Abstract Number: LPB0008
Meeting: ISTH 2021 Congress
Background: Clopidogrel treatment is effective, besides inhibiting platelet aggregation, also in impairing platelet procoagulant activity which is supported by both phosphatidylserine and Tissue Factor (TF) exposure. Platelet-ADP stimulation results in TF expression. However, the role of the 2 ADP purinergic receptors in platelet-TF modulation is still unknown.
Aims: To assess:
1) whether P2Y1 and P2Y12 receptors equally contribute to ADP-induced TF expression;
2) whether clopidogrel treatment modulate TF exposure;
3) TF intracellular localization through a pharmacological approach.
Methods: Platelets from healthy subjects were treated with the P2Y1 antagonist MRS-2250 or the P2Y12 antagonist AR-C69931MX (1pM–100nM). Expression of platelet-TF and P-selectin, as marker of alpha-granule secretion, were analyzed by flow cytometry upon ADP stimulation (10µM) and pA2 of the two P2Y-antagonists to inhibit their expression was calculated. The relationship between TF expression and the clopidogrel antiplatelet effect, assessed by the VASP-assay, was investigated in 106 coronary artery disease (CAD) patients.
Results: P2Y12-antagonist, but not P2Y1-antagonist, concentration-dependently prevented ADP-induced TF exposure. TF-activity, measured by thrombin generation assay, behaved similarly, being completely inhibited at 100nM AR-C69931MX. Platelet-TF expression in CAD patients with poor clopidogrel response (VASP>50%) was significantly higher compared to good-responders. Conversely, the extent of P-selectin inhibition was comparable between the two groups indicating that clopidogrel concentration able to inhibit P-selectin are less effective in reducing TF exposure. Indeed, AR-C69931MX pA2 to inhibit TF is 150-times lower than that of P-selectin. Inhibition of open canalicular system (OCS) by cytochalasin D impaired ADP-induced TF expression but it did not affect P-selectin, suggesting a different intracellular localization of the two proteins.
Conclusions: Data indicate that 1) P2Y12 only regulates surface expression of OCS-stored TF; 2) the therapeutic benefits of clopidogrel may also rely on inhibition of platelet-TF expression, although at higher concentrations than those required to regulate alpha-granule secretion.
To cite this abstract in AMA style:Brambilla M, Canzano P, Rovati G, Cosentino N, Becchetti A, Campodonico J, Cattaneo M, Trabattoni D, Pinna C, Tremoli E, Camera M. ADP-induced Platelet-associated Tissue Factor Expression: Unique Involvement of the P2Y12 Receptor and Modulation by Clopidogrel Treatment [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/adp-induced-platelet-associated-tissue-factor-expression-unique-involvement-of-the-p2y12-receptor-and-modulation-by-clopidogrel-treatment/. Accessed September 25, 2021.
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