA. The level of cAMP in unstimulated platelets averaged 2.1±0.7 pmol/10⁸ cells (n=4). Addition of 1.0 g/L HDL₃ to platelets for 1, 2, or 5 minutes failed to change resting cAMP levels (Figure 8). By contrast, a significant production of cAMP was observed after treatment of platelets with 10 µmol/L forskolin, which is a direct stimulator of the adenylate cyclase.

View larger version (33K): [in this window] [in a new window]   Figure 8. Effect of HDL3 on cAMP formation in human platelets. Washed platelets (1x109/mL) were preincubated with 10 µmol/L IBMX for 5 minutes and then stimulated with 1.0 g/L HDL3 for the times indicated or with 10 µmol/L forskolin for 2 minutes. cAMP was extracted and analyzed as described in "Methods." Data represent mean±SD of 3 to 4 separate experiments. *P<0.002 vs resting level as determined by Student's t test.

Effect of HDL₃ on PKC 40- to 47-kD Major Protein Substrate Phosphorylation
To further study the role of PKC in HDL₃-platelet interactions, we examined the phosphorylation of the 40- to 47-kD major protein substrate, which is considered to be a marker of PKC activation in platelets.³¹ Incubation of platelets with 1.0 g/L HDL₃ for 2 minutes resulted in the phosphorylation of the 43-kD protein (Figure 9A). The phosphorylation was concentration dependent (Figure 9B) and at the HDL₃ concentration of 1.0 g/L approached 45% to 50% of that brought about by 1 µmol/L phorbol myristate acetate. Incubation of platelets with HDL₃ for various times revealed that maximal phosphorylation of the 40- to 47-kD major protein substrate occurred within 1 minute after stimulation and decreased thereafter (Figure 9C), indicating that the HDL₃-induced phosphorylation of the PKC 40- to 47-kD major protein substrate is a transient event. Both calphostin C at a concentration of 0.2 µmol/L and bis-indolylmaleimide at concentrations of 5 µmol/L and 1 µmol/L completely inhibited HDL₃-induced phosphorylation (Figure 9D). By contrast, the phosphorylation was unaffected by bis-indolylmaleimide at a concentration of 0.1 µmol/L (Figure 9D). In 2 separate experiments, 1.0 g/L DMS-HDL failed to evoke the phosphorylation of 40- to 47-kD major protein substrate (not shown).

View larger version (43K): [in this window] [in a new window]   Figure 9. HDL3-induced phosphorylation of PKC 40- to 47-kD major protein substrate. 32P-labeled or 33P-labeled platelets (1x109 /mL) were stimulated with the indicated concentrations of HDL3. At various time points, the reactions were stopped with an equal volume of 10% TCA. The precipitate was sedimented, resuspended in a reducing sample buffer, and separated by 14% SDS-polyacrylamide gel electrophoresis. A, Autoradiography of the dried gel. Lane 1, 1.0 g/L HDL3; lane 2, unstimulated platelets. B, Concentration dependence of HDL3-induced phosphorylation of the PKC 40- to 47-kD major protein substrate, as determined by densitometry of the autoradiographed gels. The bar represents the phosphorylation of the 43-kD band brought about by 0.01 mmol/L phorbol myristate acetate in the same experiment. The phosphorylation in unstimulated platelets was set as 100%. The results are representative of 2 experiments. C and D, Effect of various incubation times with 1.0 g/L HDL3 and effects of bis-indolylmaleimide (Bis) or calphostin C on the phosphorylation of the PKC 40- to 47-kD major protein substrate, as determined by densitometry or phosphoimaging. The phosphorylation in unstimulated platelets was set as 100%. The results are mean±SD of 3 to 10 separate experiments.

Impact of Preincubation Time on HDL₃-Dependent Inhibitory Effects
From the experiments described above, the assumption arises that the HDL₃-dependent inhibitory effects should be time dependent, reaching maximum 1 to 2 minutes after stimulation and diminishing after a longer contact of platelets with HDL_3. To test this possibility, we examined the effect of various preincubation times with 1.0 g/L HDL₃ on the thrombin-induced Ins(1,4,5)P₃ formation and Ca²⁺ mobilization. The results shown in Figure 10 demonstrate that the HDL₃-dependent inhibitory effects peak 1 minute after stimulation and attenuate when platelets are incubated with HDL₃ for longer times. After 0.5, 1, 5, 10, and 30 minutes, the thrombin-induced Ins(1,4,5)P₃ increases averaged 42.3±6.9 pmol/10⁹ cells (n=4, P<0.05 versus thrombin alone), 22.3±4.6 pmol/10⁹ cells (n=4, P<0.002 versus thrombin alone), 36.1±1.9 pmol/10⁹ cells (n=5, P<0.05 versus thrombin alone), 47.3±2.5 pmol/10⁹ cells (NS versus thrombin alone), and 49.7±3.6 pmol/10⁹ cells (NS versus thrombin alone), whereas [Ca²⁺]_i increases were 219±31 nmol/L (n=8, P<0.01 versus thrombin alone), 86±26 nmol/L (n=3, P<0.001 versus thrombin alone), 256±26 nmol/L (n=6, P<0.02 versus thrombin alone), and 292±23 nmol/L (n=6, NS versus thrombin alone), respectively. After similar preincubation times, the thrombin-induced fibrinogen binding averaged 26 300±4200 (n=3, P<0.001), 12 700±2100 (n=3, P<0.001 versus thrombin), 46 500±3800 (n=3, P<0.001 versus thrombin), 65 500±4100 (n=3, P<0.01 versus thrombin), and 72 200±8900 (n=3, P<0.01 versus thrombin) bound molecules per platelet, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 10. Influence of the incubation time on the HDL3-induced inhibition of Ins(1,4,5)P3 formation, [Ca2+]i mobilization, and fibrinogen binding. Washed platelets (1x109/mL) were preincubated for the indicated times with 1.0 g/L HDL3 and then stimulated with 0.1 U/mL thrombin. Ins(1,4,5)P3 formation (A), Ca2+ mobilization (B), and fibrinogen binding (C) were determined as described in "Methods." The results are given as mean±SD of the relative inhibition and are representative of at least 3 independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
There are many critical biochemical events of platelet activation that could potentially be affected by HDL₃. The rapid turnover of inositol phospholipids is a key event occurring subsequent to the stimulation of platelets by strong agonists such as thrombin.³² Activation of PI-PLC as a consequence of agonist-receptor binding results in the generation of 2 PtdInsP₂-derived intracellular messengers, Ins(1,4,5)P₃ and DAG, accompanied by elevation of the intracellular calcium concentration. This ultimately leads to the binding of fibrinogen and aggregation. Agents interfering with PI-PLC–mediated PtdInsP₂ breakdown should therefore have a profound impact on platelet activation. In fact, several agents have been shown to exert their proaggregatory or antiaggregatory activities via modulation of PtdInsP₂ turnover.^{33 34 35 36} In this study, we examined the effect of HDL₃ on the thrombin-induced PtdInsP₂ breakdown and on the formation of the major PI-PLC products, Ins(1,4,5)P₃ and DAG. Thrombin was chosen as an agonist because of its well-characterized action on second-messenger formation. In addition, thrombin appears to play a crucial role in the pathogenesis of arteriosclerosis and thrombosis.^{37 38} We herein demonstrate that the inhibition of the thrombin-induced platelet aggregation by HDL₃ is accompanied by a decelerated turnover of PtdInsP₂, a decreased formation of Ins(1,4,5)P₃ and phosphatidylinositol-derived DAG, as well as a reduced [Ca²⁺]_i mobilization. These effects are concentration dependent and occur within a time period required for HDL₃-mediated inhibition of aggregation and fibrinogen binding. We conclude that the antiaggregatory effect exerted by HDL₃ is mediated via inhibition of InsPtdP₂-derived DAG and Ins(1,4,5)P₃ formation, most likely due to the inhibition of PI-PLC.

In addition to the inhibition of PI-PLC, the preincubation of platelets with HDL₃ induces the inhibition of thrombin-induced fibrinogen binding. At 1.0 g/L HDL₃, the inhibition of both responses was found to be maximal. Previously, it has been demonstrated that the rate and extent of the platelet aggregation is determined by the number of bound fibrinogen molecules.^{39 40} Thus, decreased fibrinogen binding observed in the presence of HDL₃ provides a plausible explanation for the HDL₃-induced inhibition of aggregation seen in this and other studies.^{6 7 8} Furthermore, despite excessive inhibition of platelet fibrinogen binding and aggregation, HDL₃ only moderately influenced the thrombin-induced expression of CD 62 on the platelet surface. Hence, the HDL₃-mediated inhibition of aggregation cannot solely reflect the effects exerted by HDL₃ on the platelet secretory responses. In a recent study, van Willigen at al⁴¹ observed no inhibition of ADP-induced platelet fibrinogen binding in the presence of HDL. This apparent discrepancy is not surprising, taking into account that PI-PLC does not appear to play a major role in the ADP-induced platelet activation.^{42 43 44 45}

Oxidative modification of lipoproteins, which may occur in vivo or during isolation, is thought to change their physicochemical and biological properties. If the inhibitory activity of HDL₃ is a consequence of oxidation, one would expect in vitro–oxidized HDL₃ to act on platelet activation more potently than native HDL₃. However, ox-HDL inhibited thrombin-induced fibrinogen binding to the same extent as unmodified lipoproteins, and even slightly decreased antiaggregatory activity of ox-HDL was noticed. Thus, the inhibitory effect of HDL₃ on platelet activation does not seem to arise as a consequence of oxidative modification. This contention is further supported by the experiments of Ardlie et al,²¹ who observed a proaggregatory rather than an antiaggregatory effect of total HDL after in vitro oxidation.

By which mechanism does HDL₃ inhibit the thrombin-induced PI-PLC activation and, in consequence, fibrinogen binding and aggregation? The direct inhibition of thrombin binding by HDL₃ is unlikely, as no inhibitory effect could be observed after short preincubation of platelets with HDL₃. Moreover, the observed reversal of HDL₃-induced PI-PLC inhibition by PKC inhibitors would not be expected if HDL₃ influenced platelet activity by inhibition of thrombin binding. Our finding that chemically modified HDL₃ does not inhibit thrombin-induced PI-PLC activation argues against the notion that platelet reactivity is reduced due to unspecific effects of HDL₃ on the physical state of the platelet membrane and strongly suggests the involvement of intracellular events. Previously, several groups demonstrated that both PKA and PKC inhibit formation of Ins(1,4,5)P₃ and DAG, as well as [Ca²⁺]_i mobilization in platelets.^{32 46 47 48 49 50} HDL₃ has been shown to trigger cAMP production in fibroblasts and to activate PKC in fibroblasts and adipocytes.^{51 52 53} However, no accumulation of cAMP was found in the present study after incubation of platelets with HDL₃ for various times. Furthermore, the inhibitor of PKA H89 failed to revert the HDL₃-induced inhibition of thrombin-stimulated Ins(1,4,5)P₃ formation and Ca²⁺ mobilization. Therefore, it appears unlikely that the effects of HDL₃ on platelet activation are mediated by PKA. On the other hand, we demonstrated that the transient phosphorylation of the 40- to 47-kD major protein substrate of PKC was brought about by incubation of platelets with native HDL₃ but not with DMS-HDL. The HDL₃-induced PKC activation closely paralleled the HDL₃-induced inhibition of Ins(1,4,5)P₃ formation and Ca²⁺ release. Calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated PKC inhibitors, completely inhibited HDL₃-induced phosphorylation of the 40- to 47-kD major protein substrate of PKC and simultaneously abrogated the HDL₃-induced inhibition of thrombin-induced PI-PLC activity. Furthermore, the reverting effect of bis-indolylmaleimide was seen only at concentrations at which inhibition of the HDL₃-induced phosphorylation was observed. Finally, the formation of the physiological PKC activator DAG, in response to native but not modified HDL₃, has been previously demonstrated,^{15 16 17 18} and another activator of PKC, lysophosphatidylcholine,⁵⁴ is intrinsically present on HDL₃ due to the activity of lecithin:cholesterol acyltransferase. Taken together, these findings suggest that the inhibition of platelet activation by HDL₃ occurs parallel to the HDL₃-induced PKC activation. The negative-feedback inhibition exerted by PKC over PI-PLC–mediated PtdInsP₂ breakdown may represent a plausible explanation for this phenomenon.

Under in vivo conditions, the PKC 40- to 47-kD major protein substrate is not in the phosphorylated state, although plasma HDL₃ concentrations are similar to those used in this study. Our results suggest that the HDL₃-induced PKC activation and consequently the inhibition of PI-PLC are transient events, which undergo desensitization at longer incubation times. Likewise, the inhibitory effect of HDL₃ on thrombin-induced fibrinogen binding decreased at longer incubation times, further supporting a role of PKC in this process. Our findings are in accordance with the results of Nazih et al,¹⁵ who observed a PKC-dependent desensitization of DAG formation in human platelets stimulated with physiologically relevant HDL₃ concentrations. Since the effect of HDL₃ on platelets is rapidly reversible, the relevance of the present results to physiological or pathophysiological situations in which platelets are constantly exposed to HDL remains unclear.

In summary, HDL₃ at physiologically relevant concentrations inhibits in vitro thrombin-induced platelet fibrinogen binding and aggregation. The HDL₃-induced inhibition of platelet function is associated with the inhibition of DAG and Ins(1,4,5)P₃ production and is paralleled by PKC activation.

	Selected Abbreviations and Acronyms

 

DAG = diacylglycerol DMS = dimethylsuberimidate IBMX = 3-isobutyl-1-methylxanthine Ins(1,4,5)P3 = inositol 1,4,5-tris-phosphate NS = not significant ox-HDL = oxidized HDL PI-PLC = phosphoinositide-specific phospholipase C PKA = protein kinase A PKC = protein kinase C PtdInsP2 = phosphatidylinositol 4,5-bis-phosphate PtdOH = phosphatidic acid TCA = trichloroacetic acid

	Acknowledgments

 
J.-R.N. was a recipient of a fellowship from the Deutscher Akademischer Austauschdienst (DAAD). The authors thank Dr Paul Cullen for the perusal of the manuscript and most helpful discussion. The excellent technical assistance of Diana Höhne is gratefully acknowledged.

Received January 27, 1997; accepted November 11, 1997.

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