(Received for publication, July 23, 1996, and in revised form, October 12, 1996)
From the University Department of Medicine, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom
We have previously reported that plasma apolipoprotein (apo) E-containing high density lipoprotein particles have a potent anti-platelet action, apparently by occupying saturable binding sites in the cell surface. Here we show that purified apoE (10-50 µg/ml), complexed with phospholipid vesicles (dimyristoylphosphatidylcholine, DMPC), suppresses platelet aggregation induced by ADP, epinephrine, or collagen. This effect was not due to sequestration of cholesterol from platelet membranes; apoE·DMPC chemically modified with cyclohexanedione (cyclohexanedione-apoE·DMPC) did not inhibit aggregation but nevertheless removed similar amounts of cholesterol as untreated complexes, about 2% during the aggregation period. Rather we found that apoE influenced intracellular platelet signaling. Thus, apoE·DMPC markedly increased cGMP in ADP-stimulated platelets which correlated with the resulting inhibition of aggregation (r = 0.85; p < 0.01, n = 10), whereas cyclohexanedione-apoE·DMPC vesicles had no effect. One important cellular mechanism for up-regulation of cGMP is through stimulation of nitric oxide (NO) synthase, the NO generated by conversion of L-arginine to L-citrulline, binds to and activates guanylate cyclase. This signal transduction pathway was implicated by the finding that NO synthase inhibitors of distinct structural and functional types all reversed the anti-platelet action of apoE, whereas a selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (100 nM), had a similar reversing action. Direct confirmation that apoE stimulates NO synthase was obtained by use of L-[3H]arginine; platelets pretreated with apoE·DMPC produced markedly more L-[3H]citrulline (0.71 ± 0.1 pmol/h/109 platelets) than controls (0.18 ± 0.03; p < 0.05). In addition, hemoglobin which avidly binds NO also suppressed the anti-aggregatory effect, indicating that apoE stimulated sufficient production of NO by platelets for extracellular release to occur. We conclude that apoE inhibits platelet aggregation through the L-arginine:NO signal transduction pathway.
Human apolipoprotein E (apoE)1 is a
299-residue protein of molecular mass 34 kDa found in the surface of
circulating triglyceride-rich lipoproteins (very low density
lipoprotein and chylomicrons, or their remnants) and certain HDL
particles (1). Its major function is to mediate hepatic clearance of
lipoproteins through interaction with two receptors, the low density
lipoprotein or B,E receptor and an apoE-specific receptor, most
probably the low density lipoprotein receptor-related protein (2). When
the apoE polypeptide is dysfunctional or absent severe hyperlipidemia
and atherosclerosis in humans or animal models ensues (1, 3, 4, 5).
Although apoE is synthesized predominantly by the liver, macrophages
also secrete apoE; this appears important for facilitating local
cholesterol redistribution, for reverse cholesterol transport, and for
restricting development of atherosclerotic lesions (6). Indeed,
atherosclerosis in apoE-deficient (apoE/
)
mice can be prevented by transplantation of normal murine bone marrow
cells (5), by macrophage-specific expression of the human apoE
transgene (7), or by adenovirus-mediated gene replacement (8).
Recently, we proposed an additional anti-atherogenic role for apoE. We found that HDL-E, the minor apoE-containing subclass of bulk plasma HDL was a powerful inhibitor of agonist-induced platelet aggregation, apparently through interaction with saturable binding sites in the platelet surface membrane (9). Indirect evidence implicated apoE as the active constituent; chemically modifying apoE blocked binding and its anti-platelet action (9), whereas abnormal apoE-enriched HDL from patients with hepatic cirrhosis had a highly potent anti-aggregatory effect that correlated with apoE content (r = 0.70, p < 0.001) (10). In the present study, we infer that apoE exerts its anti-platelet effect by enhancing production of endogenous nitric oxide (NO); apoE markedly elevated platelet NO synthase activity and intraplatelet levels of cGMP, whereas NO synthase inhibitors restricted its inhibitory action.
NG-Nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA), D-NMMA, S-nitroso-L-glutathione (GSNO), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were obtained from Alexis Corp. Ltd. (Nottingham, UK). Diphenyleneiodonium chloride (DPI), 3-isobutyl-1-methylxanthine (IBMX), and 2-ethyl-isothiopseudourea (Ethyl-ITU) were supplied by Calbiochem-Novabiochem Ltd. (Nottingham, UK). Other chemicals were from Sigma (Dorset, UK).
ApoE·DMPC ComplexesApoE was purified from triglyceride-rich lipoproteins by heparin-Sepharose affinity chromatography (11) and was incorporated into small, unilamellar vesicles of dimyristoyl phosphatidylcholine (DMPC) liposomes at a 3.75:1 phospholipid:protein ratio (12). Protein and DMPC concentrations were measured using commercially available methods (Bradford reagent (Bio-Rad Laboratories, Hemel Hempstead, UK) and phospholipid B kit (Wako, Neuss, Germany), respectively). ApoE arginine residues were modified by cyclohexanedione (11). All test samples were dialyzed against Tyrode's buffer (0.42 mM sodium phosphate, pH 7.4, containing 136.9 mM NaCl, 11.9 mM NaHCO3, and 2.7 mM KCl) before use.
Platelet PreparationsBlood was withdrawn with minimal stasis from the antecubital vein of normal volunteers and was mixed with one-ninth volume of 3.8% sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation at 200 × g for 15 min at room temperature; platelet-poor plasma was prepared by centrifugation of PRP at 2000 × g for 10 min. Washed platelets were prepared from prostacyclin-stabilized PRP (9).
Platelet AggregationPRP or washed platelets (80 µl) were preincubated for 30 s with Tyrode's buffer (20 µl) and aggregation was initiated by addition of increasing concentrations of ADP, epinephrine, collagen, or thrombin at 37 °C in a Payton dual-channel aggregometer fitted with 0.1-ml cuvettes (9). The minimum amount of each agonist required to induce secondary aggregation within 3 min was determined, and this "threshold concentration" was used in subsequent assays substituting the Tyrode's buffer for increasing amounts of free apoE, apoE·DMPC, cyclohexanedione-modified apoE·DMPC (CHD-apoE·DMPC), or DMPC liposomes alone. In experiments using NO synthase inhibitors, PRP was preincubated for 10 min at 20 °C with 300 µM of the chemical analogs of L-arginine, L-NMMA, or L-NAME or with 100 nM of hemoglobin. In the other inhibitory experiments, PRP was preincubated with 100 nM DPI or 3 µM Ethyl-ITU for 1 min at 37 °C prior to addition of ADP. To examine effects of the soluble guanylate cyclase inhibitor, ODQ, washed platelets (3 × 108/ml) were preincubated at 20 °C with 100 nM ODQ for 30 min before addition of either 200 nM GSNO or 50 µg of protein/ml of apoE·DMPC. Some variation in the extent of apoE·DMPC-induced inhibition was evident, but this presumably reflected differences in the individual platelets used or in the particular apoE·DMPC preparation, or a combination of these. However, all experiments were repeated several times and were qualitatively reproducible.
Preparation of [3H]Cholesterol-labeled PlateletsA [3H]cholesterol/albumin emulsion was prepared as before (13). Platelets were pelleted from prostacyclin-stabilized PRP, resuspended in the [3H]cholesterol/albumin emulsion for 1 h, and treated again with prostacyclin (300 nM). The mixture was then diluted 50-fold with Tyrode's buffer, centrifuged at 750 × g for 20 min, and the platelet pellet resuspended in buffer. The platelets were used for cholesterol depletion studies immediately after recovering from the prostacyclin effects.
Cholesterol RemovalAliquots of [3H]cholesterol-labeled platelet suspensions (600 µl, 3 × 108 cells/ml) were incubated in the aggregometer with buffer, apoE·DMPC, or CHD-apoE·DMPC at 37 °C. At defined time intervals up to 10 min, a portion (100 µl) was removed and rapidly centrifuged (12,000 × g, 30 s), and the [3H]cholesterol released into the supernatant (80 µl) was measured by liquid scintillation counting. In addition, at zero time an aliquot of the total platelet suspension (100 µl) was dissolved in 1 M NaOH, neutralized, and was counted similarly.
Cyclic Nucleotide AssaysIntraplatelet cGMP and cAMP concentrations were measured in PRP with and without a 10-min preincubation at 20 °C with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (1 mM). Aggregation was terminated after 3 min by addition of 40 µl of 20% HClO4; the samples were then neutralized with 1.08 M K3PO4 (80 µl), centrifuged (2000 × g for 15 min at 4 °C), and after acetylation were assayed for cGMP and cAMP contents by commercial radioimmunoassay kits (Amersham Int. Plc). All samples were corrected for the cGMP and cAMP content of platelet-poor plasma.
NO Synthase ActivityWashed platelets (109 cells) were incubated with or without apoE·DMPC (50 µg of protein/3 × 108 cells) for 10 min at 37 °C in a final volume of 1 ml, and the reaction was stopped by addition of 100 µl of 10 × homogenization buffer (250 mM Tris-HCl, pH 7.4, containing 10 mM EDTA and 10 mM EGTA). The cells were pelleted in a microcentrifuge for 30 s, resuspended in 100 µl of 1 × homogenization buffer, and lysed by two cycles of freezing in liquid nitrogen and thawing on ice. NO synthase activity was measured by the conversion of L-[3H]arginine to L-[3H]citrulline using the NOSdetect assay kit (Stratagene) and expressed as pmol/h per 109 platelets. Briefly, 25 µl of platelet extract was incubated with 25 µl of substrate buffer (50 mM Tris-HCl, pH 7.4, containing 1 mM NADPH, 6 µM tetrahydrobiopterin, 2 µM flavin adenine dinucleotide, 2 µM flavin adenine mononucleotide, 0.2 µM calmodulin, 1.2 mM CaCl2, and L-[3H]arginine (200,000 dpm)) for 1 h at 37 °C, terminating the reaction by addition of 400 µl of stop buffer (50 mM HEPES, pH 5.5, containing 5 mM EDTA) and 100 µl of cationic resin (Dowex AF 50W-X8). The mixture was transferred to a spin filter, microcentrifuged for 30 s, and the L-[3H]citrulline in the eluate was measured by liquid scintillation counting. Nonenzymic formation of citrulline was controlled for by addition of the specific NO synthase inhibitor, L-NAME (1 mM), to a parallel set of tubes.
StatisticsResults are expressed as means ± S.E., and statistical differences were determined by Student's two-tailed unpaired t test.
Aqueous
solutions of apoE had no effect on agonist-induced aggregation using
either PRP or washed platelets but were highly potent anti-platelet
agents when complexed with phospholipid (DMPC) vesicles. Thus,
ADP-induced aggregation was inhibited in a dose-dependent manner using a physiological range (10-50 µg/ml) of apoE (Fig. 1), and similar findings were seen with epinephrine and
collagen as agonists. By contrast, little inhibition was noted with
thrombin as the aggregating agent, unless either very high amounts of
apoE were added (500 µg/ml) or the incubations were prolonged (up to 30 min).
Ability of ApoE·DMPC to Remove Cholesterol from Platelet Membranes
As cholesterol-deficient platelets respond poorly to
agonists (14), we investigated whether the inhibitory action of
apoE·DMPC might reflect extraction of cholesterol from platelet
membranes. Incubation of washed [3H]cholesterol-labeled
platelets with apoE·DMPC and CHD-apoE·DMPC (both 50 µg of
protein/ml) released similar amounts of cholesterol as a function of
time, corresponding to <1% after our standard 30-s preincubation
period and only about 2% after a further 3 min when aggregation
studies would be completed (Fig. 2A).
