© 1996 American Heart Association, Inc.
Articles |
Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation
the UMR 5533 CNRS, Hopital Cardiologique, Pessac, France; Sanofi Recherche, Toulouse, France (P.S., J-M.H.); and the Department of Vascular Biology, Scripps Research Institute, La Jolla, Calif (T.J.K.).
Correspondence to Alan T. Nurden, Director, UMR 5533 CNRS, Hopital Cardiologique, 33604 Pessac, France.
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Abstract |
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Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti–ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.
Key Words: platelet aggregation • ADP • fibrinogen • GP IIb/IIIa complexes • clopidogrel
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Introduction |
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Platelets are a key element in the development of arterial thrombosis. Among the variety of soluble agonists that induce platelet aggregation, ADP is a major stimulus in vivo.1 2 3 Therefore, drugs that inhibit ADP-induced platelet function are of considerable interest in the treatment of arterial thrombosis, in which ADP may play a significant role.4 5 Recent clinical trials have shown that ticlopidine is effective in patients with a broad spectrum of thrombotic disease (data reviewed in Reference 6). Thienopyridine compounds, such as ticlopidine and its more recently developed analogue, clopidogrel, selectively inhibit ADP-induced human platelet aggregation during platelet function testing.5 7 8 9 Typically, they result in a reduced and rapidly reversible platelet aggregation even at high doses of ADP. Clopidogrel acts as an antithrombotic agent in animal models of thrombosis5 and inhibits thrombogenesis when human blood is tested in an ex vivo model.10 11 Nevertheless, despite recent advances, the precise mechanism whereby ADP activates platelets, leading to thrombus formation, and therefore the mechanism of its inhibition by thienopyridine compounds is not clearly understood.
It has been reported that thienopyridines inhibit ADP-induced binding of fibrinogen to the GP IIb/IIIa complex without inducing structural modifications of the complex.9 12 13 Other results show that clopidogrel and ticlopidine restrict the capacity of ADP to lower PGE1-elevated cAMP levels in human and animal platelets.14 15 This makes a direct action of clopidogrel on the fibrinogen receptor unlikely and suggests interference with an earlier event. 2-MeS-ADP, a stable analogue of ADP that also induces platelet shape change and aggregation while inhibiting adenylate cyclase, has been used to evaluate the number of high-affinity binding sites for ADP on platelets.16 17 18 19 20 21 22 Platelets from animals or human donors receiving clopidogrel or ticlopidine bind much-reduced amounts of this ligand.18 19 20 21 Thus, the implication is that clopidogrel interferes with ADP-induced platelet aggregation at the primary receptor level.
Our study combines immunogold labeling and electron microscopy to characterize the ultrastructure of ADP-induced platelet aggregates from a series of human volunteers who received clopidogrel. To explore the activation of GP IIb/IIIa complexes, we used AP-6, an MAb raised against the 204-to-229 amino acid sequence of GP IIIa. AP-6 has access to this sequence on ADP-stimulated platelets only when the latter have bound fibrinogen and is thus an anti-LIBS antibody.22 We studied in parallel the ultrastructure of aggregates of a patient (M.L.) with a rare congenital abnormality of an ADP-dependent activation pathway that appears to mimic the effect of thienopyridines on normal platelets.23 We found that in both situations, ADP induces small aggregates composed of loosely bound platelets with few contact sites. A ring composed predominantly of degranulated platelets found at the periphery of aggregates induced in citrated platelet-rich plasma of untreated donors was not observed. Studies with AP-6 and a polyclonal anti-fibrinogen antibody enabled us to assess the expression of activation-dependent epitopes within the residual aggregates, whereas flow cytometry was used to examine individual platelets. Our results are in agreement with the recently proposed concept that ADP-mediated activation of platelets involves two pathways,20 one of which is affected by clopidogrel, is defective in the patient, and is essential for the full activation of GP IIb/IIIa complexes and the formation of stable macroaggregates.
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Methods |
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Subjects
Ten male volunteers 23 to 35 years old were given clopidogrel (Sanofi Recherche) as a single daily oral dose of 75 mg for 7 days. All studies were performed on blood samples taken on day 0 and after the final dose was taken on day 7. Also studied was patient M.L., who has a family history of bleeding linked to an abnormal platelet response to ADP.23 This was manifested by a low initial intensity and reversibility of aggregation at all doses of ADP. Aggregation with medium to high doses of other agonists was normal. The protocol for this work was approved by the regional ethics committee, and the study was conducted according to the principles of the Declaration of Helsinki. In all cases, informed consent was obtained.
Preparation of Platelets
Blood was collected from the antecubital vein (9 vol) and anticoagulated with 3.8% (wt/vol) trisodium citrate (1 vol). PRP was obtained by centrifugation of citrated whole blood at 120g at room temperature for 15 minutes. Platelet-poor plasma was obtained by centrifugation of the residual red cells at 1200g for 30 minutes, and the platelet count was adjusted as described below.
Platelet Aggregation
Platelet aggregation was measured at 37°C by a turbidimetric method in a PAP 4 aggregometer (Bio Data Corp, Welcome Laboratories). Aliquots of control or clopidogrel-treated human PRP were stirred at 1100 rpm and tested with 5 µmol/L ADP (Sigma Chemical Co), 0.5 µmol/L 2-MeS-ADP (RBI, Bioblock), 25 µmol/L thrombin receptor activating peptide (SFLLRNPNDKYEPF, TRAP-14-mer) (Neosystem), or 25 µg/mL collagen (Diagnostica Stago).
