1. Is TRAP-6 suitable as a positive control for platelet reactivity when assessing response to clopidogrel?
Thomas Gremmel, Andreas Calatzis, Sabine Steiner, Alexandra Kaider, Daniela Seidinger, Renate Koppensteiner, Christoph W Kopp, Simon Panzer Platelets. 2010;21(7):515-21. doi: 10.3109/09537104.2010.493587.
Adenosine 5'-diphosphate (ADP) inducible aggregation is used to assess platelet response to thienopyridines. Thrombin receptor-activating peptide-6 (TRAP-6) inducible aggregation may serve as a positive control because it acts via the thrombin receptor protease-activating receptor-1, which is not blocked by thienopyridines. We therefore investigated if TRAP-6 is suitable as a positive control when assessing residual platelet reactivity to ADP. Platelet response to clopidogrel was assessed in 200 patients on dual antiplatelet therapy using ADP inducible platelet aggregation by light transmission aggregometry (LTA), multiple electrode aggregometry (MEA), and the shear-dependent Impact-R. Test specificities were monitored by TRAP-6 inducible platelet aggregation. The aggregation-independent vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay served for comparisons. ADP inducible aggregation was correlated to that by TRAP-6 (r = 0.33 to 0.72; p < 0.001 for all assays). A linear correlation was seen within MEA (r = 0.72). LTA TRAP-6 correlated weakly with the VASP assay (r = 0.19; p = 0.01), while there were no correlations of TRAP-6 responses by MEA or the Impact-R with the VASP assay (r = 0.03 and -0.09; p > 0.05). In all three assays, differences between ADP and TRAP-6 inducible aggregation varied considerably. Within MEA, TRAP-6 inducible aggregation was almost always stronger than ADP inducible aggregation, while within LTA and the Impact-R, weak responses to ADP were associated with both, weak and strong responses to TRAP-6. In conclusion, the application of TRAP-6 as a positive control for platelet reactivity has major limitations and results need to be cautiously interpreted on an individual basis.
2. Antiplatelet Activity of Isorhamnetin via Mitochondrial Regulation
Lyanne Rodríguez, Lina Badimon, Diego Méndez, Teresa Padró, Gemma Vilahur, Esther Peña, Basilio Carrasco, Hermine Vogel, Iván Palomo, Eduardo Fuentes Antioxidants (Basel). 2021 Apr 25;10(5):666. doi: 10.3390/antiox10050666.
With the diet, we ingest nutrients capable of modulating platelet function, which plays a crucial role in developing cardiovascular events, one of the leading causes of mortality worldwide. Studies that demonstrate the antiplatelet and antithrombotic potential of bioactive compounds are vital to maintaining good cardiovascular health. In this work, we evaluate the flavonol isorhamnetin's antiplatelet effect on human platelets, using collagen, thrombin receptor activator peptide 6 (TRAP-6), and phorbol myristate acetate (PMA) as agonists. Isorhamnetin induced a significant inhibition on collagen- and TRAP-6-induced platelet aggregation, with half-maximum inhibitory concentration (IC50) values of 8.1 ± 2.6 and 16.1 ± 11.1 µM, respectively; while it did not show cytotoxic effect. Isorhamnetin reduced adenosine triphosphate levels (ATP) in platelets stimulated by collagen and TRAP-6. We also evidenced that isorhamnetin's antiplatelet activity was related to the inhibition of mitochondrial function without effect on reactive oxygen species (ROS) levels. Additionally, we investigated isorhamnetin's effect on thrombus formation in vitro under flow conditions on the damaged vessel wall. In this context, we demonstrate that isorhamnetin at 20 µM induced a significant inhibition on platelet deposition, confirming its antithrombotic effect. Our findings corroborate the antiplatelet and antithrombotic potential of isorhamnetin present in many foods of daily consumption.
3. Antiplatelet Activity of Cucurbita maxima
Sigrid Sanzana, Lyanne Rodríguez, Hayleen Barraza Barrionuevo, César Albornoz Poblete, Mário Roberto Maróstica Junior, Eduardo Fuentes, Iván Palomo J Med Food. 2021 Nov;24(11):1197-1205. doi: 10.1089/jmf.2021.0006. Epub 2021 Aug 31.
Natural extracts constitute an important source in the prevention of noncommunicable diseases, such as cardiovascular diseases. The pumpkin, Cucurbita maxima, is widely consumed in Chile. Pumpkin seeds, despite having crude protein, lipids, and carbohydrates, are regarded as agro-industrial waste. In this work, we correlated the antiplatelet activity of aqueous, ethanolic, and methanolic extracts from pumpkin seeds with their bioactive compounds. In vitro platelet aggregation and activation studies were performed by turbidimetry and flow cytometry, respectively. Results reveal that the extracts inhibited, in a dose-dependent manner, platelet aggregation induced by adenosine diphosphate, thrombin receptor activator peptide 6 (TRAP-6), and collagen. Pumpkin seed extracts inhibited P-selectin secretion and glycoprotein IIb/IIIa activation on TRAP-6-activated platelets. They were found to be rich in fatty acids and a powerful source of plant-based protein, which could be related to the high antiplatelet potential identified in extracts. This research demonstrated that pumpkin seed extracts could be a candidate in the prevention of thrombotic events.
