Development of small-molecule fluorescent probes targeting neutrophils -formyl peptide receptors.

-Formyl peptide receptors (FPRs) are membrane receptors that are abundantly expressed in innate immune cells, including neutrophils and platelets, demonstrating potential new targets for immune system regulation and the treatment of inflammatory conditions. We report here the development and bio-physical validation of new FPR imaging agents as effective tools to track FPR distribution, localisation and functions, ultimately helping to establish FPR exact roles and functions in pathological and physiological conditions. The new series of probes feature a small molecule-based FPR address system conjugated to suitable fluorophores, resulting in highly specific FPR agents, including a partial agonist endowed with high affinity ( low/sub-nanomolar potency) on FPR-transfected cells and human neutrophils.

Evidence that inositol 1,4,5-trisphosphate 3-kinase and inositol 1,3,4,5-tetrakisphosphate are negative regulators of platelet function.

Background: Inositol 1,3,4,5-tetrakisphosphate (IP) is formed from inositol 1,4,5-trisphosphate (IP) by IP 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca entry, IP regulation, and phosphoinositide 3-kinase antagonism.

Objectives: To better elucidate a function for IP, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP) binding to pleckstrin-homology (PH) domain-containing proteins in platelets.

Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca(2+) elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A2 formation in human platelets.

Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+).

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood.