Abstract

Objective: Lipoprotein (a) [Lp(a)] is considered an atherogenic lipoprotein, which is also implicated in thrombosis. Lp(a) binds to platelets and modulates the effects of various platelet agonists. Platelet activating factor (PAF) is a potent platelet agonist degraded and inactivated by PAF-acetylhydrolase (PAF-AH), which in plasma is associated with lipoproteins. Lp(a) is enriched in PAF-AH, thus a functional characteristic of this lipoprotein is its capability to hydrolyze and inactivate PAF. In the present study, we investigated the effect of Lp(a) on PAF-induced platelet activation. The potential roles of the apo(a) moiety and especially of the PAF-AH content of Lp(a) on the above effect were also addressed. Methods: Lp(a) was isolated by affinity chromatography from plasma of apparently healthy fasting donors with serum Lp(a) concentrations ≥20 mg/dl. Reduced Lp(a) [Lp(a-)] was prepared by incubation of Lp(a) with dithiothreitol (DTT), whereas inactivation of Lp(a)-associated PAF-AH was performed by incubation of Lp(a) with pefabloc [pefa-Lp(a)]. Platelet-rich plasma (PRP) or washed platelets were prepared from peripheral venous blood samples of normolipidemic, apparently healthy fasting donors with their serum Lp(a) levels lower than 0.8 mg/dl. The surface expression of the platelet integrin-receptor αIIbβ3 and the fibrinogen binding to the activated αIIbβ3 was studied by flow cytometry. Results: Lp(a), at doses higher than 20 μg/ml, inhibits PAF-induced platelet activation in a dose-dependent manner. Pefa-Lp(a), lacking PAF-AH activity, exhibited a similar to Lp(a) inhibitory effect. Importantly, the Lp(a) inhibitory effect was not influenced by the apo(a) isoform size, whereas Lp(a-) was a more potent inhibitor compared to Lp(a). Similarly to PAF, Lp(a) inhibits platelet aggregation induced by ADP or Calcium ionophore A23187. Lp(a), pefa-Lp(a) or Lp(a-) effectively inhibited PAF- or ADP-induced surface expression of αIIbβ3, the Lp(a-) being more potent compared to Lp(a) or to pefa-Lp(a). Finally, Lp(a) significantly inhibited fibrinogen binding to platelets activated with PAF. Conclusions: Lp(a) inhibits PAF-induced platelet activation in a non-specific manner. The Lp(a)-associated PAF-AH does not play any important role in this effect, whereas the apo(a) moiety of Lp(a) significantly reduces its inhibitory effect. The inhibition of αIIbβ3 activation and fibrinogen binding to the activated platelets may represent the major mechanism by which Lp(a) inhibits PAF-induced platelet aggregation.

1. Introduction

Lipoprotein (a) [Lp(a)] is composed of a low density lipoprotein (LDL)-like particle to which a large, highly glycosylated apolipoprotein (a) [apo(a)] is linked by a disulfide bond [1]. Several studies have demonstrated a significant correlation between increased plasma levels of Lp(a) (>20 to 30 mg/dl) and coronary artery disease as well as stroke [2–5]. Lp(a) is considered an atherogenic lipoprotein, which is also implicated in thrombosis, although the underlyning mechanisms remain incompletely understood [6]. Early studies showed that Lp(a) plays a potential role in thrombogenesis, interfering with several steps in the fibrinolytic pathway [7,8]. In this regard, more recent studies have demonstrated that Lp(a) binds to platelets, although there are conflicting reports as to which platelet protein or Lp(a) component plays a major role in this interaction [9–12]. Through its interaction with platelets, Lp(a) may impair platelet-mediated fibrinolysis by reducing the binding of both plasminogen and t-PA to the platelet surface and by increasing the Km values of t-PA for plasminogen [13]. In addition, intact Lp(a) or its apo(a) moiety may modulate the effects of various platelet agonists. In this context, there are marked differences concerning the results from various studies, which have examined the effects of Lp(a) on platelet activation induced by various agonists. Thus, it has been shown that intact Lp(a) does not influence platelet activation induced by collagen or thrombin [14]. Moreover, other studies demonstrated that intact Lp(a) inhibits collagen-induced platelet aggregation [10,12,15] or ADP-induced platelet aggregation [10]. Furthermore, it has been shown that intact Lp(a) or recombinant apo(a) promotes the aggregation response of platelets to subaggregant doses of arachidonic acid, whereas recombinant apo(a) does not affect the platelet response to low doses of collagen or thrombin [11]. Finally, intact Lp(a) or recombinant apo(a) increases thrombin receptor-activating hexapeptide (SFLLRN, TRAP)-induced platelet activation, whereas they do not affect ADP- or thrombin-induced platelet activation [16].