Antibodies Used
AP-2, a murine monoclonal IgG specific for GP IIb/IIIa complexes, and AP-6, an anti-LIBS murine monoclonal IgM raised against a synthetic peptide of the 204-to-227 sequence of GP IIIa,22 23 24 were produced in the laboratory of one of the authors (T.J.K.). F-26, an anti-RIBS monoclonal murine IgG,25 was kindly provided by Dr H. Gralnick (Bethesda, Md). VH10, an anti–P-selectin murine monoclonal IgG, has been reported previously.23 Purified IgG of a rabbit antiserum to fibrinogen was purchased from Dakopatts.
Flow Cytometry
Preparation of FITC-Conjugated Fibrinogen
Human fibrinogen was conjugated with FITC by the procedure developed by Xia and Frojmovic.26 In brief, purified fibrinogen (Kabi AB) was dissolved at 2 mg/mL in PBS, pH 7.2, and the pH was adjusted to 8 with a 5% wt/vol Na2CO3 solution. Then, FITC (Molecular Probes) was added at a concentration of 1 mg FITC/mg fibrinogen, and the mixture was incubated for 10 minutes at room temperature with intermittent shaking. The sample was applied to a Sephadex G-25 column (Pharmacia LKB) equilibrated with PBS, pH 7.2, and FITC-conjugated fibrinogen was recovered in the void volume. The fluorescein/protein molar ratio was 
2.5. The FITC-conjugated fibrinogen was dialyzed overnight against a modified Tyrode's buffer consisting of (in mmol/L) NaCl 137, KCl 2, NaHCO3 12, NaH2PO4 0.3, MgCl2 1, glucose 5.5, and HEPES 5, and 0.35% (wt/vol) BSA (Sigma), pH 7.4 (HEPES-Tyrode's buffer). Studies performed with washed platelets prepared as described by Nurden et al23 showed that the binding of FITC-fibrinogen to ADP-stimulated platelets was competitively inhibited by the unlabeled protein according to normal kinetic parameters.
Fibrinogen Binding to Platelets
The platelet count in PRP was adjusted with platelet-poor plasma, and a volume (10 µL) containing 2.5x108 platelets/mL was added to polystyrene tubes containing 10 µL FITC-conjugated fibrinogen (500 µg/mL) and 70 µL HEPES-Tyrode's buffer. Also present was 10 µmol/L ADP or 0.5 µmol/L 2-MeS-ADP added in a 10-µL volume. In control incubations, the agonist was replaced by an additional 10-µL volume of HEPES-Tyrode's buffer. After an initial mixing, the unstirred suspensions were diluted at 30 seconds, 5 minutes, or 30 minutes with 750 µL of HEPES-Tyrode's buffer and analyzed immediately in the flow cytometer.
MAb Binding Assay
A volume (10 µL) of PRP containing 2.5x108 platelets/mL was added to polystyrene tubes containing 90 µL HEPES-Tyrode's buffer and a predetermined saturating concentration of one of the following MAbs: AP-2, IgG ascites 1/500; AP-6, IgM ascites 1/1000; F-26, purified IgG 5 µg/mL; or VH10, purified IgG 5 µg/mL, as previously described.27 Unstirred samples were incubated with 10 µmol/L ADP, 0.5 µmol/L 2-MeS-ADP, or 10 µL HEPES-Tyrode's buffer for 15 minutes at room temperature after the initial mixing. Then, 10 µL of a predetermined concentration of FITC-conjugated F(ab')2 fragments (1/40) of a sheep antibody to mouse IgG (Silenus) or DTAF-conjugated F(ab')2 fragments (1/100) of a donkey antibody to mouse IgM (Jackson Immunoresearch) was added.27 The tubes were left in the dark for an additional 15 minutes before dilution with 750 µL HEPES-Tyrode's buffer and immediate analysis.
Passage in the Flow Cytometer
A Becton-Dickinson FACScan flow cytometer was used. The samples were first analyzed by forward and wide-angle light scatter, and the gate was set so as to include the majority of platelets and exclude larger particles. FITC-conjugated antibodies or FITC-fibrinogen was detected with a 530-nm detection wavelength. Fluorescence histograms were obtained for 10 000 cells, and data were compiled and analyzed with Lysys II software (Becton-Dickinson).27
Electron Microscopy
Immunostaining on Ultrathin Sections of Platelet Aggregates
Aliquots of control or clopidogrel-treated human PRP at 2.5x108 platelets/mL were stirred at 1100 rpm in an aggregometer cuvette with 10 µmol/L ADP or 0.5 µmol/L 2-MeS-ADP. Aggregation was stopped either at the peak of the aggregation as determined on the aggregometer tracing or at maximal disaggregation (see Fig 1
) by rapid transferral of the contents of the cuvette to 20 mL 1.25% wt/vol glutaraldehyde (Fluka AG) diluted in 0.1 mol/L PB, pH 7.4, containing 0.2% (wt/vol) picric acid and 0.5 mmol/L CaCl2. Fixation was for 2 hours at room temperature. After centrifugation, samples were twice incubated for 1 hour in 0.1 mol/L PB, pH 7.4, containing 0.5 mmol/L CaCl2 and 3.5% (wt/vol) sucrose (washing buffer) followed by 1 hour at room temperature in washing buffer containing 50 mmol/L glycine (Sigma) to quench residual aldehyde groups. To remove PB, samples were incubated four times for 15 minutes in 0.1 mol/L maleate buffer, pH 6.5, containing 3.5% sucrose, after which they were postfixed in 2% wt/vol uranyl acetate (Fluka) diluted in 0.1 mol/L maleate buffer, pH 6, for 2 hours at 0°C. The samples were washed three times in 0.1 mol/L maleate buffer, pH 6.5, preembedded in a low-gelling agarose, and dehydrated through a series of graded acetones at -20°C before being embedded in Lowicryl K4M (Taab) as described by Heilmann et al.28 Photopolymerization of the hydrophilic resin was continued at 4°C for 2 days. Ultrathin sections were obtained with an Ultracut E Ultramicrotome (Reichert Jung) and mounted on collodion carbon–coated nickel grids. These were placed onto a drop of a solution containing primary antibody (AP-6, ascites 1/2000; IgG of rabbit antisera to human fibrinogen, 1/200), diluted in 20 mmol/L Tris containing 150 mmol/L NaCl, pH 8.2 (Tris buffer), and incubated overnight at room temperature in a moist chamber. Grids were washed by floating three times onto Tris buffer containing 0.5% (wt/vol) BSA (Tris-alb). They were then placed onto a drop of Tris buffer containing a 1/100 dilution of 5 or 10 nm gold–labeled goat anti-mouse IgM or 5 nm gold–labeled anti-rabbit IgG (Amersham) for 2 hours that had been centrifuged at low speed before use to remove any aggregates. The sections were rinsed with Tris-alb and contrasted with 2% wt/vol osmium tetroxide and lead citrate. Grids were observed at 80 kV in a Philips EM 201 electron microscope.28 29
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Standard Procedures
On occasion, samples containing platelet aggregates were fixed by addition to 20 mL 1.25% (vol/vol) glutaraldehyde diluted in 0.1 mol/L PBS, pH 7.4, at room temperature. After 20 minutes, platelets and aggregates were sedimented and washed 3 times in PBS, pH 7.4. Platelets were postfixed with 1% wt/vol osmium tetroxide. After washing, the pellet was dehydrated in a series of graded ethanol solutions and embedded in epoxy resin (Taab). Ultrathin sections were obtained with the Ultracut E Ultramicrotome, placed onto copper grids, and stained in uranyl acetate and lead citrate.29 30 Grids were observed at 80 kV in a Philips EM 201 electron microscope as described above.
Measurement of [Ca2+]i
Suspensions of gel-filtered platelets were obtained by addition of citrated PRP to a Sepharose CL2B column (Pharmacia LKB) equilibrated with HEPES-Tyrode's buffer as previously described.31 Platelets (2x108/mL) were incubated with 2.5 µmol/L fluo-3/AM (Molecular Probes) at 37°C for 30 minutes in the dark before being washed twice and resuspended in buffer containing (in mmol/L) NaCl 137, KCl 4, MgCl2 0.5, NaH2PO4 0.5, HEPES 10, and probenecid (Sigma) 2.5, pH 7.4, supplemented with 0.1% (wt/vol) glucose and 0.1% (wt/vol) BSA. Probenecid is an ion transport inhibitor that prevents fluo-3 leakage from cells (see Reference 31). Its presence does not interfere with the Ca2+ response as measured here. Fluorescence measurements were performed in a Spex Fluoromax spectrofluorimeter (Jobin Yvon). The excitation and emission wavelengths were 495 and 530 nm, respectively. Platelets (1.5 mL at 2.5x107 platelets/mL) in a quartz cuvette were incubated for 15 minutes at 37°C with 2 mmol/L CaCl2 or 2 mmol/L EDTA before stimulation with 10 µmol/L ADP. Variations in fluorescence were followed over a period of 10 minutes. In experiments in which Ca2+ influx was determined on samples suspended and stimulated in 2 mmol/L EDTA, 4 mmol/L CaCl2 was added and the incubation was continued for a further 10 minutes. [Ca2+]i was calculated from the equation [Ca2+]i=Kdx(F-Fmin)/(Fmax-F), where Kd is the dissociation constant for Ca2+-bound fluo-3 and is 864 nmol/L at 37°C. F represents the fluorescence of the experimental sample. The calibration procedure consists of first obtaining Fmax by lysing platelets with 0.05% Triton X-100 (Sigma) and then Fmin in the presence of 4 mmol/L EGTA in 30 mmol/L Tris-HCl, pH 8.7.31 This was performed for each sample under study.
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Results |
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Platelet Aggregation
Platelet aggregation in citrated PRP was compared for each of 10 donors before and after they had taken clopidogrel (75 mg/d) for 7 days. After clopidogrel treatment, aggregation induced by both 5 µmol/L ADP and 0.5 µmol/L 2-MeS-ADP was of normal initial velocity, but maximal intensity reached a peak at about 1 minute, and this was followed by a partial dissociation of the aggregates (Fig 1
). The inhibitory effect of clopidogrel on the maximum intensity of aggregation induced by 0.5 µmol/L 2-MeS-ADP tended to be greater (77.6±16.5%) than that observed for 5 µmol/L ADP (60.5±26%) (Table 1). Such levels of inhibition were highly significant (P<.001). Clopidogrel had a similar inhibitory effect on aggregation induced by 10 µmol/L ADP (59.5±4%, n=5), the dose used in the rest of the study. In contrast, clopidogrel had little or no effect on platelet aggregation induced by 25 µmol/L TRAP-14-mer peptide (Table 1) or by 0.25 mg/mL arachidonic acid (data not shown). Finally, a small but highly variable inhibitory effect of clopidogrel on aggregation induced by 25 µg/mL collagen was observed (33±32%) (Table 1). These results agreed well with previous studies on the effect of clopidogrel on human platelets (see "Discussion") and closely resembled those we reported for patient M.L.23
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Electron Microscopy and Immunogold Staining
A morphological assessment of the structure of ADP-induced platelet aggregates was combined with an analysis of the distribution of fibrinogen and of ligand-bound GP IIb/IIIa complexes within them. Results were compared for platelets stimulated with 10 µmol/L ADP or 0.5 µmol/L 2-MeS-ADP before and after clopidogrel treatment.
Unstimulated Platelets
Immunogold labeling of sections of Lowicryl K4M–embedded samples of unstimulated platelets was as previously reported from our laboratory. With a polyclonal anti-fibrinogen antibody, heavy staining of the storage pool of fibrinogen within the 
-granules was accompanied by little or no staining of the surface membrane.28 With the anti-LIBS MAb AP-6, gold particles were again found primarily inside the platelet and with a tendency to be most abundant over the membrane of -granules.22 This staining is highlighted later in the text (see Fig 6C). Clopidogrel treatment had no effect on the labeling of sections of unstimulated platelets with these antibodies, and platelet morphology was also unmodified by the drug (data not shown).
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ADP-Induced Aggregates of Untreated Normal Subjects
Electron microscopy was first used to assess the ultrastructure of the large macroaggregates formed when 10 µmol/L ADP was stirred with control citrated PRP. As shown at high-power magnification by standard transmission electron microscopy in Fig 2, an intriguing aspect of the morphology of the aggregate is that platelets at the periphery generally appeared to be degranulated, whereas those within the central zone of what is a compact mass often still contained secretory organelles. In this experiment, the maximum intensity of the aggregation as measured in the platelet aggregometer was 87%, and the aggregates were fixed after 7 minutes. Fig 3 shows the distribution of fibrinogen and of the AP-6 epitope within the aggregate. Immunolabeling of the peripheral layer with anti-fibrinogen antibody showed that clusters of gold particles were detected on the platelet membrane and between adjacent platelets (Fig 3A). Deeper within the aggregate, immunogold staining for fibrinogen was also found in the interior of individual platelets, where residual -granules and/or fused secretory vesicles continued to be recognized despite the loss of morphology that accompanies the labeling of Lowicryl-embedded sections (see Fig 3C). The fact that staining with anti-fibrinogen antibody was found only in the interplatelet spaces at the periphery of the aggregate suggests a contribution of secreted fibrinogen to the platelet-to-platelet cohesion in this zone. Immunogold labeling with the anti-LIBS MAb AP-6 was also clearly most abundant over the plasma membrane in the peripheral region of the aggregate (Fig 3B). Labeling with AP-6 in the interplatelet spaces localized in the center of the aggregate was more heterogeneous than at the periphery, and visual inspection suggested a lower level of labeling, although this was not quantified (Fig 3D). Overall, our results imply a special organization within these aggregates with, in particular, a layer of platelets at the periphery often devoid of -granules and with a high level of GP IIb/IIIa complex occupancy. Platelets within aggregates induced by 0.5 µmol/L 2-MeS-ADP showed identical ultrastructural features and similar immunolabeling with both anti-fibrinogen and AP-6 antibodies to those illustrated for 10 µmol/L ADP (results not illustrated).
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ADP-Induced Aggregates of Clopidogrel-Treated Normal Subjects
A decreased maximal intensity of aggregation and a rapid disaggregation were a constant feature of ADP- and 2-MeS-ADP–stimulated platelets from clopidogrel-treated subjects (Table 1). In the experiment illustrated in Fig 4, the maximum intensity of aggregation was 50%, with samples being fixed 1 minute after the addition of 10 µmol/L ADP. Electron microscopy showed that the aggregates were composed of loosely packed platelets and were now much smaller, with the majority containing between 10 and 50 platelets. Notwithstanding, most of the platelets had formed pseudopods and had undergone a partial centralization of -granules showing that they had responded to the ADP (Fig 4A). A peripheral zone of degranulated platelets was never observed, and fibrinogen was uniformly present in -granules throughout the aggregate. As visualized at higher magnification, labeling with antifibrinogen antibody at the platelet surface and within the interplatelet space was low (see arrowheads, Fig 4B) compared with that seen for the control platelet aggregates (see above). Staining of the -granules remained abundant, confirming that little secretion had occurred. Surface labeling with AP-6 was also low but not totally absent (see arrowheads, Fig 4C). A feature of these aggregates was that platelet-to-platelet interactions were particularly heterogeneous, with occasional areas of close contact separated by zones with no contact at all. Intriguingly, some tight junctions were seen without intervening fibrinogen or occupied GP IIb/IIIa complexes being detectable (see arrows on Fig 4B and 4C). When 2-MeS-ADP was used instead of ADP, the effect of clopidogrel on aggregate size was, if anything, slightly more pronounced than after ADP stimulation (data not shown). Immunolabeling of surface membranes by anti-fibrinogen antibody and AP-6 remained low after clopidogrel ingestion, whereas labeling of internal pools was unchanged compared with that of unstimulated platelets.
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A feature of the clopidogrel-treated samples was the instability of the aggregates formed with ADP (or 2-MeS-ADP). Electron microscopy showed that disaggregation resulted in (1) a much increased number of single platelets and (2) a decrease in size of the residual aggregates, the bulk of which contained from 3 to 20 platelets after 7 minutes in the aggregometer. In the experiment illustrated in Fig 5, the intensity of aggregation at the time of fixation had returned to 20%. Many isolated platelets now had a morphology similar to that of unstimulated platelets; for others, pseudopods persisted (Fig 5A). The surface of these platelets showed areas with little labeling with anti-fibrinogen antibody; nevertheless, the internal pools continued to be revealed. The ultrastructure of a residual aggregate is shown in Fig 5B; immunogold staining with the anti-fibrinogen antibody showed few gold particles on the platelet surface or within the interplatelet space. The platelets had clearly retained -granules, and internal labeling for fibrinogen was abundant. Results with AP-6 for both isolated platelets and the residual aggregates paralleled those obtained with the anti-fibrinogen antibody (not illustrated). Ultrastructural features of the disaggregation phase of 2-MeS-ADP–induced aggregates included many single discoid platelets and the presence of small aggregates whose ultrastructure was indistinguishable from that shown for ADP (data not illustrated).
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ADP-Induced Aggregates of Patient M.L. With a Congenital Deficiency of the ADP Activation Pathway
We also examined the aggregates obtained when platelets from patient M.L. were stimulated with 10 µmol/L ADP (Fig 6). Again, at the peak of the aggregation (1 minute), the aggregates were of smaller than normal size and contained loosely packed platelets with limited zones of contact. A peripheral layer of degranulated platelets was never observed. With the anti-fibrinogen antibody, the -granules were well stained, but labeling was low both on the exposed platelet surface and at contact sites between platelets in the aggregate (Fig 6A). Surface staining with AP-6 was also low, with the occasional gold beads in the interplatelet spaces well spaced (see arrows, Fig 6B). Granule membranes continued to be labeled (see arrowheads). As was seen for clopidogrel-treated normal platelets, these aggregates were not stable, and disaggregation was rapid (typical aggregation curves for this patient are shown in Reference 23). Samples taken after 7 minutes showed a large proportion of single platelets. The ultrastructure of the residual aggregates was similar to that seen after 1 minute, although the aggregates were now smaller. A typical section of an isolated platelet as labeled with AP-6 is illustrated in Fig 6C. As with all dissociated platelets in the 7-minute sample, -granules clearly remained, showing that secretion was minimal. It should be noted that the staining of the -granule membrane by AP-6 is particularly well illustrated on this section (see arrowheads). On the plasma membrane, the labeling was weak and well spaced.
Flow Cytometry
Flow cytometry was used to assess fibrinogen binding or the expression of the AP-6 epitope when platelet suspensions were stimulated with ADP or 2-MeS-ADP.
Binding of FITC-Fibrinogen
Fig 7 shows that binding of FITC-fibrinogen increased with time when unstirred samples of control citrated PRP were stimulated with either 10 µmol/L ADP or 0.5 µmol/L 2-MeS-ADP. The whole platelet population showed a positive fluorescence at 30 seconds, and this increased progressively, reaching a maximum at 30 minutes for both agonists. After clopidogrel treatment, the binding of FITC-conjugated fibrinogen to the stimulated platelets was greatly reduced, with, for the 10 subjects, an average 70% inhibition of the mean fluorescence intensity attained after 30 minutes for ADP (Student's t test, P<.001) and 77% for 2-MeS-ADP (P<.001). No evidence was obtained for a normal initial binding followed by dissociation of FITC-fibrinogen from the clopidogrel-treated platelets.
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Platelet Activation Assessed With MAbs
An identical binding of AP-2 (a GP IIb/IIIa complex-dependent MAb) to unstimulated or ADP- or 2-MeS-ADP–stimulated platelets before or after clopidogrel treatment (Fig 8) confirmed previous findings that clopidogrel neither affected the level of GP IIb/IIIa complex expression on platelets nor induced its dissociation.12 13 Expression of occupied GP IIb/IIIa complexes was assessed with AP-6 and F-26 (an anti-RIBS MAb; see "Methods"). Fig 8 confirms that stimulation of untreated platelets in diluted PRP by both ADP and 2-MeS-ADP produced a single major population of platelets positive for both antibodies. For each of the 10 subjects tested, clopidogrel treatment resulted in a marked inhibition of the expression of these activation-dependent epitopes. The mean percentage reductions in the binding of AP-6 and F-26 to clopidogrel-treated platelets were 88% and 78%, respectively, a result in good agreement with the experiments with FITC-conjugated fibrinogen. Fig 8 also shows that ADP and 2-MeS-ADP induced the expression of some P-selectin on the surface of platelets in citrated PRP, amounting to 20% to 30% of the levels of VH10 binding seen with 0.5 U/mL thrombin (data not illustrated). Clopidogrel completely abolished the expression of P-selectin on the activated platelet surface (Fig 8). These results confirm that ADP and 2-MeS-ADP can induce some -granule secretion from platelets under our experimental conditions, although this was insufficient to increase the surface pool of GP IIb/IIIa as detected with AP-2.
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Ca2+ Influx and Mobilization
Table 2 shows ADP-induced changes in intraplatelet Ca2+ levels in platelets isolated from donors both before and after clopidogrel treatment. Data were obtained from fluorescence measurements of gel-filtered platelets loaded with fluo-3. To measure both Ca2+ mobilization from internal stores and Ca2+ influx across the membrane, experiments were performed in the presence of 2 mmol/L EDTA, in the presence of 2 mmol/L Ca2+, and in the presence of 2 mmol/L EDTA followed 10 minutes later by the addition of 4 mmol/L Ca2+. Clopidogrel treatment at a therapeutic dose influenced neither Ca2+ influx nor Ca2+ mobilization from intracellular stores when the platelets were stimulated with 10 µmol/L ADP.
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Discussion |
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A large number of previous studies have shown that thienopyridine compounds selectively inhibit an ADP-dependent pathway of platelet aggregation.4 5 6 7 8 9 10 11 12 13 14 15 18 19 20 21 32 In agreement with these reports, we found that clopidogrel treatment (75 mg/d for 7 days) resulted in, for each of 10 subjects tested, a reduced and reversible platelet aggregation when ADP was added to citrated PRP. We also confirmed that for other agonists, the platelet response was modified only under conditions in which the aggregation response of human platelets is known to be dependent on released ADP and has previously been shown to be decreased when platelets are stimulated in the presence of ADP scavengers (see References 32 and 33). It should be emphasized that clopidogrel, like ticlopidine, has no effect when added to PRP alone and that the active derivative is a metabolite produced in the liver.34 Thus, studies on samples taken after ingestion are a necessary part of understanding its mode of action. Here, we have defined how clopidogrel downregulates the extent to which ADP can induce platelet-to-platelet cohesion and activate their GP IIb/IIIa complexes. We have previously shown by flow cytometry how clopidogrel modifies the capacity of ADP to induce conformational changes in GP IIb/IIIa in unstirred platelet suspensions as detected with the MAb PAC-1,21 and we have extended our results here to the use of the anti-LIBS MAb AP-6 and an anti-fibrinogen antibody, both of which permit the visualization of fibrinogen-occupied GP IIb/IIIa complexes within the loosely bound aggregates that characterize the response of clopidogrel-treated donors to ADP. Studies were performed on citrated PRP rather than on washed platelets,20 as in vitro activation during platelet isolation is a recognized hazard with regard to the detection of activation-dependent markers on platelets.35 36
We also included in our study a patient with a congenital disorder whose platelets show a ticlopidine-like functional response to ADP. The first such patient was described by Cattaneo et al.33 Patient M.L. was reported more recently.23 Specific defects common to thienopyridine-treated normal platelets13 14 15 18 19 20 21 and the above-described patients20 23 33 include (1) an inability of ADP to decrease cAMP levels raised by preincubating platelets with PGE1 or PGI2 and (2) a much decreased binding of radiolabeled 2-MeS-ADP. Clearly, the abnormalities in each situation concern a platelet activation pathway used by ADP. Previously, using PAC-1, AP-6, and two anti-RIBS antibodies in flow cytometry, we had shown that GP IIb/IIIa complexes in unstirred suspensions of platelets of patient M.L. showed a much decreased activation and fibrinogen binding in response to ADP.23 The results presented here for unstirred suspensions of clopidogrel-treated platelets are virtually identical to those previously obtained for M.L., thus providing further evidence that the same pathway is affected. Furthermore, electron microscopy and immunogold localization of occupied GP IIb/IIIa complexes within the ADP-induced aggregates showed that results for the patient and clopidogrel-treated platelets of normal donors were identical with the presence of aggregates composed of a small number of loosely bound platelets with reduced surface AP-6 binding and fewer contact points containing fibrinogen.
Features of the aggregates obtained for untreated normal donors in citrated PRP included their large size, the relatively regular appearance of the close interplatelet spaces between adjoining platelets, and the frequent presence of a peripheral ring composed of degranulated platelets. This layer could also contain the outer portions of platelets that have undergone morphological changes, with the granules being confined to a central body located more deeply within the aggregate. The majority of the labeling with AP-6 and the anti-fibrinogen antibody was found in the interplatelet spaces within the peripheral zone, suggesting that the internal pool of GP IIb/IIIa complexes was now participating in the platelet-to-platelet cohesion. The absence of degranulated platelets from aggregates after clopidogrel treatment and from aggregates obtained for patient M.L. could be a factor in explaining the reversibility of the aggregation that characterizes both situations. Ticlopidine has previously been reported to inhibit ADP-induced dense body secretion from platelets, as assessed by measurement of serotonin and ATP release.7 8 9 We now show by flow cytometry that clopidogrel also inhibits the surface expression of P-selectin when platelets are stimulated by ADP. In citrated PRP, the release of granule contents and irreversible aggregation induced by ADP are thought to be dependent on feedback mechanisms resulting from the initial platelet-to-platelet binding and to result in thromboxane A2 formation.37 38 The latter then binds to surface receptors to react synergistically with the ADP and amplify the signal. We speculate that the contact interactions in the small aggregates formed by ADP after clopidogrel treatment or with platelets from patient M.L. are insufficient to induce this amplification step. Thromboxane A2 formation is known to be favored by the low Ca2+ levels in citrated PRP.37 38 With this in mind, studies are planned to compare the morphology of normal platelet aggregates in citrated PRP with those formed by ADP in hirudin- or heparin-anticoagulated blood, in which physiological levels of divalent cations are maintained.
