Inhibition of protein tyrosine phosphatases suppresses P-selectin exocytosis in activated human platelets

The cytolethal distending toxin (CDT) of Haemophilus ducreyi is encoded by the cdtABC genes, but the composition of active CDT is not known. Both immunoaffinity and metal affinity chromatographic methods were used to purify H. ducreyi CDT from recombinant Escherichia coli strains bearing wild-type or mutated H. ducreyi cdtABC genes. Both affinity-purified preparations contained CdtA, CdtB, and CdtC proteins. These purification efforts also revealed that the formation of a noncovalent CdtB-CdtC complex and production of a fully active CDT complex required the presence of a functional CdtA protein. When purified recombinant CdtB and CdtC proteins were mixed, only very slight CDT activity was detected. In contrast, when a bacterial cell extract containing CdtA was mixed with purified preparations of both CdtB and CdtC, full CDT activity was reconstituted in vitro. These results indicate that CdtA is essential for normal H. ducreyi CDT activity and that CdtA likely modifies or alters either CdtB or CdtC or both to form the active CDT complex.     

Inhibition of protein tyrosine phosphatases suppresses P-selectin exocytosis in activated human platelets

P-selectin (CD62P), a cell adhesion molecule for most leukocytes, is stored in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Upon thrombogenic and inflammatory challenges, P-selectin is rapidly expressed, by exocytosis, on activated platelets and stimulated endothelial cells. However, little is known for the molecular mechanisms governing the regulation of the rapid mobilization of P-selectin in these cells. Here we show that phenylarsine oxide (PAO) and diamide (both were inhibitors for protein tyrosine phosphatases), but not genistein (an inhibitor for protein tyrosine kinases), adenosine, wortmannin and LY294002 (all were inhibitors for phosphatidylinositol 3- and 4-kinases), could inhibit P-selectin exocytosis on activated platelets and could abolish the P-selectin mediated aggregation of activated platelets to neutrophils. However, PAO did not attenuate the P-selectin mediated adhesion of human promyeloid HL-60 cells on the stimulated endothelial cells under flow conditions. Further, PAO had no detectable effects on the exocytosis of P-selectin in the stimulated endothelial cells. These results indicate that protein tyrosine phosphatases are necessary for P-selectin exocytosis on the activated platelets, but not on the stimulated endothelial cells, and suggest that inhibitors for protein tyrosine phosphatases may be potentially valuable for treatment of platelet/leukocyte aggregation.     

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets

Background: P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate.     

Perspectives on the nanocarriers with miRNAs for targeting melanoma stemness through epigenetic regulation

Several research reports delineated the significant role of miRNAs in cancer proliferation, and their modulatory role in cancer mitigation, and drug resistance. Melanoma cells have been acquiring stemness to several chemotherapeutic agents through drug efflux proteins, epigenetic modulation, and DNA repair. miRNAs could be applied as novel therapeutic modalities for treating several kinds of cancers to modulate these mechanisms involved in stemness. Nanocarriers to carry these tumor-targeting miRNAs to modulate stemness are a prominent strategy to overcome their low penetrability, minimal stability, and nonspecificity. We have searched several public databases such as PubMed, Medline, Google scholar, and NLM and obtained the information pertinent to the miRNA-based nanocarrier systems to target stemness through epigenetic modulation in melanomas. This review delineates that various miRNAs can modulate the stemness in melanomas by specific intricate epigenetic signaling, and other cell-based signaling mechanisms. Specific nanocarrier formulations with specific miRNAs are optimal methods to deliver these miRNAs in order to achieve significant entrapment efficiency, loading efficiency, and stability. Furthermore, the combinatorial regimen of FDA-approved chemotherapeutic molecules with tumor-targeting miRNAs and chemotherapy combined with nanocarriers can efficiently deliver the utmost therapeutic window by targeting tumor matrix, invasion, metastasis, and angiogenesis in melanomas. Substantial research should focus on the clinical application of this gene therapy in melanomas using these low immunogenic, highly degradable, and biocompatible combinatorial nanotherapeutic regimens.     