Platelet activating factor (PAF) is a proinflammatory phospholipid mediator and a potent platelet agonist [17]. PAF is degraded and inactivated by PAF-acetylhydrolase (PAF-AH), an enzyme that exhibits a Ca2+-independent phospholipase A2 activity and in plasma, it is associated with lipoproteins [18]. We have previously shown that Lp(a) is enriched in PAF-AH, thus, a functional characteristic of this lipoprotein is its capability to hydrolyze and inactivate PAF [19]. According to our knowledge, there is a paucity of data concerning the effect of Lp(a) on PAF-induced platelet activation. This information could be of particular interest, since unlike other platelet agonists, PAF-induced platelet activation could be influenced not only through the apo(a) moiety or the LDL-like component of Lp(a), but also via its PAF-AH content. Thus, in the present study, we investigated the effect of Lp(a) on PAF-induced platelet activation. The potential roles of the apo(a) moiety and especially of the PAF-AH content of Lp(a) on the above effects were also addressed.

2. Methods

2.1. Isolation of Lp(a)

Lp(a) was prepared from EDTA-containing plasma samples of apparently healthy fasting donors with serum Lp(a) concentrations ≥20 mg/dl. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–4). Lp(a) was isolated by affinity chromatography using a Lysine-Sepharose 4B column, as previously described [20]. The purity of isolated Lp(a) was evaluated by agarose gel electrophoresis (Hydradel Lipo and Lp(a) kit, Sebia), gradient SDS-PAGE in 5–19% SDS-gradient polyacrylamide gels and by double rocket electrophoresis. Lp(a) was extensively dialyzed against 10 mM PBS containing 0.01% EDTA, pH 7.4, and stored at 4 °C under nitrogen for up to 2 weeks. Before use in experiments with platelets, Lp(a) was dialyzed overnight in 10 mM PBS to remove EDTA. No oxidation was observed in each Lp(a) preparation, as it was revealed by agarose gel electrophoresis and the determination of the TBARS values (0.9±0.4 malondialdehyde equivalents per mg of protein for all lipoprotein preparations). In some experiments, we pre-incubated Lp(a) with 1 mM pefabloc for 30 min at 37 °C, a procedure that completely and irreversibly inactivates the endogenous PAF-AH [21]. The pefabloc-treated Lp(a) [pefa-Lp(a)] was stored as above and it was extensively dialyzed in 10 mM PBS before use.

2.2. Reductive cleavage of Lp(a)

Lp(a) containing 600 μg protein/ml HEPES was submitted to reductive cleavage by incubation with 10 mM dithiothreitol (DTT) at 37 °C for 3 h [22], a procedure that completely dissociates apo(a) from Lp(a) [23]. Reduced Lp(a) [Lp(a-)] was then fractionated by density gradient ultracentifugation as previously described [19]. After ultracentrifugation, 28 fractions of 400 μl were collected and analyzed for their content in cholesterol and PAF-AH activity [19]. Subsequently, fractions 11–15 containing Lp(a-) were pooled, and used in platelet activation studies after an overnight dialysis in 10 mM PBS. The Lp(a-) preparations were characterized by agarose gel electrophoresis and by 3.75% SDS-PAGE followed by immunoblotting as described previously [19]. In some experiments, unreduced Lp(a) [(Lp(a) incubated in the absence of DTT] was also submitted to ultracentrifugation and used as a control.

2.3. Preparation of platelet-rich plasma

Platelet-rich plasma (PRP) was prepared from peripheral venous blood samples of normolipidemic, apparently healthy fasting donors with serum Lp(a) levels lower than 0.8 mg/dl, as previously described [24]. ACD solution, consisting of 0.07 M citric acid, 0.11 M sodium citrate and 0.11 M D(+) glucose was used as an anticoagulant. The platelet count of PRP was adjusted to a final platelet concentration of 2.5×108/ml with homologous plasma.

2.4. Preparation of washed platelets

Peripheral venous blood samples were collected from the same donors using ACD as an anticoagulant. Washed platelets were prepared over an erythrocyte pellet as previously described by Lotner et al. [25] with some modifications. Briefly, the anti-coagulated blood was centrifuged at 3900×g for 5 min at room temperature to remove plasma. The pelleted cells were then resuspended in Tyrode's pH 7.0 and washed twice using identical centrifugations at 650×g for 10 min. The pelleted cells were then resuspended in Tyrode's buffer pH 7.4 and centrifuged at 120×g for 10 min at room temperature to obtain the washed platelet suspension. The platelet count was adjusted to a final platelet concentration of 2.5×108/ml.

2.5. Aggregation studies

Platelet aggregation studies in either PRP or washed platelets were performed in aliquots of 0.5 ml, in a platelet aggregometer (model 560, Chronolog) at 37 °C, with continuous stirring at 1200 rpm. Platelets were pre-incubated at 37 °C with the various Lp(a) preparations for several time intervals up to 30 min before the initiation of aggregation. The maximal aggregation, achieved within 3 min after the addition of the agonist, was determined and expressed as a percentage of 100% light transmission calibrated for each specimen (maximal percentage of aggregation) [24]. In some experiments with PRP, platelets were pre-incubated with aspirin (0.1 mM) for 15 min before the addition of Lp(a). In these experiments, a combination of creatine phosphate (CP) and creatine kinase (CPK) (4 mM and 40 U ml−1, respectively) was added 1 min before PAF-induced aggregation [24]. In experiments with washed platelets, fibrinogen (0.5 mg/ml) was added to the samples, 30 s before addition of the agonist.

2.6. Secretion studies

The effect of Lp(a) on PAF-induced ATP (a platelet dense granule component) secretion was studied. ATP secretion was determined in parallel to platelet aggregation in aliquots of 0.45 ml PRP along with 50 μl of Luciferin/Luciferase (to measure ATP secretion, Chrono-lume reagent, Chronolog) [26], in a platelet aggregometer (model 560, Chronolog) at 37 °C, with continuous stirring at 1200 rpm.

Effect of Lp(a) on the expression of the platelet integrin αIIbβ3

The expression of the platelet integrin-receptor αIIbβ3 was studied by flow cytometry in washed platelets or PRP using the FITC-labeled specific monoclonal antibody PAC-1 (Becton Dickinson, San Jose, CA) as previously described [27]. Briefly, platelets (2.5×108/ml) were incubated with 30 ng/ml of PAC-1 in the absence or in the presence of Lp(a), pefa-Lp(a) or Lp(a-) for 10 min prior to activation with PAF (80 nM final concentration). Activation was performed for 10 min at 37 °C without stirring. This incubation time is necessary to obtain the maximum PAC-1 binding under our experimental conditions. Platelets were then diluted with PBS (1:5 v/v) and immediately analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson) [28]. To quantify non-specific binding, control samples containing FITC-conjugated IgG were also assayed. In some experiments, the effect of Lp(a) or Lp(a-) on ADP-induced PAC-1 binding to platelets in PRP was studied with the same method, using 100 μM ADP final concentration [28]. Finally, in control experiments, the peptide RGDS (1 mM final concentration), instead of Lp(a), was added to platelets prior to activation.

2.8. Effect of Lp(a) on fibrinogen binding

The effect of Lp(a) on the binding of FITC-labelled fibrinogen (FITC-Fg) to platelets in PRP was studied by flow cytometry, as previously described [28]. Fluorescein labelling of fibrinogen was performed as previously described [29]. PRP with platelet number ranging from 2.5×108/ml to 4.5×108/ml was diluted 10-fold with Walsh-albumin buffer. Diluted PRP was then mixed with FITC-Fg (500 nM final concentration), in the presence or absence of Lp(a). Platelet activation was performed with 80 nM PAF at room temperature for 60 min in the dark without stirring. This incubation time is necessary to obtain the maximum FITC-Fg binding under our experimental conditions. Then platelets were immediately analyzed by flow cytometry, using 10,000 cell events. The mean fluorescence intensity (MFI) values for both the non-activated and activated platelets, in the presence or absence of Lp(a), were calculated. The MFI values of non-activated platelets, in the presence or absence of Lp(a) (non-specific binding), were subtracted from those obtained after platelet activation (total binding), respectively, thus obtaining the specific binding of FITC-Fg [30]. To quantify non-specific binding, control samples containing FITC-conjugated albumin were also assayed. The effect of 1 mM RGDS on FITC-Fg binding to activated platelets was also studied using the same procedure. Numeric data were processed with the Cellquest software (Becton-Dickinson).

2.9. Biochemical determinations

PAF-AH activity in Lp(a) preparations was measured by the trichloroacetic acid precipitation procedure using 1-O-hexadecyl-2-[3H-acetyl]-sn-glycero-3-phosphocholine [3H]-PAF (10 Ci/mmol; DuPont-New England Nuclear, Boston, MA, USA) as a substrate and 5 μg protein of Lp(a) as the source of the enzyme [31]. Plasma Lp(a) levels were measured by a sandwich enzyme immunoassay method (Macra Lp(a), Terumo Medical, Elkton, MD, USA) [32]. The apo(a) isoform size of the various Lp(a) preparations was determined in total plasma prior to the isolation of Lp(a), by agarose gel electrophoresis and expressed as number of K4 repeats, as previously described [4]. The cholesterol content of Lp(a) was analyzed by commercially available enzymatic reagent (BioMerieux), whereas the Lp(a) protein content was measured by the bicinchoninic acid (BCA) method (PIERCE).

2.10. Statistical analysis

Results are expressed as mean±S.D. and were compared using analysis of variance (ANOVA) followed by the least significant difference test (LSD). Statistically significant values were defined at a value of p<0.05.

3. Results

3.1. Effect of Lp(a) on PAF-induced platelet aggregation in PRP

The effect of Lp(a) on PAF-induced platelet aggregation was first studied in PRP from normolipidemic, apparently healthy volunteers in which Lp(a) levels were lower than 0.8 mg/dl. The PRP was initially incubated in the absence or in the presence of various Lp(a) concentrations (10 to 80 μg protein/ml) for 1 min at 37 °C and then platelet aggregation was initiated by 25 nM PAF [a concentration that induces an irreversible platelet aggregation with a maximal percentage of aggregation of about 80%, in the absence of Lp(a)]. As shown in Fig. 1A, Lp(a) inhibits PAF-induced platelet aggregation in a dose-dependent manner. Similar results were obtained when PRP was pre-incubated with Lp(a) for 5, 10 or 30 min before the initiation of the aggregation (data not shown). Typical aggregation curves illustrating the dose-dependent inhibitory effect of Lp(a) are illustrated in Fig. 1B. An inhibition by Lp(a) of the secondary aggregation, which is due to the effect of other agonists secreted from activated platelets, is also evident in this figure. Thus, in order to determine the specific effect of Lp(a) on PAF-induced platelet aggregation, with respect to the primary aggregation, we pre-incubated PRP with CP/CPK (a scavenger of ADP produced during platelet activation) as well as with aspirin, which inhibits the agonist-induced formation of thromboxane A2[33]. Activation was initiated with 25 nM PAF, a concentration, which induced a maximal percentage of aggregation of about 40% in the presence of aspirin and CP/CPK. All aggregation curves were monophasic and reversible. Under these conditions, Lp(a) inhibited platelet aggregation in a dose-dependent manner (Fig. 1A). Typical aggregation curves are illustrated in Fig. 1C. One of the platelet components secreted during PAF-induced platelet aggregation is ADP and this agonist significantly contributes to the platelet secondary aggregation. Thus, we next studied the effect of Lp(a) on ADP-induced platelet aggregation. Platelet aggregation was initiated by 5 μM ADP, a concentration that induced a maximum percentage of aggregation of about 70%. All aggregation curves were irreversible. As shown in Fig. 2, Lp(a) significantly inhibits ADP-induced platelet aggregation in a dose-dependent manner. Similar results were obtained when PRP was pre-incubated with Lp(a) for 5, 10 or 30 min before the initiation of the aggregation (data not shown). Finally, we studied the effect of Lp(a) on platelet aggregation induced by 5 μM Calcium ionophore A23187, a concentration that induces an irreversible platelet aggregation with a maximum percentage of aggregation of about 50%. Lp(a), at a concentration of 40 μg protein/ml, inhibited platelet aggregation by 61±12%.

Fig. 2

Effect of Lp(a) on ADP-induced platelet aggregation in PRP. Platelets in PRP were incubated with various concentrations of Lp(a) in 37 °C for 1 min under stirring and then platelet aggregation was initiated with the addition of 5 μM ADP. Values represent the mean±S.D. from four different platelet and Lp(a) preparations.

Fig. 2

Effect of Lp(a) on ADP-induced platelet aggregation in PRP. Platelets in PRP were incubated with various concentrations of Lp(a) in 37 °C for 1 min under stirring and then platelet aggregation was initiated with the addition of 5 μM ADP. Values represent the mean±S.D. from four different platelet and Lp(a) preparations.

Fig. 1

Effect of Lp(a) on PAF-induced platelet aggregation in PRP. (A) Bar-graph showing the % inhibition of platelet aggregation in the absence (open bars) or in the presence of aspirin and CP/CPK (closed bars). Values represent the mean±S.D., from four different platelet and Lp(a) preparations. (B) Representative aggregation curves illustrating the dose-dependent inhibitory effect of Lp(a) on platelet aggregation induced by 25 nM PAF. (C) Representative aggregation curves illustrating the dose-dependent inhibitory effect of Lp(a) on platelet aggregation induced by 25 nM PAF, in the presence of aspirin and CP/CPK.

Fig. 1

Effect of Lp(a) on PAF-induced platelet aggregation in PRP. (A) Bar-graph showing the % inhibition of platelet aggregation in the absence (open bars) or in the presence of aspirin and CP/CPK (closed bars). Values represent the mean±S.D., from four different platelet and Lp(a) preparations. (B) Representative aggregation curves illustrating the dose-dependent inhibitory effect of Lp(a) on platelet aggregation induced by 25 nM PAF. (C) Representative aggregation curves illustrating the dose-dependent inhibitory effect of Lp(a) on platelet aggregation induced by 25 nM PAF, in the presence of aspirin and CP/CPK.

3.2. Effect of Lp(a) on PAF-induced ATP secretion

To further investigate the inhibitory effect of Lp(a) on the PAF-induced secondary wave of aggregation, we studied the effect of Lp(a) on ATP secretion (a marker of dense granule secretion) from activated platelets. PAF at a concentration of 25 nM induced a secretion of 1.9±0.4 nmol ATP. Lp(a) at doses higher than 20 μg/ml significantly inhibited ATP secretion, at a dose-dependent manner. This inhibition reached the 100% at an Lp(a) concentration of 80 μg protein/ml.

3.3. Effect of Lp(a) on PAF-induced washed platelet aggregation

In order to investigate the role of the Lp(a)-associated PAF-AH on the inhibitory effect of this lipoprotein on PAF-induced platelet activation, we performed studies with washed platelets, i.e. in the absence of plasma which is enriched in PAF-AH activity, mainly associated with LDL [18]. In these experiments, we used Lp(a) pre-incubated with pefabloc [pefa-Lp(a)], a treatment that completely and irreversibly inhibits the endogenous PAF-AH activity [21]. PAF, at a concentration of 10 nM, induced a maximal percentage of aggregation of about 50%. Under these conditions, Lp(a) (containing PAF-AH activity of 13±5 nmol/mg protein/min) at a concentration higher than 20 μg protein/ml, significantly inhibited PAF-induced washed platelet aggregation. Surprisingly, pefa-Lp(a), lacking PAF-AH activity, exhibited a similar inhibitory effect to that of Lp(a) with active PAF-AH (Fig. 3).

Fig. 3

Role of endogenous PAF-AH and apo(a) on Lp(a)-mediated inhibition of PAF-induced platelet aggregation. Washed platelets (2.5×108/ml) were incubated with Lp(a) (expressing PAF-AH activity of 13±5 nmol/mg protein/min) (open bars) or pefa-Lp(a) (inactive PAF-AH) (gray bars) or Lp(a-) (closed bars) at 37 °C for 1 min under stirring. Fibrinogen, 0.5 mg/ml was added 30 s prior to aggregation with 10 nM PAF. Values represent the mean±S.D. from three different experiments. *p<0.03 compared to either Lp(a) or pefa-Lp(a).

