Synthesis and evaluation of platelet aggregation inhibitory activity of some 3-phenyl-pyrroloquinazolinones.

A series of 3-phenyl-2,7-dihydro-1H-pyrrolo[3,2-f]quinazolin-1-one derivatives (3-PPyQZ) was synthesized starting from 5-amino-indoles, via condensation with N-ethoxycarbonylthiobenzamides followed by thermal cyclization. On the basis of their structural analogy with reported anti-thrombin pyrroloquinazolines, the derivatives were first tested for their capacity to inhibit platelet aggregation. Some of them had in vitro inhibitory effects on collagen and thrombin-induced aggregation in the micromolar range, and much higher inhibition than that shown by some phenyl-pyrroloquinolinones.

Mitochondrial oxidative stress induced by Ca2+ and monoamines: different behaviour of liver and brain mitochondria in undergoing permeability transition.

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca(2+) concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria.

Regulation of serotonin transport in human platelets by tyrosine kinase Syk.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation.

Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients.

PTPepsilon has a critical role in signaling transduction pathways and phosphoprotein network topology in red cells.

Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. Here, we report that red blood cells (RBCs) from mice lacking PTPepsilon (Ptpre(-/-)) exhibit (i) abnormal morphology; (ii) increased Ca(2+)-activated-K(+) channel activity, which was partially blocked by the Src family kinases (SFKs) inhibitor PP1; and (iii) market perturbation of the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating an alteration of RBC signal transduction pathways. Using the signaling network computational analysis of the Tyr-phosphoproteomic data, we identified seven topological clusters.

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

Background/aim: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation.

Methods: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions.

Inhibitory effect by new monocyclic 4-alkyliden-beta-lactam compounds on human platelet activation.

In the present study some new beta-lactam compounds were screened for their ability to inhibit human platelet activation. In particular four compounds differing in the group on the nitrogen atom of the azetidinone ring were investigated. A beta-lactam having an ethyl 2-carboxyethanoate N-bound group was demonstrated to inhibit, in the micromolar range, both the Ca(2+) release from endoplasmic reticulum, induced either by thrombin or by the ATPase inhibitor thapsigargin, and the Ca(2+) entry in platelets driven by emptying the endoplasmic reticulum.

Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability.

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine.

Thrombin-induced tyrosine phosphorylation of HS1 in human platelets is sequentially catalyzed by Syk and Lyn tyrosine kinases and associated with the cellular migration of the protein.

Thrombin stimulation of platelets triggers Tyr phosphorylation of several signaling proteins, most of which remain unidentified. In this study, we demonstrate for the first time that hematopoietic lineage cell-specific protein 1 (HS1) undergoes a transient Tyr phosphorylation in human platelets stimulated with thrombin. The protein is synergistically phosphorylated by Syk and Lyn tyrosine kinases according to a sequential phosphorylation mechanism.

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

Background/aim: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+.

Methods: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes.

Green tea epigallocatechin-3-gallate inhibits platelet signalling pathways triggered by both proteolytic and non-proteolytic agonists.

Epigallocatechin-3-gallate (EGCG), a component of green tea, inhibits human platelet aggregation and cytosolic [Ca(2+)](c) increases more strongly when these processes are induced by thrombin than by the non-proteolytic thrombin receptor activating peptide (TRAP), thromboxane mimetic U46619, or fluoroaluminate. In line with the previously demonstrated EGCG anti-proteolytic activity, a marked inhibition on aggregation is obtained by pre-incubation of thrombin with EGCG prior to addition to cellular suspension. The catechin also reduces cellular Ca(2+) influx following thapsigargin-induced calcium emptying of endoplasmic reticulum, and the agonist-promoted cellular protein tyrosine phosphorylation.