Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y₁₂ and Link to Residual Platelet Reactivity

Background: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y₁ and P2Y₁₂ receptors in ADP-induced TF exposure; (2) modulation of TF^pos-platelets in anti-P2Y₁₂-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets.

Methods: The effects of P2Y₁ or P2Y₁₂ antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y₁₂^-/-, and patients with gray platelet syndrome. Ex vivo, P2Y₁₂ inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization.

Results: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TF^pos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y₁₂ inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y₁₂ is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TF^pos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TF^pos-platelets similar to the poor-responder group. Indeed, a stronger P2Y₁₂ inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TF^pos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome.

Conclusions: Data show that TF expression is regulated by P2Y₁₂ and not P2Y₁; P2Y₁₂ antagonists downregulate the percentage of TF^pos-platelets. In clopidogrel good-responder patients, assessment of TF^pos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.