Fig. 3

Role of endogenous PAF-AH and apo(a) on Lp(a)-mediated inhibition of PAF-induced platelet aggregation. Washed platelets (2.5×108/ml) were incubated with Lp(a) (expressing PAF-AH activity of 13±5 nmol/mg protein/min) (open bars) or pefa-Lp(a) (inactive PAF-AH) (gray bars) or Lp(a-) (closed bars) at 37 °C for 1 min under stirring. Fibrinogen, 0.5 mg/ml was added 30 s prior to aggregation with 10 nM PAF. Values represent the mean±S.D. from three different experiments. *p<0.03 compared to either Lp(a) or pefa-Lp(a).

We next asked whether the apo(a) moiety of Lp(a) could play any role in the inhibitory effect of Lp(a). Initially, we investigated whether the length of the apo(a) influences the Lp(a) inhibitory effect. In these experiments, we used Lp(a) preparations containing low ≤21K4 or high ≥27K4 apo(a) size. No significant differences were observed between Lp(a) preparations containing low or high apo(a) isoform size (data not shown). Subsequently, we treated Lp(a) with DTT, a procedure that completely removes apo(a) from Lp(a), and then purified reduced Lp(a) by ultracentrifugation [19]. This treatment did not significantly influence the endogenous PAF-AH activity. The effect of such Lp(a) preparations [Lp(a-)] was studied on PAF-induced washed platelet aggregation in comparison to that of unreduced Lp(a). As shown in Fig. 3, both Lp(a) preparations significantly inhibited platelet aggregation in a dose-dependent manner; however, Lp(a-) was a more potent inhibitor compared to Lp(a).

3.4. Effect of Lp(a) on PAF-induced PAC-1 binding

To further investigate the mechanism of Lp(a)-induced inhibition of platelet aggregation in the presence of PAF, as well as the role of the PAF-AH content and the apo(a) moiety, we studied the effect of Lp(a), pefa-Lp(a) and Lp(a-) on the binding of the specific monoclonal antibody PAC-1, which recognizes the activated form of the platelet integrin receptor αIIbβ3. In these experiments, the above Lp(a) preparations were used at concentrations of 40 and 80 μg protein/ml. As shown in Fig. 4A, all forms of Lp(a) studied, at both concentrations, effectively inhibited the PAC-1 binding to activated platelets in the presence of 80 nM PAF, the Lp(a-) being more potent compared either to Lp(a) or to pefa-Lp(a), a finding which accords to the results from the aggregation studies. No difference in their inhibitory potency was observed between Lp(a) and pefa-Lp(a) (Fig. 4A). Representative flow cytometry curves illustrating the effect of 80 μg protein/ml Lp(a) or Lp(a-) on PAC-1 binding are shown in Fig. 4B,C. Similarly to PAF, Lp(a) and Lp(a-), at concentrations of 40 and 80 μg protein/ml, significantly inhibited ADP-induced PAC-1 binding, the Lp(a-) being more potent than Lp(a) (24±9% versus 15±6% inhibition at 40 μg protein/ml and 68±12% versus 43±14% inhibition at 80 μg protein/ml, p<0.03 for both comparisons). Finally, as expected, the RGDS tetrapeptide at a concentration of 1 mM completely inhibited PAC-1 binding induced by either PAF or ADP.

Fig. 4

Role of endogenous PAF-AH and apo(a) on Lp(a)-mediated inhibition of PAF-induced activation of αIIbβ3. (A) Bar-graph showing the %inhibition of PAC-1 binding in the presence of Lp(a) (expressing PAF-AH activity of 13±5 nmol/mg protein/min) (open bars) or pefa-Lp(a) (inactive PAF-AH) (gray bars) or Lp(a-) (closed bars). Values represent the mean±S.D. from three different experiments. *p<0.04 compared to either Lp(a) or pefa-Lp(a). (B, C) Representative histograms, obtained by FACS analysis, illustrating the effect of 80 μg protein/ml of either Lp(a) (B) or Lp(a-) (C) on PAC-1 binding on activated platelets.

