iloprost and cangrelor

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Blood Platelets; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Humans; Iloprost; Phosphorylation; Phosphotyrosine; Platelet Activation; Protein Processing, Post-Translational; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Signal Transduction; src Homology Domains; Tandem Mass Spectrometry

The rationale for monitoring platelet inhibition by thienopyridines for the identification of patients at risk for future recurrent arterial thrombosis or ischemic events is intensively discussed, as well as which monitoring systems are appropriate, robust and reliable. Flow cytometric measurement of phosphorylated VASP (vasodilator-stimulated phosphoprotein), expressed as platelet reactivity index (PRI), is presently "the gold standard method" for evaluating P2Y(12) receptor inhibition. The PFA-100 system, a commercially available and clinically widely used platelet test system, is based on a different principle, not that of VASP phosphorylation. The aim of the present study was to compare the two methods and evaluate whether the conventional PFA-100 collagen/ADP cartridge could be pharmacologically improved to enable its routine clinical use for detection of platelet P2Y(12) receptor inhibition. The effects of increasing concentrations of the competitive P2Y(12) receptor antagonist cangrelor (AR-C69931MX) and the time-dependent effects of a single oral loading dose of clopidogrel (600 mg) were analysed with human whole blood. P2Y(12) receptor inhibition was measured by the VASP/PRI assay and the PFA-100 collagen/ADP cartridge system, with and without preincubation with the prostacyclin analog iloprost (Ilomedin). In vitro addition of iloprost (0.5 nM) enabled PFA-100 collagen/ADP cartridge system detection of P2Y(12) receptor inhibition in whole blood by cangrelor in vitro or by clopidogrel treatment of volunteers. The addition of a prostacyclin analog facilitates PFA-100 collagen/ADP system detection of P2Y(12) receptor inhibition, achieving a sensitivity similar to that of the VASP/PRI reference method. Future studies should now evaluate whether this modified PFA-100 system, like the VASP assay, is a reliable test system for monitoring P2Y(12) receptor inhibition under clinical conditions.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adult; Blood Platelets; Cell Adhesion Molecules; Clopidogrel; Female; Flow Cytometry; Humans; Iloprost; Male; Microfilament Proteins; Middle Aged; Phosphoproteins; Phosphorylation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Receptors, Epoprostenol; Receptors, Purinergic P2Y12; Ticlopidine; Young Adult