Search

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI2). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI2. After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alphaIIbbeta3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to alphaIIbbeta3, particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alphaIIbbeta3. The distance between these sites was estimated to be 6.1 +/- 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta3-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alphaIIbbeta3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in alphaIIbbeta3 and initiate different functional consequences within the receptor itself and within platelets.

Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency. Thus, proper prodrugs carrying the 5'-pdFdC moiety may help to overcome this problem. A series of new derivatives of gemcitabine possessing N-acyl(thio)phosphoramidate moieties were prepared and their cytotoxic properties were determined. N-Acyl-phosphoramidate derivatives of gemcitabine have similar cytotoxicity as gemcitabine itself, and have been found accessible to the cellular enzymes. The nicotinic carboxamide derivative of gemcitabine 5'-O-phosphorothioate occurred to be the best inhibitor of bacterial DNA polymerase I and human DNA polymerase α.

Efficient Pd(0)-catalysed synthesis of 5-alkynyl-1-β-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide depends on the presence of different protecting groups of the ribose moiety. Peracetylated 5-iodo substrate (15) couples with terminal alkynes or trimethyl-[(tributylstannyl)ethynyl]silane in 50-71% and 72% yield (ETCAR), respectively, although its hydrodehalogenation to 19 is noticeable. On the other hand, hydrodehalogenation of acetonide (16) predominates over coupling with terminal alkyne and slightly decreases a yield of cross-coupling reaction with trimethyl[(tributylstannyl)ethynyl]silane. Alternative conditions of reaction with terminal alkynes, to exclude so far identified hydride sources to produce hydridopalladium species, have been established for acetonide 16 and allowed to achieve 72% of coupling. Fluoromethyl derivative (42) was prepared from its 5-hydroxymethyl precursor by fluorination with DAST. Additionally, X-ray structural analysis of 42 was performed. All 1,2,3-triazolonucleosides and two synthesized cycloSal-pronucleotides were evaluated for cytotoxic activity against K562, HeLa and HUVEC cells.

A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture. Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin. The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2. These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture. The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP).

The intracellular ability of the “10–23” DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mm Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10–23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mm Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mm Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mm Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.

Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.