In affecting an early step in the ADP-induced aggregation pathway (see below), clopidogrel (and ticlopidine) thus appear to stop the formation (or transmission) of the message leading to granule release by ADP. We show this to be so both in unstirred suspensions of citrated PRP (flow cytometry) and after aggregation has occurred (electron microscopy). A partial platelet degranulation in nonaggregating conditions was assumed by Janes et al39 to be associated with ADP-induced shape change and/or Ca2+ mobilization. In our study with clopidogrel, however, shape-changed platelets with pseudopods were clearly visible on the electron micrographs of the aggregates, and normal Ca2+ influx was quantified for all 10 donors after platelet stimulation with ADP (see below). Our results therefore appear to discount such an association. Interestingly, release of -granules has been observed when ADP was added to unstirred platelets anticoagulated with hirudin and analyzed in the presence of a physiological concentration of Ca2+, suggesting that thrombin is not the cause.39 The phenomenon has also been reported with aspirin-treated platelets, implying that a thromboxane-independent pathway may play a role.40 Although some -granule secretion occurred when we incubated unstirred suspensions of platelets with ADP, this was without substantial mobilization of the internal pools of GP IIb/IIIa complexes (results with AP-2 in Fig 8). In contrast, major morphological changes were seen in the peripheral region of ADP-induced aggregates (Figs 2 and 3), thus suggesting that surface contact interactions participate in the signaling process within the aggregate.
The characteristic morphological appearance of the aggregates from clopidogrel-treated platelets (or from patient M.L.) should also be compared with that observed by others41 in conditions of reversible aggregation induced by 2 µmol/L ADP in citrated PRP or with washed platelets stimulated with 5 µmol/L ADP in the presence of 1 to 2 mmol/L Ca2+.42 The common final step in platelet aggregation is fibrinogen (or another adhesive protein) binding to GP IIb/IIIa complexes, and we have studied this event within aggregates using both anti-fibrinogen antibodies and AP-6, an IgM MAb produced against the 204-to-227 sequence of GP IIIa.22 24 This antibody recognizes a unique LIBS epitope on ADP-stimulated platelets whose expression requires the binding of fibrinogen but is not induced by the dodecapeptide (
400-411) or RGD peptides.22 AP-6 is the only such antibody that we have tested to label ultrathin sections of Lowicryl K4M–embedded samples, permitting the recognition of ligand-bound GP IIb/IIIa in internal membrane systems, including those of -granules.22 It is now accepted that fibrinogen is incorporated by megakaryocytes and platelets into -granules by endocytosis through a GP IIb/IIIa–mediated process.43 Ligand-bound GP IIb/IIIa complexes in the -granule membrane may represent complexes that have yet to release their ligand before recycling.44 Although quantitative determinations have not been made, immunogold labeling with antifibrinogen antibody of platelet -granules from patient M.L. or from donors receiving clopidogrel for 7 days was indistinguishable from that of normal platelets. Labeling of intracellular membranes with AP-6 was unchanged in both situations. Such results suggest that the ADP-dependent activation pathway(s) defective in these platelets are not involved in the incorporation of fibrinogen into platelets or megakaryocytes.
A common conclusion from our studies is that ADP-induced aggregates of clopidogrel-treated normal platelets and those of patient M.L. are composed of a small number of "loosely packed" platelets with a lower-than-normal number of contact points due to the limited activation of GP IIb/IIIa complexes by ADP. Notwithstanding the importance of the inhibition, it should be emphasized that fibrinogen binding was not zero and that the induced defect was not an absence of platelet aggregation, as in Glanzmann's thrombasthenia.45 Although we cannot totally exclude the possibility that a rapidly reversible fibrinogen binding to GP IIb/IIIa was a factor in limiting aggregate size, our results with FITC-fibrinogen point more to a low degree of activation of GP IIb/IIIa and to a level of fibrinogen binding insufficient to permit the formation of the macroaggregates. Nevertheless, the initial rate of aggregation both of clopidogrel-treated platelets and of those from patient M.L.23 was normal, suggesting that the number of occupied receptors required to start the aggregation process and bring platelets together is low. Another interesting feature highlighted in the aggregates of clopidogrel-treated platelets was the presence of occasional tight junctions with little or no staining with antifibrinogen antibody or AP-6. Such junctions have previously been observed in aggregates of washed human platelets,28 42 and the mechanism leading to their formation remains a mystery. Although others, in experiments on rabbit or washed human platelets, have observed a reversible binding of 125I-fibrinogen to ADP-stimulated platelets,46 47 we obtained no evidence to suggest that the disaggregation observed with high-dose ADP– and clopidogrel-treated platelets (or those from patient M.L.) is related to the loss of fibrinogen from activated GP IIb/IIIa complexes. Nevertheless, it is interesting to speculate that the absence of secondary clustering of occupied receptors48 brought about by the reduced fibrinogen binding contributes to aggregate instability.
Others have shown by quantification of ADP-induced shape change in aggregometry that it is unchanged by thienopyridines.12 18 32 Because shape change occurs in the presence of EDTA, this process can proceed through Ca2+ mobilization from internal pools alone. Our results provide morphological evidence for the presence of pseudopods on platelets within ADP-induced aggregates obtained after clopidogrel treatment. Studies on both human and rat platelets have shown that ADP-induced Ca2+ influx is unchanged by ticlopidine and clopidogrel.13 20 We have confirmed this finding under our experimental conditions for human platelets stimulated with both ADP and 2-MeS-ADP. Thus, two independent ADP-induced platelet responses, shape change and Ca2+ influx, are unaffected by clopidogrel. Although Feliste et al49 showed that in rat platelets, high doses of PCR 4099 inhibit Ca2+ mobilization from internal stores, up to 100 mg/kg of thienopyridine were used. This is something that we did not see with a therapeutic dose of clopidogrel. Although we did not measure ADP-induced mobilization of Ca2+ in the platelets of patient M.L., a somewhat decreased total level was seen for the patient studied by Cattaneo et al,33 making possible a defective ADP-induced release from internal stores. This aspect of platelet Ca2+ metabolism and the effect of high doses of thienopyridines require further study.
Binding studies using [3H]-2-MeS-ADP have demonstrated a residual population of 2-MeS-ADP binding sites unaffected by clopidogrel in both human18 21 and rat platelets,19 20 and this situation is repeated for both reported patients with a ticlopidine-like inherited defect of the ADP activation pathway.20 23 For example, results for our laboratory showed that the number of high-affinity binding sites for [3H]-2-MeS-ADP on platelets was reduced to about 30 for the patient compared with 836±126 (mean±SD, n=3) for normal subjects.23 This was very similar to the value of 32±5 (mean±SD, n=12) that we previously determined for platelets from another series of donors who had received clopidogrel.21 Thus, as described by Gachet et al,20 we favor a two-receptor model in which the initial platelet response to ADP is linked to a ligand-gated ion channel and aggregation to a second receptor. The latter has been described as belonging to the P2T class of purinergic receptor.50 Although Gachet et al showed that ADP-induced aggregation of rat platelets is totally inhibited after the administration of much increased doses of clopidogrel (25 mg/kg), some caution is needed in interpreting results obtained with high doses of the drug. For example, at high doses, clopidogrel has been shown to have nonspecific effects on platelet aggregation in a thrombosis model.51 Our results leave open the possibility that a limited activation of GP IIb/IIIa and a reversible platelet aggregation can be mediated by ADP through direct activation of an ion channel. As an alternative explanation, the reversible aggregation may be mediated by a small population of P2T receptors unaffected by clopidogrel and residual in the platelets of M.L.23 Nevertheless, our results suggest that it is the P2T receptor pathway, which includes a link to adenylate cyclase, that is required for the full activation of GP IIb/IIIa complexes and the formation of large stable aggregates.
Recently, clopidogrel has been demonstrated to impair thrombus growth at different shear rates without diminishing platelet adhesion to collagen in an experimental model of thrombogenesis based on the use of flowing nonanticoagulated human blood.10 11 As with our results, these authors observed a loosely packed thrombus after clopidogrel intake. It was suggested that in this model, the antithrombotic effect of clopidogrel was due to its inhibition of ADP released during the initial platelet-collagen interaction. The theory that ADP plays an important role in both thrombus formation and stabilization is also supported by studies using a rat model4 and by the results obtained with human blood in a perfusion chamber showing that it is not the initial but rather the later phases of thrombus growth that require thrombin generation.52 In conclusion, it is likely that ADP plays a key role in thrombogenesis in humans, explaining the antithrombotic effects of drugs such as clopidogrel. We show that the latter interfere with a specific ADP-dependent step of GP IIb/IIIa complex activation. This impairs the formation of the platelet-to-platelet contacts needed for normal thrombus growth and stabilization.
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Acknowledgments |
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The authors thank the CNRS, the University of Bordeaux II (DRED), CRAMA/INSERM (contract No. 9.263), the Conseil Regional de l'Aquitaine, the MENESR (ACC-SV9), and the Centre Hospitalier Regional de Bordeaux for providing financial support for their work. Dr Kunicki also acknowledges support from the National Heart, Lung, and Blood Institute (grant HL-46979). We also thank Professor J.C. Faverel-Garrigues and Dr F. Penouil (CHR Hopital Pellegrin) for supervising the administration of clopidogrel to selected volunteers. Christine Gaud and Mari-Claude Pinchart acted as monitors for the study. Joelle Winckler and Annie Paponneau provided excellent technical assistance at various times during this work.
Received September 18, 1995;
revision received April 26, 1996;
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C. Bihour, C. Durrieu-Jais, L. Macchi, C. Poujol, P. Coste, P. Besse, P. Nurden, and A. T. Nurden Expression of Markers of Platelet Activation and the Interpatient Variation in Response to Abciximab Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 212 - 219. [Abstract] [Full Text] [PDF]
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J. A. Remijn, Y.-P. Wu, E. H. Jeninga, M. J.W. IJsseldijk, G. van Willigen, P. G. de Groot, J. J. Sixma, A. T. Nurden, and P. Nurden Role of ADP Receptor P2Y12 in Platelet Adhesion and Thrombus Formation in Flowing Blood Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 686 - 691. [Abstract] [Full Text] [PDF]
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