Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets

U73122 ((1-[6-(( 17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)exyl]-1H-p yrrole-2,5-dione)) is generally used as a selective inhibitor of phospholipase C (PLC) and the related rise in cytosolic Ca2+. Recently, by using hepatocytes, it was suggested that its action sites are different for PLC activation and increase in Ca2+ concentration. To verify whether U73122 has different sites for inhibiting PLC activation and calcium responses in human platelets, aggregation, Mn2+ influx, cytosolic Ca2+ increase and PLC activation were studied in response to thrombin and the synthetic agonist of the thromboxane receptor U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha). With both agonists, U73122 inhibited aggregation, Mn2+ influx and the enhancement of cytosolic calcium at concentrations of 2 microM or lower, while 10 microM was necessary to inhibit PLC activation. Our results suggested that U73122 is much more active in antagonizing Ca2+ channels, both the intracellular ones, which are activated by formation of inositol 1,4,5 P3 and those present on plasma membrane, than in reducing the activation of PLC.     

Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).     

The effect of different molecular species of sphingomyelin on phospholipase C delta 1 activity

Bovine brain sphingomyelin was separated into different molecular species using a reverse phase column. PLC delta 1 was inhibited by all molecular species of sphingomyelin. The extent of this inhibition was dependent on the hydrophobicity. Based on fatty acid analysis, we conclude that the inhibition of PLC delta 1 depends on the chain length and degree of unsaturation of the fatty acid moiety of SM. N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin inhibited PLC delta 1 less then N-oleoyl-D-sphingomyelin. In the absence of Ca2+ (1 mM EGTA) all tested molecular species of SM inhibited weakly the enzyme. The sensitivity of PLC delta 1 to inhibition by SM increased with increasing Ca2+ concentration. The shape of calcium curve differed for molecular species with saturated and unsaturated fatty acids. Inhibition of PLC delta 1 by N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin reached a maximum at 0.2 microM Ca2+, while inhibition by N-oleoyl-D-sphingomyelin reached maximum at 2 microM Ca2+. PLC delta 1 is more sensitive to inhibition by SM when it is maximally activated by spermine and calcium and the extent of this inhibition depends on the length and degree of fatty acid unsaturation of the molecular species.     

Chelerythrine inhibits the secretory response of human blood platelets without specifically inhibiting protein kinase C

Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of the serine/threonine-specific protein kinase C (PKC). In this study, it was shown that 10 microM chelerythrine completely inhibited serotonin secretion and partially inhibited phosphatidic acid formation in human blood platelets activated by thrombin (1U/ml). However, there was no effect on PKC activity as assessed by the level of phosphorylation of the 47K protein. Therefore, chelerythrine has been shown not to be a specific inhibitor of PKC. Without specifically affecting PKC activity, it is nevertheless capable of completely inhibiting platelet secretion, indicating that it may affect the signal transduction pathway responsible for platelet secretion at a point downstream or independent of PKC.     

Effect of electric charge on the adhesion of human blood platelets

The paper presents the results of research into the effect of the size and depth of the implanted electric charge on the adhesion of human blood platelets. The experiments were carried out on polyethylene terephthalate PET foil of 36 microns thickness. The electret formation process was carried out in an electron-beam device. The electrization conditions were such that electrets with the excess electric charge accumulated at various depths were obtained. The selection of conditions was verified by investigating the space charge distribution with the use of the virtual electrode method. The microscopic observation of non-electrified foils and electrets as well as the quantitative examination of the adhesion of human blood platelets has explicitly confirmed the positive influence of the electret effect on the thrombogenesis of PET foil. This made it possible to define the optimum electrization conditions. The research has additionally indicated that the relationship between the amount of adherent blood platelets and the size of the electric charge is not a simple relation of the kind: the larger negative charge, the more thrombogenic material. The decisive and positive effect of the space charge has been confirmed by analysing the effectiveness of the surface and space charge.     

The effect of platelets on invasiveness and protease production of human mammary tumor cells

Interaction of tumor cells with platelets facilitates metastasis of tumor cells. It has been proposed that platelets protect tumor cells against the host's immune defense and enhance tumor-cell extravasation. In the present work we show that platelets increase the invasiveness of 3 mammalian cell lines (MCF-7, ZR-51 and MDA-MB231) through extracellular matrix, and propose this as an additional mechanism by which platelets facilitate metastasis. Since gelatinase and urokinase have both been implicated in degradation of the extracellular matrix and cell migration, and therefore in tumor invasion, we have also analyzed whether the interaction of platelets with tumor cells can modify the secretion of these proteases by tumor cells. MDA-MB231, which was the most invasive cell line among the 3 tested and was the most potent in inducing platelet aggregation, secreted the highest level of urokinase and was the only one in which gelatinase was detected. While platelets had no significant effect on the urokinase activity expressed by these cells, they induced in MDA-MB231 an important increase in the secretion of gelatinase, which can be reproduced by both platelet membrane and platelet releasate of activated platelets. This increase in gelatinase could be responsible, at least in part, for the increased invasiveness of these cells, since added TIMP-1 significantly reduced the number of cells which traversed matrigel.     

