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 TFpos-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 TFpos-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%; TFpos-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 TFpos-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 TFpos-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 TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Heterotrimeric G proteins demonstrate differential sensitivity to beta-arrestin dependent desensitization.

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing beta2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and delta opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 alpha subunit (as opposed to Galphaq and Galphas) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by beta-arrestin. Interestingly, co-expression of Galpha15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced beta2 adrenergic receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.

Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone.

Aging is associated with remodeling of the immune system, contributing to increased incidence of infections, autoimmune diseases, and cancer among the elderly. Alterations in several signal transduction pathways have been reported to play an important role in immunosenescence. We show that peripheral blood leukocytes obtained from old donors (> or =65 years) have a significantly reduced expression of receptor for activated C kinase 1 (RACK-1), a protein required for protein kinase C (PKC)-beta signaling, as compared with young donors (< or =40 years), both in males and females. The decline in RACK-1 immunoboth in reactivity was age-related (Spearman correlation, r=-0.278, P=0.012). All leukocyte subpopulations, namely lympho-monocytes, granulocytes, and B and T cells, showed a similar defect. We also observed a direct correlation between circulating dehydroepiandrosterone (DHEA) and RACK-1 expression in leukocytes (Spearman correlation, r=0.388, P=0.001). Furthermore, in vitro treatment with DHEA resulted in increased RACK-1 expression in leukocytes and lymphocyte proliferation, confirming the role of this hormone in the modulation of its expression and immune functions. A relevant consequence of RACK-1-reduced expression was the observation that release of tumor necrosis factor alpha following lipopolysaccharide challenge and mitogen-induced lymphocye proliferation, which involves PKC-beta activation, was significantly reduced in elderly subjects. Overall, our findings contribute to the understanding of the complex process of immunosenescence and identify age-related loss in immunological responses as partially associated with decreased RACK-1 expression.

Two Distinct P2Y Receptors Are Involved in Purine- and Pyrimidine-Evoked Ca2+ Elevation in Mammalian Brain Astrocytic Cultures.

ATP and 2-methyl-thio-ATP (2-Me-SATP) increase cytosolic calcium concentrations ([Ca2+]i) in rat striatal astrocytes (Centemeri et al. [1997] Br J Pharmacol 121:1700-1706). The aim of the present study was to: (1) characterize pyrimidine-induced [Ca2+]i increases in the same experimental system, and (2) try to identify the multiple P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP triggered a concentration-dependent [Ca2+]i elevation (EC50s = 0.58 µM ± 0.4 and 31 µM ± 6, respectively).Pyrimidine-evoked [Ca2+]i elevation was solely due to mobilization from intracellular stores, because: (1) removing calcium from extracellular medium or (2) blocking its influx with Ni2+ did not modify UTP responses; (3) the store-depleting agent thapsigargin completely abolished UTP-evoked [Ca2+]i increments. Guanosine-5'-O-(2-thiodiphosphate) partially inhibited the UTP response, whereas pertussis toxin (PTx) had no effect. The phospholipase C inhibitor U-73122 significantly reduced the UTP-evoked [Ca2+]i rise. Computer-assisted analysis indicated that the UTP and UDP responses are mediated by a single receptor, while ATP and 2-Me-SATP interact with two distinct receptors. The selective P2Y1 receptor antagonist MRS2179 abolished the ATP higher potency component. Sequential challenges with the same nucleotides resulted in almost complete homologous desensitization. Pre-exposure to UTP lowered the subsequent responses to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and ATP were not additive. In conclusion, [Ca2+]i elevation in astrocytes by purines and pyrimidines is mediated by two distinct P2Y receptors, likely the P2Y1 and P2Y6 subtypes.