In vitro Research Concerning Effect of Clopidogrel Alone and on Combination with Aspirin and Dypiridamoleon Sedimentation of Erythrocytes

Abstract

Based on extended theory of Derjaguin, Landau and Overbeeck (xDLVO) concerning aggregation of colloids and biological cells it was hypothesized that platelet antiaggregant agents have to reduce the aggregation of erythrocytes also. Applying Einstein-Stokes theory of sedimentation of spheres in viscous media it was concluded that sedimentation of erythrocytes is in fact sedimentation of aggregates of the approximately same size. Consequently, an expected outcome was that addition of antiaggregants in vitro to blood samples from patients with rheumatic or cardiovascular diseases will be the decrease of erythrocytes sedimentation. Starting from usual practice of dual antiaggregant therapy (aspirin and clopidogrel) effects of clopidogrel were compared with effects of clopidogrel plus small concentrations of aspirine and dipyridamole (smaller that their concentrations in plasma after in vivo administration) in order to put in evidence a possible synergic effect at platelet membrane level. Whole blood (0.8ml) was collected on 0.1ml 1% EDTA and then was added 25 or 50µl normal saline solution of clopidogrel or of the combination acetylosalycilic acid, clopidogrel and dypyridamole. The final concentrations were 1, 2 and µg/ml, of the same order as cumulated concentration of clopidogrel and its metabolites in clinical pharmacokinetics. Experiments were performed on a number of 40 human blood samples obtained from 2 groups of 20 patients. Sedimentation of erythrocytes was recorded using a camera and captured data were stored on a computer. Global analysis evidenced that in presence of antiaggregants the clusters of the sedimentation curves shifted down and into right, indicating a decrease and delay of sedimentation. Initial slopes and extent of sedimentation decreased linearly on clopidogrel concentration within the 1-3µg/ml range. For comparison of mean curves corresponding to different clopidogrel concentrations it was applied a metric from biopharmacy: areas under plasma concentrations curves (AUC) of drugs. The areas under average sedimentation curves decreased linearly at clopidogrel concentration within the 1-3µg/ml range. The same experiments were performed and similar results were obtained with the triple antiaggregant combination (clopidogrel, acetylosalycilic acid and dipyridamole). Apparently, a synergism between the tested antiaggregants appeared at studied concentration but the number of data was not sufficient to prove the statistical significance of the difference between clopidogrel alone and in triple combination.

Keywords: Bioequivalence Metrics; Blood Cells Aggregation; Clopidogrel; Erythrocyte Sedimentation Curves; xDLVO.

Fig. 1.

Schematic representation of clopidogrel metabolism

Fig. 2.

Effect of clopidogrel on sedimentation curves

Fig. 3.

Effect of clopidogrel on mean sedimentation curves

Fig. 4.

AUC (c) dependence on 1-,2- and 3µg/ml CLO

Fig. 5.

Liniarity of the sedimentation process

Fig. 6.

CLO+ASA+DYP. Dependence of slope on antiaggregant concentration during the first interval

Fig. 7.

CLO+ASA+DYP dependence of maximum sedimentation on concentration

Fig. 8.

CLO+ASA+DYP.Dependence of AUC normalized to control, on antiaggregant concentration

Fig. 9.

Orthokinetic aggregation by capture of the smaller complexes by the large ones

Fig. 10.

Aggregates of approximately same size in sedimenting blood