A novel mechanism of sustained platelet αIIbß3 activation via PEAR1.

Because single nucleotide polymorphisms (SNPs) in platelet endothelial aggregation receptor 1 (PEAR1) are associated with differential functional platelet responses in healthy subjects, we studied the function of PEAR1 in human platelets. During platelet aggregation by various agonists, the membrane expression of PEAR1 and its tyrosine phosphorylation increased. The recombinant PEAR1 EMI domain (GST-EMI) competitively reduced platelet adhesion to surface-coated PEAR1, diminished platelet aggregation, and eliminated PEAR1 phosphorylation. Polyclonal antibodies against the extracellular PEAR1 domain triggered PEAR1 phosphorylation in a src family kinase (SFK)-dependent manner. Such resulted in downstream signaling, culminating in extensive platelet degranulation and irreversible aggregation reactions interrupted by excess monovalent anti-GST-EMI F(ab) fragments. In resting platelets, the cytoplasmic tail of PEAR1 was found complexed to c-Src and Fyn, but on its phosphorylation, phospho-PEAR1 recruited p85 PI3K, resulting in persistent activation of PI3K and Akt. Thus, αIIbß3 activation was amplified, hence stabilizing platelet aggregates, a signaling cascade fully interrupted by the SFK inhibitor PP1 and the PI3K inhibitor LY294002. This study is the first demonstration of a functional role for PEAR1 in platelet activation, underpinning the observed association between PEAR1 and platelet function in genome-wide association studies.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) impairs the regulation of apoptosis in megakaryocytes by activating NF-κB: a proteomic study.

We previously showed that the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptor VPAC1 are negative regulators of megakaryopoiesis and platelet function, but their downstream signaling pathway that inhibits this process still remained unknown. A combined proteomic, transcriptomic, and bioinformatic approach was here used to elucidate the molecular mechanisms underlying PACAP signaling via VPAC1 in megakaryocytes. Two-dimensional difference gel electrophoresis and tandem MS were applied to detect differentially expressed proteins in megakaryocytic CHRF cells stimulated with PACAP. The majority of the 120 proteins modulated by PACAP belong to the class of "cell cycle and apoptosis" proteins. The up- or down-regulated expression of some proteins was confirmed by immunoblot and immunohistochemical analysis. A meta-analysis of our data and 12 other published studies was performed to evaluate signaling pathways involved in different cellular models of PACAP response. From 2384 differentially expressed genes/proteins, 83 were modulated by PACAP in at least three independent studies and Ingenuity Pathway Analysis further identified apoptosis as the highest scored network with NF-κB as a key-player. PACAP inhibited serum depletion-induced apoptosis of CHRF cells via VPAC1 stimulation. In addition, PACAP switched on NF-κB dependent gene expression since higher nuclear levels of the active NF-κB p50/p65 heterodimer were found in CHRF cells treated with PACAP. Finally, a quantitative real time PCR apoptosis array was used to study RNA from in vitro differentiated megakaryocytes from a PACAP overexpressing patient, leading to the identification of 15 apoptotic genes with a 4-fold change in expression and Ingenuity Pathway Analysis again revealed NF-κB as the central player. In conclusion, our findings suggest that PACAP interferes with the regulation of apoptosis in megakaryocytes, probably via stimulation of the NF-κB pathway.

Thrombopoietic effect of VPAC1 inhibition during megakaryopoiesis.

Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.

Thrombogenic changes in young and old mice upon subchronic exposure to air pollution in an urban roadside tunnel.

Epidemiological studies indicate that elderly persons are particularly susceptible to the cardiovascular health complications of air pollution, but pathophysiological mechanisms behind the increased susceptibility remain unclear. Therefore, we investigated how continuous traffic-related air pollution exposure affects haemostasis parameters in young and old mice. Young (10 weeks) and old (20 months) mice were placed in an urban roadside tunnel or in a clean environment for 25 or 26 days and markers of inflammation and endothelial cells or blood platelet activation were measured, respectively. Plasma microvesicles and pro/anticoagulant factors were analysed, and thrombin generation analysis was performed. Despite elevated macrophage carbon load, tunnel mice showed no overt pulmonary or systemic inflammation, yet manifested reduced pulmonary thrombomudulin expression and elevated endothelial von Willebrand factor (VWF) expression in lung capillaries. In young mice, soluble P-selectin (sP-sel) increased with exposure and correlated with soluble E-selectin and VWF. Baseline plasma factor VIII (FVIII), sP-sel and VWF were higher in old mice, but did not pronouncedly increase further with exposure. Traffic-related air pollution markedly raised red blood cell and blood platelet numbers in young and old mice and procoagulant blood platelet-derived microvesicle numbers in old animals. Changes in coagulation factors and thrombin generation were mild or absent. Hence, continuous traffic-related air pollution did not trigger overt lung inflammation, yet modified pulmonary endothelial cell function and enhanced platelet activity. In old mice, subchronic exposure to polluted air raised platelet numbers, VWF, sP-sel and microvesicles to the highest values presently recorded, collectively substantiating a further elevation of thrombogenicity, already high at old age.