Production of soluble P-selectin by platelets and endothelial cells

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.     

Azo-reductase activated budesodine prodrugs for colon targeting

Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis.     

Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets

The role of platelets in thrombotic vascular disease has been widely studied in rabbits. Yet, in rabbit platelets, there is little known about the α-granules, which contain many of the key effector molecules for thrombosis. In this comparative study of rabbit platelets, we have characterized the structure and expression of P-selectin, an α-granule membrane protein that mediates leukocyte adhesion and thrombus propagation. The sequences of tryptic peptides of rabbit P-selectin show an overall sequence identity of 74% with human P-selectin, and 69–77% identity with cow, dog, mouse, rat and sheep P-selectins. The mean (±S.D.) apparent molecular mass of reduced rabbit P-selectin is 117±7 kDa which is 8 kDa larger than the unreduced protein (109±5 kDa). Rabbit P-selectin appears smaller than human P-selectin, but is comparable to other species P-selectins, that have fewer ‘complement regulatory protein’ repeat domains. Cell membrane labeling experiments and antibody binding studies indicate that rabbit P-selectin is nearly absent from the surface of resting platelets (290±30 molecules cell⁻¹). However, cellular activation with thrombin causes nearly a 30-fold increase in expression to 14 200±1100 molecules cell⁻¹. P-selectin is also be expressed on the surface of rabbit platelets activated by other agonists like ADP, A23817 and epinephrine. This selective expression is explained by immunoelectronmicroscopic studies, which show that rabbit P-selectin is sequestered in the intracellular granules of resting platelets. After cell activation by thrombin, P-selectin is found decorating the external membranes of platelet pseudopodia and the surface connected canalicular system. In summary, these studies of P-selectin in rabbit platelets indicate that it is similar in structure, cell localization and expression to human and other species P-selectins. This suggests that studies of P-selectin in thrombosis in rabbits are likely to provide useful insights into the role of this molecule in human thrombotic vascular disease and related conditions.     

D-d-lactoylglutathione accumulation in activated human platelets

• 1.1. S-d-Lactoylglutathione accumulates in human platelets activated by agonists. Among the tested inducers thrombin is the most active.
• 2.2. The effect is dose and time-dependent. S-d-Lactoylglutathione, corresponding to depleted pool of reduced glutathione, can also be detected in platelets incubated with exogenous methylglyoxal.
• 3.3. A further significant increase was observed in platelets stimulated with trombin in the presence of methylglyoxal.
• 4.4. No change in glyoxalase activities upon platelet stimulation with thrombin was shown.

     

5-Caffeoylquinic acid and caffeic acid orally administered suppress P-selectin expression on mouse platelets

Caffeic acid and 5-caffeoylquinic acid are naturally occurring phenolic acid and its quinic acid ester found in plants. In this article, potential effects of 5-caffeoylquinic acid and caffeic acid on P-selectin expression were investigated due to its significant involvement in platelet activation. First, the effects of 5-caffeoylquinic acid and caffeic acid on cyclooxygenase (COX) enzymes were determined due to their profound involvement in regulating P-selectin expression on platelets. At the concentration of 0.05 microM, 5-caffeoylquinic acid and caffeic acid were both able to inhibit COX-I enzyme activity by 60% (P<.013) and 57% (P<.017), respectively. At the same concentration, 5-caffeoylquinic acid and caffeic acid were also able to inhibit COX-II enzyme activity by 59% (P<.012) and 56% (P<.015), respectively. As expected, 5-caffeoylquinic acid and caffeic acid were correspondingly able to inhibit P-selectin expression on the platelets by 33% (P<.011) and 35% (P<.018), at the concentration of 0.05 microM. In animal studies, 5-caffeoylquinic acid and caffeic acid orally administered to mice were detected as intact forms in the plasma. Also, P-selectin expression was respectively reduced by 21% (P<.016) and 44% (P<.019) in the plasma samples from mice orally administered 5-caffeoylquinic acid (400 microg per 30 g body weight) and caffeic acid (50 microg per 30 g body weight). These data suggest that both 5-caffeoylquinic acid and caffeic acid orally administered can be absorbed and suppress P-selectin expression on mouse platelets.     

Polymeric systems with antithrombin activity for thermally activated targeting

Copolymers of N,N-diethylacrylamide and N-acryloylphthalimide with lower critical solution temperature (LCST) were synthesized by radical copolymerization. Polymeric systems with antithrombin activity and LCST were prepared via a reaction of amino groups of hirudin with phthalimide groups of the copolymers. On increasing hirudin content, LCST of the polymeric systems increased. The antithrombin activity of polymeric systems obtained by hirudin immobilization on copolymer carriers was inversely related to the content of the copolymer, amounting to 6% of the activity of native hirudin.     

Functional dendritic polymer architectures as stimuli-responsive nanocarriers

Stimuli-responsive polymer architectures are molecular systems which evolve with an external signal. The observed changes are mainly decomposition, isomerization, polymerization, activation, supramolecular aggregation, and structural modifications of these molecules. The external stimuli, which can be combined in order to provoke these molecular changes, are numerous. In this review, we have chosen to present an overview on different mechanisms to impart responsiveness to dendritic polymers, with the particular aim of delivery and release of bioactive molecules.     

Stimulus-aggregation coupling in platelets activated with PAF-acether

Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.     

Tyrosine phosphorylation of alpha-actinin in activated platelets

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.     

SNAP-23 is a target for calpain cleavage in activated platelets

The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.     

Selectins ligand decorated drug carriers for activated endothelial cell targeting

New active particulate polymeric vectors based on branched polyester copolymers of hydroxy-acid and allyl glycidyl ether were developed to target drugs to the inflammatory endothelial cell surface. The hydroxyl and carboxyl derivatives of these polymers allow grafting of ligand molecules on the polyester backbones at different densities. A known potent nonselective selectin ligand was selected and synthesized using a new scheme. This synthesis allowed the grafting of the ligand to the polyester polymers, preserving its binding activity as assessed by docking simulations. Selectin expression on human umbilical cord vascular endothelial cells (HUVEC) was induced with the pro-inflammatory bacterial lipopolysaccharide (LPS) or with the nonselective inhibitor of nitric oxide synthase L-NAME. Strong adhesion of the ligand decorated nanoparticles was evidenced in vitro on activated HUVEC. Binding of nanoparticles bearing ligand molecules could be efficiently inhibited by prior incubation of cells with free ligand, demonstrating that adhesion of the nanoparticles is mediated by specific interaction between the ligand and the selectin receptors. These nanoparticles could be used for specific drug delivery to the activated vascular endothelium, suggesting their application in the treatment of diseases with an inflammatory component such as rheumatoid arthritis and cancer.     

pH-responsive polysaccharide-based polyelectrolyte complexes as nanocarriers for lysosomal delivery of therapeutic proteins

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ～ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.     

Stimuli-responsive nanocarriers for intracellular delivery

The emergence of different nanoparticles (NPs) has made a significant revolution in the field of medicine. Different NPs in the form of metallic NPs, dendrimers, polymeric NPs, carbon quantum dots and liposomes have been functionalized and used as platforms for intracellular delivery of biomolecules, drugs, imaging agents and nucleic acids. These NPs are designed to improve the pharmacokinetic properties of the drug, improve their bioavailability and successfully surpass physiological or pathological obstacles in the biological system so that therapeutic efficacy is achieved. In this review I present some of the current approaches used in intracellular delivery systems, with a focus on various stimuli-responsive nanocarriers, including cell-penetrating peptides, to highlight their various biomedical applications.     

GMP-140: a receptor for neutrophils and monocytes on activated platelets and endothelium.

GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet-leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.