However, CHD-apoE·DMPC was an ineffective inhibitor of platelet
aggregation (Fig. 2B) compared with its unmodified control
(2 ± 5% versus 63 ± 4% inhibition,
respectively; p < 0.001), implying that an alternative
mechanism to cholesterol sequestration must explain the anti-platelet
effect of apoE.
Effects of ApoE·DMPC on Intraplatelet cGMP and cAMP Levels
Because attenuation of platelet responsiveness is
frequently accomplished by changes in intraplatelet cGMP or cAMP levels (15), we measured the influence of apoE on these cyclic nucleotides during ADP-induced aggregation of PRP. The basal levels of platelet cGMP and cAMP (3.7 ± 1.1 and 11.7 ± 1.9 pmol/109 platelets, respectively) were not significantly
altered by incubation with 50 µg of protein/ml of apoE·DMPC
vesicles (1.9 ± 0.7 and 9.5 ± 1.5 pmol/109
platelets, respectively). However, with threshold concentrations of
ADP, the same apoE·DMPC complexes produced marked
dose-dependent increases in both cGMP (33.9 ± 3.2 versus 13.6 ± 1.5 pmol/109 platelets at 50 µg of protein/ml of apoE·DMPC; p < 0.001, n = 3) and cAMP (23.5 ± 3.3 versus
7.4 ± 1.1 pmol/109 platelets at 50 µg of protein/ml
of apoE·DMPC, p < 0.001, n = 3)
(Fig. 3); these correlated with the observed concomitant
inhibition of aggregation after 3 min (r = 0.85 and
0.81 for cGMP and cAMP, respectively; both p < 0.01, n = 10). No changes in cyclic nucleotides were noted
with CHD-apoE·DMPC (Fig. 3A), free apoE or DMPC alone.
Effects of IBMX and ODQ on ApoE·DMPC-treated Platelets
Levels of cGMP and cAMP are controlled directly by the
activities of their synthesizing enzymes, guanylate cyclase and
adenylate cyclase, respectively, and catabolizing enzymes, cGMP and
cAMP phosphodiesterases (16, 17). When platelets were preincubated with
the general phosphodiesterase inhibitor, IBMX (1 mM) (18), basal cAMP levels were increased 2-fold (14.8 ± 3.0 versus 7.4 ± 1.1 pmol/109 platelets) as a
consequence of inhibition of platelet cAMP phosphodiesterase. However,
addition of apoE·DMPC vesicles to the system did not invoke a further
rise in cAMP (14.7 ± 3.4 versus 14.8 ± 3.0 pmol/109 platelets at 50 µg of protein/ml of apoE·DMPC;
Fig. 3B), implying that apoE had no effect on adenylate
cyclase. By contrast, apoE·DMPC still elicited a
dose-dependent rise in cGMP levels in the presence of IBMX
(10.8 ± 0.8 versus 2.6 ± 1.1 pmol/109 platelets at 50 µg of protein/ml; Fig.
3A), indicating an apoE-induced increase in synthesis of
this cyclic nucleotide rather than a decrease in its catabolism.
Further support for this pre-eminent role of cGMP was obtained by use
of ODQ, a potent and specific inhibitor of soluble guanylate cyclase
(19, 20). This reagent impaired, as expected, the anti-aggregatory
action of the NO donor, GSNO, but also effectively reversed the
anti-platelet effect of 50 µg of protein/ml of apoE·DMPC vesicles
(7.5 ± 8.9 versus 68.7 ± 4.4% inhibition;
p < 0.001, n = 3) (Fig.
4).
Effects of NO Synthase Inhibitors on the Aggregation of ApoE·DMPC-treated Platelets
One important cellular mechanism
for up-regulation of cGMP is through stimulation of NO synthase (21,
22); this enzyme acts on L-arginine to produce NO which
then binds to, and hence activates, soluble heme-containing guanylate
cyclase, its physiological target (23, 24). When we preincubated
platelets with L-NMMA or L-NAME, chemical
analogs of L-arginine and competitive inhibitors of NO
synthase (21, 22), the anti-platelet action of apoE·DMPC vesicles was
essentially blocked (Fig. 5). These inhibitory reactions were enantiomer-specific since D-NMMA was ineffective.
Moreover, two additional inhibitors of NO synthase, Ethyl-ITU (25, 26) and DPI (27, 29), also reversed the anti-platelet effect of apoE (Fig.
5B) without discernible effect themselves on
aggregation. Finally, hemoglobin, a competitor for NO binding (29),
suppressed the anti-aggregatory effect of apoE·DMPC (Fig.
5A).
Stimulation of Platelet NO Synthase Activity by ApoE·DMPC Vesicles
Platelet NO synthase is the constitutive, strictly
Ca2+-dependent form of the enzyme which
generates only trace amounts of NO (21, 22, 30). Indeed, a highly
specialized, noncommercial porphyrinic microsensor was required to
detect the transient release of low pmol amounts of NO following
agonist activation, whereas basal synthesis and release of NO by human
platelets was not detected (30). We monitored, therefore, release of NO
by measuring the stoichiometric production of
L-[3H]citrulline from
L-[3H]arginine. When platelets were incubated
with apoE·DMPC vesicles for 10 min, NO synthase activity was markedly
increased as judged by a 4-fold rise in conversion of
L-[3H]arginine to
L-[3H]citrulline (0.71 ± 0.17 pmol of
citrulline produced per h per 109 apoE·DMPC-treated
platelets versus 0.18 ± 0.03 in untreated platelets; p < 0.05, n = 4) (Fig.
6). This was considered specific NO synthase activity
since L-NAME (1 mM) negated any increase.
This study substantiates our previous work implicating apoE as the active anti-platelet constituent of HDL-E. The metabolic activity of apoE is sensitive to its lipid environment; purified apoE does not interact with the low density lipoprotein receptor (12), whereas the apoE in surfaces of very low density lipoprotein and chylomicrons is also inactive unless these lipoproteins are from hypertriglyceridemic subjects (31) or have undergone substantial lipolysis to form remnant particles (32). Similarly, apoE in free solution did not inhibit platelet aggregation, although apoE-phospholipid complexes, which mimic the form secreted by macrophages (1), were potent inhibitors; presumably the DMPC allowed the apoE polypeptide to assume an appropriate orientation and conformation for biological activity (12). Recently, apoE·DMPC complexes were reported to inhibit collagen- or thrombin-induced aggregation (33), apparently by avidly extracting cholesterol from platelet plasma membranes and hence impairing arachidonate release and conversion to thromboxane A2 (35). However, sequestration of cholesterol in our experiments was much lower, <1% versus 10%, reflecting our shorter preincubation times (30 s versus 30 min), an amount insufficient to be anti-aggregatory with ADP as agonist (13). Indeed, we showed this explanation to be implausible by use of chemically modified apoE; CHD-apoE·DMPC failed to suppress platelet aggregation but still removed the same amount of platelet cholesterol as inhibitory apoE·DMPC.
Calcium is central to control of platelet reactivity, interacting with
diverse second messengers through a myriad of complex, but tightly
regulated, signaling pathways (35). Two important control elements for
suppression of platelet activation are the cyclic nucleotides, cAMP and
cGMP, and agents that increase their intraplatelet levels exert
anti-aggregatory effects both in vitro and in
vivo. For example, prostacyclin and adenosine limit platelet activation by raising cAMP (36, 37), while NO or NO-donating compounds
are similarly restrictive by increasing cGMP (19, 38). Although apoE
induced increases in both cAMP and cGMP, additional experiments
implicated a specific stimulation of guanylate cyclase activity and a
rise in cGMP as prerequisites for the anti-platelet action of apoE.
