Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis-derived platelet concentrates.

BACKGROUND: Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)-dependent signaling systems in stored PLTs was investigated. STUDY DESIGN AND METHODS: Whole blood samples (WB) and apheresis-derived PLT concentrates (APCs) were obtained from healthy volunteers. Washed PLTs were prepared from WB on Day 0 and from APCs on Days 0, 2, and 5. The basal phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and phosphodiesterase 5A (PDE5A) levels were quantified by Western blot. CN and PDE5A activity were measured by enzyme-linked immunoassay kits. Fibrinogen binding and aggregation were measured in PLT-rich plasma of WB or APC samples. Unpaired t test was used for statistical analysis. RESULTS: Basal VASP phosphorylation levels were comparable in WB and APCs on Day 0. VASP phosphorylation increased significantly during storage of APCs, more pronounced at Ser(239) than at Ser(157) . Similarly, intracellular cGMP, but not cAMP, concentration continuously increased in stored PLTs, whereas PDE5A levels and activity significantly decreased accompanied by diminished thrombin receptor activator peptide 6-induced fibrinogen binding and aggregation. CONCLUSION: Storage of APCs leads to intracellular cGMP accumulation that could be caused by degradation of PDE5A. Enhanced cGMP level supports subsequent cGMP-dependent protein kinase-mediated increase of VASP phosphorylation resulting in reduced fibrinogen binding and aggregation.

Dasatinib enhances migration of monocyte-derived dendritic cells by reducing phosphorylation of inhibitory immune receptors Siglec-9 and Siglec-3.

The SRC family of kinases (SFKs) is crucial to malignant growth, but also important for signaling in immune cells such as dendritic cells (DCs). These specialized antigen-presenting cells are essential for inducing and boosting specific T-cell responses against pathogens and malignancies. Targeted therapy with SFK inhibitors holds great promise as a direct anti-cancer treatment, but potentially also as an indirect treatment via immunomodulation. Here, we investigated whether the BCR-ABL/SRC inhibitor dasatinib would modulate the major effector functions of DCs, especially their migration, a prerequisite to interaction with lymphocytes in secondary lymphoid organs. We report for the first time that dasatinib more than doubled the number of mature human monocyte-derived DCs (moDCs) migrating toward a CCL19 gradient despite unchanged CCR7 expression when used for pretreatment. These effects were caused by dephosphorylation of SFKs, as confirmed by the specific SFK inhibitor SRC inhibitor 1, leading to dephosphorylation of the inhibitory immunoreceptors Siglec-9 and Siglec-3. The specific blocking of the latter also enhanced migration and underlined the importance of these SFK-dependent receptor systems for migration of moDCs. Dasatinib hampered the secretion of interleukin-12 by moDCs at clinically relevant concentrations. In contrast, endocytosis or boosting of cytomegalovirus-specific CD8(+) T-cell responses remained unaltered when applying dasatinib-pretreated moDCs, in line with minor effects on the expression of co-stimulatory molecules essential for DC-T cell interaction. The induction of enhanced migration of moDCs may potentially be useful in chemo-immunotherapeutic applications. Thus, the use of dasatinib or blocking Siglec antibodies as adjuvants in this setting to induce stronger immune responses is worthy of further study.