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Drug-Free Platelets Can Act as Seeds for Aggregate Formation During Antiplatelet Therapy.

Hoefer T, Armstrong PC, Finsterbusch M, Chan MV, Kirkby NS, Warner TD - Arterioscler. Thromb. Vasc. Biol. (2015)

Bottom Line: To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite.Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists.They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation.

View Article: PubMed Central - PubMed

Affiliation: From The William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, United Kingdom (T.H., P.C.A., M.F., M.V.C., T.D.W.); and National Heart and Lung Institute, Imperial College London, London, United Kingdom (N.S.K.).

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Interaction between aspirin- or prasugrel active metabolite (PAM)–inhibited platelets and a drug-free subpopulation of platelets. Confocal images of platelet aggregates formed from combined platelet populations containing 80% aspirin-inhibited (red, A) or PAM-inhibited (red, B) platelets and 20% drug-free (green) platelets. Washed platelet aggregates were obtained at the end of 5-minute light transmission aggregometry (LTA) responses stimulated by arachidonic acid (AA; 250 µmol/L), ADP (20 µmol/L), or ristocetin (2 mg/mL). For experiments, platelet suspensions were pretreated with aspirin (30 µmol/L), PAM (3 µmol/L), or corresponding vehicle for 20 minutes, washed and labeled with either PKH67 (green) or PKH26 (red) before mixing and stimulation. Images were processed with Imaris software to show images of inhibited (red) and drug-free (green) platelets. Bottom row of both panel sets show 5-µm median focal sections. Scale bars indicate 20 µm. Each image is representative of images from platelets prepared from at least 4 different individuals.
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Figure 2: Interaction between aspirin- or prasugrel active metabolite (PAM)–inhibited platelets and a drug-free subpopulation of platelets. Confocal images of platelet aggregates formed from combined platelet populations containing 80% aspirin-inhibited (red, A) or PAM-inhibited (red, B) platelets and 20% drug-free (green) platelets. Washed platelet aggregates were obtained at the end of 5-minute light transmission aggregometry (LTA) responses stimulated by arachidonic acid (AA; 250 µmol/L), ADP (20 µmol/L), or ristocetin (2 mg/mL). For experiments, platelet suspensions were pretreated with aspirin (30 µmol/L), PAM (3 µmol/L), or corresponding vehicle for 20 minutes, washed and labeled with either PKH67 (green) or PKH26 (red) before mixing and stimulation. Images were processed with Imaris software to show images of inhibited (red) and drug-free (green) platelets. Bottom row of both panel sets show 5-µm median focal sections. Scale bars indicate 20 µm. Each image is representative of images from platelets prepared from at least 4 different individuals.

Mentions: To elucidate the contribution of drug-free platelets to platelet aggregation, the morphologies of aggregates containing mixed platelet subpopulations were characterized by confocal imaging. Platelet aggregates comprising aspirin-inhibited platelets with a 20% subpopulation of drug-free platelets were characterized by intermingling of platelet populations (Figure 2A). Conversely, aggregates formed from combinations of 80% PAM-inhibited platelets and 20% drug-free platelets demonstrated clear clustering of drug-free platelets to the cores of platelet aggregates on stimulation by ADP or AA. Aggregates formed by stimulation of the mixed platelet populations with ristocetin were always characterized by intermingled platelet populations (Figure 2B). Analysis of confocal images revealed 5× greater clustering of drug-free platelets in the presence of PAM after ADP stimulation than the clustering after stimulation by ristocetin.


Drug-Free Platelets Can Act as Seeds for Aggregate Formation During Antiplatelet Therapy.

Hoefer T, Armstrong PC, Finsterbusch M, Chan MV, Kirkby NS, Warner TD - Arterioscler. Thromb. Vasc. Biol. (2015)

Interaction between aspirin- or prasugrel active metabolite (PAM)–inhibited platelets and a drug-free subpopulation of platelets. Confocal images of platelet aggregates formed from combined platelet populations containing 80% aspirin-inhibited (red, A) or PAM-inhibited (red, B) platelets and 20% drug-free (green) platelets. Washed platelet aggregates were obtained at the end of 5-minute light transmission aggregometry (LTA) responses stimulated by arachidonic acid (AA; 250 µmol/L), ADP (20 µmol/L), or ristocetin (2 mg/mL). For experiments, platelet suspensions were pretreated with aspirin (30 µmol/L), PAM (3 µmol/L), or corresponding vehicle for 20 minutes, washed and labeled with either PKH67 (green) or PKH26 (red) before mixing and stimulation. Images were processed with Imaris software to show images of inhibited (red) and drug-free (green) platelets. Bottom row of both panel sets show 5-µm median focal sections. Scale bars indicate 20 µm. Each image is representative of images from platelets prepared from at least 4 different individuals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587545&req=5

Figure 2: Interaction between aspirin- or prasugrel active metabolite (PAM)–inhibited platelets and a drug-free subpopulation of platelets. Confocal images of platelet aggregates formed from combined platelet populations containing 80% aspirin-inhibited (red, A) or PAM-inhibited (red, B) platelets and 20% drug-free (green) platelets. Washed platelet aggregates were obtained at the end of 5-minute light transmission aggregometry (LTA) responses stimulated by arachidonic acid (AA; 250 µmol/L), ADP (20 µmol/L), or ristocetin (2 mg/mL). For experiments, platelet suspensions were pretreated with aspirin (30 µmol/L), PAM (3 µmol/L), or corresponding vehicle for 20 minutes, washed and labeled with either PKH67 (green) or PKH26 (red) before mixing and stimulation. Images were processed with Imaris software to show images of inhibited (red) and drug-free (green) platelets. Bottom row of both panel sets show 5-µm median focal sections. Scale bars indicate 20 µm. Each image is representative of images from platelets prepared from at least 4 different individuals.
Mentions: To elucidate the contribution of drug-free platelets to platelet aggregation, the morphologies of aggregates containing mixed platelet subpopulations were characterized by confocal imaging. Platelet aggregates comprising aspirin-inhibited platelets with a 20% subpopulation of drug-free platelets were characterized by intermingling of platelet populations (Figure 2A). Conversely, aggregates formed from combinations of 80% PAM-inhibited platelets and 20% drug-free platelets demonstrated clear clustering of drug-free platelets to the cores of platelet aggregates on stimulation by ADP or AA. Aggregates formed by stimulation of the mixed platelet populations with ristocetin were always characterized by intermingled platelet populations (Figure 2B). Analysis of confocal images revealed 5× greater clustering of drug-free platelets in the presence of PAM after ADP stimulation than the clustering after stimulation by ristocetin.

Bottom Line: To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite.Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists.They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation.

View Article: PubMed Central - PubMed

Affiliation: From The William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, United Kingdom (T.H., P.C.A., M.F., M.V.C., T.D.W.); and National Heart and Lung Institute, Imperial College London, London, United Kingdom (N.S.K.).

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Related in: MedlinePlus