The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes.

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic β cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans.

Folic acid-conjugated 4-amino-phenylboronate, a boron-containing compound designed for boron neutron capture therapy, is an unexpected agonist for human neutrophils and platelets.

Boron neutron capture therapy (BNCT) is an anticancer treatment based on the accumulation in the tumor cells of (10) B-containing molecules and subsequent irradiation with low-energy neutrons, which bring about the decay of (10) B to very toxic (7) Li(3+) and (4) He(2+) ions. The effectiveness of BNCT is limited by the low delivery and accumulation of the used (10) B-containing compounds. Here, we report the development of folic acid-conjugated 4-amino-phenylboronate as a novel possible compound for the selective delivery of (10) B in BNCT.

Biocompatibility of functionalized boron phosphate (BPO4) nanoparticles for boron neutron capture therapy (BNCT) application.

Unlabelled: Boron neutron capture therapy (BNCT) is a radiotherapy treatment based on the accumulation in the tumor of a (10)B-containing drug and subsequent irradiation with low energy neutrons, which bring about the decay of (10)B to (7)Li and an α particle, causing the death of the neoplastic cell. The effectiveness of BNCT is limited by the low delivery and accumulation of the used boron-containing compounds. Here we report the development and the characterization of BPO4 nanoparticles (NPs) as a novel possible alternative drug for BNCT.

Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca(2+)-dependent activation of platelet Rap1b GTPase.

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A).

Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: a study with bio-orthogonal chemical probes.

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C.

Nanoparticles induce platelet activation in vitro through stimulation of canonical signalling pathways.

Unlabelled: Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)β3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2.

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling.

Integrin α2β1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3Kβ(KD)), but occurred normally in PI3Kγ(KD) platelets.

Epinephrine-mediated protein kinase C and Rap1b activation requires the co-stimulation of Gz-, Gq-, and Gi-coupled receptors.

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A.

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A).

Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples.

Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/10⁶ RBCs.

On the association of lipid rafts to the spectrin skeleton in human erythrocytes.

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs).

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V.

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibalpha of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization.

Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes.

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes.

Resistance of human erythrocyte membranes to Triton X-100 and C12E8.

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8.

The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets.

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase.

Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets.

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction.

Neutrophil granulocytes uniquely express, among human blood cells, high levels of Methionine-sulfoxide-reductase enzymes.

L-Methionine (Met), in its free form or when inserted in proteins, is sensitive to oxidation of its thioether group by reactive oxygen species from exogenous or endogenous sources. Two stable diastereomers of Met sulfoxide [Met-(O)] may be formed [Met-S-(O) and Met-R-(O)], but these can be reduced by two classes of Methionine-sulfoxide-reductase (Msr) enzymes: MsrA, which reduces the S, and MsrB, which reduces the R sulfoxide. In this study, we have examined the levels of expression of Msr in human blood cells by enzymatic activity assay, Western blotting, and RT-PCR of purified populations of polymorphonuclear neutrophils and eosinophils, mononuclear cells, platelets, and erythrocytes.

The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgammaRIIA-mediated cell activation.

Activation of human platelets by cross-linking of the low-affinity receptor for immunoglobulin G (FcgammaRIIA) is initiated by Src kinase-mediated phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) within the receptor, but the identity of the enzyme responsible for its dephosphorylation and inactivation is unknown. Here we report that the 18-kDa low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) is expressed in human platelets and undergoes subcellular redistribution upon FcgammaRIIA cross-linking. In vitro, LMW-PTP was found to efficiently dephosphorylate activated FcgammaRIIA and LAT, but not Syk or phospholipase Cgamma2.

Epinephrine induces intracellular Ca2+ mobilization in thrombin-desensitized platelets: a role for GPIb-IX-V.

In this work we have investigated the ability of epinephrine to trigger the release of intracellular Ca2+ in thrombin-desensitized platelets. Addition of thrombin to platelets in the presence of extracellular EGTA caused a rapid and transient release of Ca2+ from intracellular stores and rendered platelets unresponsive to a second addition of the same agonist. Although epinephrine alone had no effect on intracellular Ca2+ mobilization, its addition to thrombin-desensitized platelets was associated to a rapid and evident secondary release of intracellular Ca2+.

Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation.

Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F.

The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3.

The involvement of the small GTPase Rap1b in platelet integrin alpha2beta1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin alpha2beta1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)-derived second messengers.

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins.

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody.