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

Abstract

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.

Novel Therapeutic Targets at the Platelet Vascular Interface

“…Electron micrographs of aggregated platelets show the close proximity of the plasma membranes of adjacent platelets. [1][2][3] Estimates for the width of the gap between adjacent platelets range from as little as zero to as much as 50 nm. 4 In theory, the short distance between platelets makes it possible for molecules on the surface of one platelet to bind to molecules on an adjacent platelet and for information to be passed, much like a neurological or immunologic synapse.…”

Minding the gaps to promote thrombus growth and stability

“…Electron micrographs show the close proximity of the plasma membranes of adjacent platelets but do not show adherens or tight junctions such as those formed by epithelial and endothelial cells (1)(2)(3). Estimates for the width of the gap between adjacent platelets range from as little as 0 to as much as 50 nm (4).…”

Expression of Markers of Platelet Activation and the Interpatient Variation in Response to Abciximab

“…25 Platelets were stimulated with 0.5 to 8 mol/L ADP, 0.72 mg/mL arachidonic acid, 25 mol/L TRAP 14-mer (SFLLRNPNDKYEPF) purchased from Neosystem, or 1.5 mg/mL ristocetin. Results were expressed as percentage light transmission at maximum aggregation.…”

“…10,25 Fluorescence histograms were obtained for 10 000 cells, data being analyzed using Lysys II software (Becton-Dickinson). Histograms were composed from fluorescence data obtained using a logarithmic mode of amplification.…”