Fig. 4

Role of endogenous PAF-AH and apo(a) on Lp(a)-mediated inhibition of PAF-induced activation of αIIbβ3. (A) Bar-graph showing the %inhibition of PAC-1 binding in the presence of Lp(a) (expressing PAF-AH activity of 13±5 nmol/mg protein/min) (open bars) or pefa-Lp(a) (inactive PAF-AH) (gray bars) or Lp(a-) (closed bars). Values represent the mean±S.D. from three different experiments. *p<0.04 compared to either Lp(a) or pefa-Lp(a). (B, C) Representative histograms, obtained by FACS analysis, illustrating the effect of 80 μg protein/ml of either Lp(a) (B) or Lp(a-) (C) on PAC-1 binding on activated platelets.

3.5. Effect of Lp(a) on PAF-induced fibrinogen binding

To investigate whether the inhibition of PAF-induced αIIbβ3 expression and activation (PAC-1 binding) by Lp(a) influences the association of fibrinogen with the receptor, we performed binding studies using FITC-Fg. As shown in Fig. 5A, Lp(a) at a concentration of 40 and 80 μg protein/ml, significantly inhibited FITC-Fg binding to platelets activated with 80 nM PAF. Representative flow cytometry curves illustrating the effect of Lp(a) at 40 μg protein/ml, on PAF-induced FITC-Fg binding are shown in Fig. 5B. Finally, as expected, the PAF-induced FITC-Fg binding was completely inhibited by 1 mM RGDS (Fig. 5A,C).

Fig. 5

Lp(a) mediated inhibition of PAF-induced FITC-Fg binding. (A) Bar-graph showing the %inhibition of FITC-Fg binding in the presence of Lp(a) (open bars or 1 mM RGDS (black bars)). Values represent the mean±S.D. from three different experiments. (B, C) Representative histograms, obtained by FACS analysis, illustrating the effect of Lp(a) (40 μg protein/ml) (B), or RGDS (C) on FITC-Fg binding to platelets activated with 80 nM PAF.

Fig. 5

Lp(a) mediated inhibition of PAF-induced FITC-Fg binding. (A) Bar-graph showing the %inhibition of FITC-Fg binding in the presence of Lp(a) (open bars or 1 mM RGDS (black bars)). Values represent the mean±S.D. from three different experiments. (B, C) Representative histograms, obtained by FACS analysis, illustrating the effect of Lp(a) (40 μg protein/ml) (B), or RGDS (C) on FITC-Fg binding to platelets activated with 80 nM PAF.

4. Discussion

The results of the present study show for the first time that Lp(a) inhibits PAF-induced platelet activation in a dose-dependent manner. Importantly, even in the presence of all plasma components including lipoproteins (i.e. in PRP), Lp(a) at a concentration higher than 20 μg protein/ml, which corresponds to plasma Lp(a) concentration >8 mg/dl, significantly inhibits PAF-induced platelet activation. A similar inhibitory effect of Lp(a) was observed in washed platelets, an indication that the plasma components do not significantly influence the Lp(a)-mediated inhibition of platelet aggregation.

An important observation of the present study is that the PAF-AH component of Lp(a) did not significantly contribute to the inhibitory effect of Lp(a) on PAF-induced platelet activation, thus suggesting that in plasma, which is enriched in PAF-AH activity mainly associated with LDL [31], the Lp(a)-PAF-AH may not play any significant role in the Lp(a)-mediated inhibition of platelet aggregation induced by PAF. However, we cannot exclude the possibility that this enzyme may significantly influence other Lp(a) functions, which implicate this lipoprotein in atherothrombosis. Thus, the pathophysiological role of the Lp(a)-associated PAF-AH remains to be established.

According to our results, Lp(a) inhibits both the primary and the secondary aggregation as well as the secretion of dense granules (measured by determining their ATP content) induced by PAF. A content of the platelet dense granules and one of the endogenous platelet agonists that significantly contributes to the secondary aggregation induced by PAF is ADP [35]. Lp(a) inhibited ADP-induced platelet aggregation; consequently, the inhibition of the PAF-induced secondary aggregation by Lp(a) can be at least partially attributed both to the inhibition of ADP secretion and ADP-induced platelet aggregation.

The inhibition of platelet aggregation induced by different agonists (i.e. PAF, ADP and Calcium ionophore A23187) by Lp(a) suggests that it may not be a specific effect of this lipoprotein towards a certain agonist but rather that Lp(a) influences a common step through which all agonists lead to platelet aggregation. This common step concerns the surface expression and conformational change of the platelet integrin receptor αIIbβ3, which is observed during platelet activation induced by any agonist [34]. The αIIbβ3, in its activation state, expresses several binding sites for fibrinogen, which binds to the receptor, thus leading to platelet aggregation [34]. Our results showed that Lp(a) inhibits both the PAF- and the ADP-induced activation of αIIbβ3, measured by the capacity of the receptor to bind PAC-1, which recognizes only the activated form of this integrin [36]. In addition, Lp(a) significantly inhibited the binding of fibrinogen to activated platelets. These results suggest that Lp(a) inhibits platelet aggregation by influencing the expression of αIIbβ3 and fibrinogen binding to the activated receptor. Several studies over the last years have shown that Lp(a) interacts with human platelets at their resting state in a specific, saturable and reversible manner, although a specific Lp(a) receptor on platelets has not been described yet [9,10,12,13]. In this context, it has been suggested that Lp(a) binds to the αIIb subunit of the αIIbβ3 in its resting state. Thus, this subunit may represent the major Lp(a) binding protein on platelets [9], although other studies showed that αIIbβ3 in its resting state may not be implicated in Lp(a) binding [10,13]. Taking into account the above results, we may suggest that binding of Lp(a) to resting platelets results in the inhibition (through a yet unidentified mechanism) of αIIbβ3 surface expression and activation induced by any agonist, thus leading to the inhibition of both fibrinogen binding and platelet aggregation. Alternatively, in addition to its association with the resting platelets, Lp(a) may bind to the activated form of αIIbβ3, thus inhibiting the binding of PAC-1 and fibrinogen. This hypothesis is currently under investigation in our laboratory.

An important observation of the present study is that removal of apo(a) from Lp(a) significantly enhances the antiaggregatory effect of Lp(a) as well as its inhibitory effect on PAC-1 binding to activated platelets. Previous studies have demonstrated that apo(a) is not necessary for the interaction of Lp(a) with resting platelets (9). Furthermore, Lp(a-) can also bind to platelets although with lower affinity [9,10]. However, it has been shown that recombinant apo(a) is also capable of binding to resting platelets, in an αIIbβ3-independent manner, implying that another unidentified yet platelet protein may be involved in this process [11]. The interaction of apo(a) with platelets enhances their response to arachidonic acid or SFLLRN [11,16] thus, in contrast to Lp(a), its apo(a) moiety may play a proaggregant role. This suggests that Lp(a) and apo(a) may modulate agonist-induced platelet activation, interacting with distinct binding sites on the platelet surface. In accordance to that, it has been shown that lysine residues on apo(a) may play an important role in the apo(a)-platelet interaction [11], whereas it was recently suggested that the lysine residues of apo(a) may not mediate the Lp(a)-induced inhibition of platelet aggregation [12]. Based on our results as well as on the results of previous studies, we may suggest that the inhibitory effect of Lp(a) on PAF-induced platelet aggregation is primarily due to its Lp(a-) moiety, since it exhibits a more potent inhibitory effect compared to Lp(a). Overall, we may speculate that Lp(a) interacts with distinct binding sites on resting platelets, i.e. through its apo(a) moiety and through its LDL-like particle [Lp(a-)]. The binding of Lp(a) through its Lp(a-) moiety leads to the inhibition of platelet activation, whereas its binding through the apo(a) moiety may enhance agonist-induced platelet activation. Thus, the effect of Lp(a) on PAF- or ADP-induced platelet aggregation, described in the present study, could be the result of the Lp(a-) inhibitory effect and the apo(a) stimulatory effect. This hypothesis may also explain the controversial results published in the literature concerning the effects of Lp(a) or apo(a) on platelet aggregation.

In conclusion, the present study demonstrates that Lp(a) inhibits PAF-induced platelet activation in a non-specific manner. The Lp(a)-associated PAF-AH does not play any important role in this effect, whereas the apo(a) moiety of Lp(a) significantly reduces its inhibitory effect in an apo(a) isoform size-independent manner. We suggest that the inhibition of αIIbβ3, activation and fibrinogen binding to the activated platelets may represent the major mechanism by which Lp(a) inhibits PAF-induced platelet aggregation.

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Author notes

Time for primary review 26 days