The effect of chromatographically pure sample of adenosine 5''-tetraphosphate on sheep and rabbit blood platelets

1. The commercially available trisodium salt of adenosine 5'-tetraphosphate (ATetraP) (Sigma) was found to be contaminated with ATP, ADP and AMP, and therefore unsuitable for use in platelet studies. 2. The more stable barium salt of ATetraP was converted to the ammonium salt and found to be chromatographically homogeneous. This sample was tested for its influence on sheep blood platelets in citrated-rich plasma by the photometric method. 3. The ammonium salt of ATetraP (5-105 mumol.1(-1)) induced platelet aggregation which showed no tendency towards disaggregation. 4. The log dose-response lines for ATetraP and for adenosine diphosphate were parallel. On a molar basis, the tetraphosphate and only 1.5% of the aggregating activity of ADP. 5. The initial rate of aggregation induced by the tetraphosphate was inhibited by adenosine 5'-monophosphate analogues which are selective ADP-antagonists. These compounds also dispersed aggregates produced by ATetraP. 6. Platelets made refractory to ADP were also refractory to ATetraP. 7. Like ADP, ATetraP induced the change in shape of rabbit platelets and in this respect had only 3.4% the activity of ADP. 8. It is concluded that ATetraP per se can induced platelet aggregation and platelet shape change, and appears to exert its effect at the same site on the platelet surface as does ADP.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

The effect of heparin on the interaction of factor VIII and human platelets in vitro

Heparin was found to inhibit the interaction between human factor VIII and platelets. This was noted in two different systems, namely, platelet aggregation induced by neuraminidase-treated human cryoprecipitate and platelet aggregation induced by ristocetin-human factor VIII complex. Heparin appeared to have an inhibitory effect on the above systems similar to that reported on bovine factor VIII-induced platelet aggregation.     

Genetic mapping of the human and mouse phospholipase C genes

To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human x rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8.     

U73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets

The effect of the putative phospholipase C inhibitor U73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione) on platelet phosphoinositide metabolism was examined. In unstimulated rabbit platelets prelabeled with [32P]phosphate and [3H]glycerol, U73122 caused decreases of up to 50% in the amount and labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), but not phosphatidylinositol, within 1 min of addition and inhibited incorporation of [32P]phosphate and [3H]glycerol into PIP2 and PIP during incubations of up to 1 hr. These results point to inhibition by U73122 of the phosphatidylinositol and PIP kinases, although stimulation of the PIP and PIP2 phosphomonoesterases could be involved. In platelets stimulated with thrombin, U73122 blocked the thrombin-induced increases in PIP and phosphatidic acid; most increases in the inositol phosphates were blocked, but significant formation of inositol phosphate was found at 120 sec. The effects on inositol phosphates and phosphatidic acid were consistent with U73122 inhibiting phospholipase C; however, parallel dose-response curves with U73122 for the decreases in PIP2 and inhibition of thrombin-stimulated formation of inositol phosphates indicate that the inhibition of phospholipase C by U73122 may be due to decreased substrate availability rather than direct inhibition. Thrombin-stimulated decreases in PIP2 and PIP, found in the presence of U73122, could be explained by the action of phospholipase C in the absence of resynthesis. Although the changes were not as large, U73122 had a similar effect on PIP2 and PIP in unstimulated and stimulated human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta-adrenomimetic effect of human and animal blood serum

The conventional analysis and design of highway bridges ignore the contribution of sidewalks and∕or railings in a bridge deck when calculating the flexural strength of superstructures. The presence of sidewalks and railings or parapets acting integrally with the bridge deck have the effect of stiffening the outside girders and attracting more load while reducing the load effects in the interior girders. This paper presents the results of a parametric study showing the influence of typical sidewalks and railings on wheel load distribution as well as on the load-carrying capacity of highway bridges. A typical one-span, two-lane, simply supported, composite steel girder bridge was selected in order to investigate the influence of various parameters such as: span length, girder spacing, sidewalks, and railings. A total of 120 bridges were analyzed using three-dimensional finite-element analysis. American Association of State Highway and Transportation Officials (AASHTO) HS20 design trucks were positioned in both lanes to produce the maximum moments. The finite-element analysis results were also compared with AASHTO wheel load distribution factors. The AASHTO load and resistance factor design (LRFD) wheel load distribution formula correlated conservatively with the finite-element results and all were less than the typical empirical formula (S∕5.5). The presence of sidewalks and railings were shown to increase the load-carrying capacity by as much as 30% if they were included in the strength evaluation of highway bridges.     

Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.     

UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.