Claude Bihour

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

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.

Clopidogrel: An Antithrombotic Drug Acting on the ADP-dependent Activation Pathway of Human Platelets

The aim of the study was to determine the effect of clopidogrel on adenosine diphosphate (ADP)- induced platelet activation in human volunteers. Platelets from human volunteers before and after a 7-day treatment with clopidogrel (75 mg/kg), were tested for their sensi tivity to ADP by measuring ADP-induced aggregation, adenylyl cyclase downregulation, and [3H]-2-MeS-ADP binding. Platelet membrane glycoprotein (GP IIb-IIIa; GP Ib, GMP-140) expression was measured by flow cy tometry using fluorescent-labeled antibodies or fibrino gen. After oral administration to human volunteers (75 mg/day for 7 days), clopidogrel, a novel ADP-selective antiplatelet agent, inhibited ADP-induced aggregation of platelets ex vivo. This effect was irreversible in nature, and no activity could be detected in the plasma of treated subjects. Although clopidogrel did not modify ADP- induced shape change, it prevented the inhibitory effect of ADP (but not that of epinephrine) on the prostoglandin- E 1 (PGE1)-induced increase in platelet cAMP. The num ber of binding sites for [ 3H]-2-MeS-ADP, a stable ana logue of ADP that labels ADP binding sites linked to the inhibition of stimulated adenylyl cyclase, was reduced from 525 ± 62 sites/cell in the controls to 32 ± 5 sites/cell after treatment with clopidogrel (p < 0.001). This effect occurred with no consistent change in the binding affinity of [3H]-2-MeS-ADP, indicating that inhibition of platelet functions by clopidogrel was mainly due to a selective and irreversible reduction of ADP binding sites on plate lets. Flow cytometry experiments showed that clopi dogrel selectively inhibited ADP-inducing binding of fi brinogen to platelets. This effect occurred through a ma jor reduction of the ADP-induced activation of the GP IIb-IIIa complex. These findings therefore indicate that clopidogrel downregulates platelet responses via a selec tive and direct interaction with the ADP receptors, me diating the inhibition of stimulated adenylyl cyclase ac tivity in human platelets.

Markers of platelet activation in coronary heart disease patients.

Abstract. We have applied flow cytometry to the detection of activated platelets in patients with coronary heart disease. Paraformaldehyde‐fixed platelets were incubated with one of the following monoclonal antibodies (MAbs): Bx‐1 (anti‐GP Ib), AP‐2 (anti‐GP IIb‐IIIa complex), VH10 (anti‐GMP‐140, a glycoprotein of the α‐granule membrane), or PAC‐1 (directed against an activation‐dependent determinant on GP IIb‐IIIa complexes). Bound antibody was quantitated after the addition of FITC‐conjugated anti‐immunoglobulin. This report highlights studies on 16 unstable angina patients undergoing transluminal angioplasty. Blood samples were taken at different periods before and after the angioplasty. Levels of activated platelets were variable, remaining in the 2–4% range of control donors for some, but increasing to 10–30% postangioplasty for others (despite all patients receiving heparin and aspirin). Maximum numbers of activated platelets were detected at 24 or 48 h. Nonetheless, the amount of antibody bound to individual platelets rarely reached the levels seen when control platelets were stimulated with thrombin in vitro. Results with VH10 and PAC‐1 often, but not always, correlated suggesting different pathways of platelet activation.

Platelet activation and thrombosis: Studies in a patient with essential thrombocythemia

Recent advances permit the detection of activated platelets using specific monoclonal antibodies and flow cytometry. Nevertheless, there are few reports in which activated platelets have been studied over a period of time in patients at risk for thrombosis. Our patient S.D. has essential thrombocythemia and a prothrombotic state manifested in two major thrombotic episodes involving the portal vein and a mesenteric artery. Investigation revealed both spontaneous aggregation and hyperaggregability in response to ADP and the presence of activated platelets in platelet‐rich plasma as revealed by flow cytometry. Interestingly, the activated platelets were recognized by an anti‐RIBS (“receptor‐induced binding site”) monoclonal antibody that recognized bound fibrinogen but not by antibodies reactive with antigens whose presence on the platelet surface was secretion dependent. Treatment with aspirin inhibited spontaneous platelet aggregation but had little effect on the activated platelet profile. A change of therapy to ticlopidine suppressed expression of platelet activation markers. Treatment with ticlopidine has continued for 1 year so far without further thrombotic complications.

Confirmation that GP Ib-IX complexes have a reduced surface distribution on platelets activated by thrombin and TRAP-14-mer peptide

Summary. In 1990 we reported that GP Ib‐IX complexes accumulated within the surface‐connected canalicular system (SCCS) of thrombin‐stimulated platelets. This conclusion was reached following investigations using monoclonal antibodies (MAbs) in flow cytometry and a polyclonal antibody to GP Iba in electron microscopy with immunogold staining performed on ultrathin sections of resin‐embedded platelets. Recent controversy concerning these results has prompted us to perform further studies using 14 anti‐GP Ib‐IX MAbs obtained from the 1993 Boston Workshop on Leukocyte Antigens. Features were the use of the MAbs in mixtures and the fact that immunogold staining was performed on frozen thin sections. Platelets were stimulated with either a‐thrombin or TRAP‐14‐mer peptide. In all cases a decreased density of GP Ib‐IX complexes on exposed areas of the activated platelet surface was accompanied by an increased expression within the SCCS. At the same time we noted that when platelets were stimulated with TRAP‐14‐mer they progressively exhibited a different internal morphology in comparison to that seen with thrombin. In particular, the dense central mass disappeared and large vacuoles were present throughout the cytoplasm. Overall, these studies confirm that changes in the distribution of GP Ib‐IX complexes which follow thrombin‐induced platelet activation (i) are indeed observed when antibody mixtures are used to detect them, and (ii) are mediated through the receptor recognized by the TRAP‐14‐mer peptide.

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb–IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63‐year‐old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb‐IIIa complex (AP2, P2). The patients platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patients serum inhibited ADP‐induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patients platelets. Levels of GP Ib‐IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb‐IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ibα in addition to the band in the normal position of GP Ibα. Finally, ADP‐stimulated (E.B.) platelets failed to express activation‐dependent epitopes on GP IIb‐IIIa as recognized by PAC‐1, AP6, or F26 and additionally gave a reduced P‐selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex‐dependent determinant on GP IIb–IIIa and inhibitory of platelet aggregation. Am. J. Hematol. 57:164–175, 1998.