Thus, ODQ, a potent and selective inhibitor of soluble guanylate
cyclase (19, 20), was able to reverse the anti-aggregatory action of
apoE. Similarly, studies with the general phosphodiesterase inhibitor,
IBMX, supported a primary role for cGMP since this reagent abolished
the apoE·DMPC-induced rise in cAMP but not the
dose-dependent increase in cGMP. Interestingly, these
findings were consistent with second messenger "cross-talk" (15),
namely that the increase in cGMP invoked by apoE in the absence of IBMX
may cause inhibition of cAMP phosphodiesterase (39, 40), permitting
levels of cAMP to rise (Fig. 3B and Fig. 7).
Several experiments implicated platelet NO synthase in mediating the anti-aggregatory action of apoE through production of NO, a physiological activator of guanylate cyclase. Thus, NO synthase inhibitors of distinct structural and functional types, the amino acid analogs of L-arginine, L-NMMA, and L-NAME, a nonamino acid analog, ethyl-ITU, and the flavoprotein inhibitor, DPI, all reversed the anti-platelet action of apoE. These chemicals inhibit cellular production of NO by binding to NO synthase to displace either L-arginine or, in the case of DPI, essential cofactors (26). Because all reagents were used at concentrations close to their quoted IC50 values for specific NO synthase inhibition, it seems improbable that they act via diverse NO-independent mechanisms to block the apoE·DMPC effects. Indeed, we demonstrated direct involvement of the L-arginine:NO pathway by measuring platelet NO synthase activity; lysates from platelets pretreated with apoE·DMPC had a 4-fold increased ability to convert L-arginine to L-citrulline which could be abolished by L-NAME. Furthermore, hemoglobin, which strongly inhibits NO activation of guanylate cyclase by forming a hemoglobin-NO adduct, suppressed the anti-platelet action of apoE. As hemoglobin does not penetrate platelets (41), this implies that apoE generated sufficient NO for secretion; presumably binding of NO by hemoglobin in the extracellular medium created a concentration gradient to reduce NO inside platelets (28) and hence allow aggregation. Interestingly, our observation that apoE·DMPC vesicles do not inhibit thrombin-stimulated aggregation provides indirect support for the functional importance of platelet NO synthase; although the reasons are obscure, NO synthase is not activated during platelet aggregation induced by this agonist (21, 30).
In summary, as indicated in Fig. 7, we believe that our findings provide clear evidence for the L-arginine:NO signal transduction pathway as the mechanism by which apoE exerts its anti-platelet effect. Less clear are the initial events required to activate this pathway. However, the ability of platelets to bind saturably HDL-E (9) and apoE·DMPC complexes (33) suggests a distinctive apoE receptor in platelet surface membranes, a proposal consistent with the noninhibitory action of CHD-apoE·DMPC. Note also that the failure of apoE to increase basal levels of cGMP was expected; platelet NO synthase requires Ca2+-calmodulin for activation, and an agonist such as ADP is essential to supply the initial burst of Ca2+ needed for up-regulation (21). Intriguingly, this pathway allows us to speculate that occupation of specific receptors by apoE may "prime" platelets to help attenuate activation when challenged by agonists or other agents.
Although NO synthesized by the constitutive enzyme NO synthase is now well-recognized as an important regulatory mechanism for platelet hemostasis (21, 22, 23, 24), and hence prevention of thrombosis, our finding that apoE substantially augments its production is novel and, potentially, important. Clinically, it may be directly relevant to reports that platelet reactivity increases prevalence (42) and incidence (43) of coronary heart disease and that low apoE and HDL-E are important risk factors (44, 45, 46). But we can also speculate on wider implications. First, that the extracellular NO released from platelets by apoE stimulation of NO synthase activity has the potential to function in a paracrine manner. Second, that apoE might act directly on other cell types to elicit production of NO, including neuronal cells (47, 48, 49) and T-lymphocytes (50, 51). Clearly, further experiments are needed to ascertain the biochemical and clinical consequences of this newly identified link in platelets between apoE and NO, two molecules increasingly implicated in cardiovascular disease and neurological disorders (47, 52).