Membrane dynamic alterations associated with activation of human platelets by thrombin.

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.

Lysine binding to activated human platelets and its similarity to fibrinogen binding.

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.

Mechanism of interaction between interferon-gamma and antineoplastic agent on the differentiation of HL-60 promyelocytic cells.

Exposure of HL-60 promyelocytic leukemia cells to a combination of interferon-gamma (IFN gamma) and 5-fluorouracil (5-FU), at concentrations ineffective by themselves, induced a significant differentiation into monocyte-like cells. This phenomenon was accompanied by a synergistic antiproliferative effect. Further characterization of these two activities of the IFN gamma/5-FU combination on HL-60 cells was carried out. Whereas a brief pretreatment of the cells with IFN gamma followed by 5-FU was sufficient to exert the synergistic antiproliferative action, the effect on differentiation was dependent on a prolonged concomitant exposure to both drugs. In an attempt to gain more insight into the biochemical mechanisms of these phenomena, we have examined the effects of RNA and protein synthesis inhibitors and of cytoskeleton disrupting agents on the actions of IFN gamma. Inhibition of RNA or protein synthesis by actinomycin D or cycloheximide did not prevent the antiproliferative action of IFN gamma nor the induction of monocytic differentiation, yet these two compounds blocked the priming effect of IFN gamma on the potentiation of 5-FU action. Actinomycin D synergistically potentiated the antiproliferative action of IFN gamma. Colchicine, vinblastine, and cytochalasin B, disrupting the microtubular and microfilament structure, did not interfere with the actions of IFN gamma; higher concentrations of the drugs even improved the priming effect. Exogenous thymidine, known to counteract the antiproliferative effect of 5-FU, also blocked the antigrowth action but not the differentiation induced by the IFN gamma/5-FU combination. The results suggest the existence of two different mechanisms of the IFN gamma/5-FU synergism: one governing the antiproliferative action via an effect on thymidine synthetase, inducible by a short-term IFN gamma pretreatment and dependent on de novo RNA and protein synthesis; and the other mediating the induction of differentiation requiring a long-term exposure of the cells to both drugs. From a clinical point of view, drug combinations such as IFN gamma and 5-FU, inducing differentiation as well as inhibiting proliferation, may suggest a new approach to the treatment of leukemia.

The roles of protein phosphorylation/dephosphorylation in tumor necrosis factor antitumor effects.

The roles of protein phosphorylation and dephosphorylation in the tumor necrosis factor (TNF) cytotoxic and antiproliferative effects on L-929-transformed fibroblasts were explored. Genistein and erbstatin, specific inhibitors of tyrosine kinase, had antiproliferative but not cytotoxic effects on the cells by themselves and synergistically enhanced the cytotoxic and antiproliferative effects of TNF-alpha. Immunoblot analysis with a monoclonal antiphosphotyrosine antibody revealed that TNF, administered for 5-180 min, induced tyrosine dephosphorylation of two pairs of membranal proteins, 34-36 kDa and 50-52 kDa, and potentiated tyrosine phosphorylation of a 115-kDa protein in both the cytosolic and membranal fractions of the cells. A very brief exposure (30 sec) to TNF induced rapid phosphorylation of several proteins, whereas genistein, but not inhibitors of other protein kinases, enhanced this effect of TNF. The results suggest that TNF activity could be potentiated by the inhibition of tyrosine phosphorylation and point to specific proteins that are dephosphorylated on tyrosine in response to TNF.

Impairment of platelet aggregation by Echis colorata venom mediated by L-amino acid oxidase or H2O2.

Echis colorata bites cause impairment of platelet aggregation and hemostatic disorders. The mechanism by which the snake venom inhibits platelet aggregation was studied. Upon fractionation, aggregation impairment activity and L-amino acid oxidase activity were similarly separated from the crude venom, unlike other venom enzymes. Preparations of L-amino acid oxidase from E. colorata and from Crotalus adamanteus replaced effectively the crude E. colorata venom in impairment of platelet aggregation. Furthermore, different treatments known to inhibit L-amino acid oxidase reduced in parallel the oxidase activity and the impairment potency of both the venom and the enzyme preparation. H2O2 mimicked characteristically the impairment effects of L-amino acid oxidase and the venom. Catalase completely abolished the impairment effects of the enzyme and the venom. It is concluded that hydrogen peroxide formed by the venom L-amino acid oxidase plays a role in affecting platelet aggregation and thus could contribute to the extended bleeding typical to persons bitten by E. colorata.

Rapid interferon-gamma-stimulated tyrosine phosphorylation of cytosolic and membranal proteins in HL-60 promyelocytic cells.

The involvement of tyrosine phosphorylation in the early stages of interferon-gamma (IFN gamma)-induced monocytic differentiation of HL-60 cells was studied. Immunoblotting analysis demonstrated that IFN gamma induced rapid changes in the tyrosine phosphorylation of several endogenous cytosolic and membranal proteins. The most prominent of these polypeptides was a 84 kDa protein. In membranes, the IFN gamma-induced phosphorylation of this protein was detectable in 5 min, remained elevated for 3 h and declined thereafter, while a gradual decrease in the phosphotyrosine content was observed in cytosols. In parallel, a 40% increase in the phosphotyrosine phosphatase activity was detected in the later stages of IFN gamma treatment. Rapid changes in tyrosine phosphorylation were detected also in a 64 kDa protein. In contrast, 2-day exposure to IFN gamma was needed to potentiate significantly the tyrosine phosphorylation of a 36 kDa membranal polypeptide. These data support the involvement of tyrosine phosphorylation in the early stages of IFN gamma-induced monocytic differentiation of HL-60 cells.

Effect of radiation on red cell membrane and intracellular oxidative defense systems.

Ionizing radiation is currently used for prevention of transfusion associated graft versus host disease (TAGVHD). As radiation damage is associated with the production of activated oxygen species, the aim of this study was to observe the immediate effect of ionizing radiation on red cell membrane and intracellular oxidative defense systems. Neonatal and iron deficiency (IDA) cells, known for their increased sensitivity to oxidative stress, were chosen and compared with normal cells. Irradiation was performed in doses of 1500 cGy, 3000 cGy and 5000 cGy. GSH and methemoglobin levels and the activity of different antioxidant enzymes, measured under optimal in vitro conditions, were preserved in all cells after irradiation. Only radiation at the highest does of 5000 cGy, caused significant potassium leakage in neonatal cells and insignificant increase in IDA cells. Thus, cells with increased sensitivity to oxidative stress are more susceptible to damage by ionizing radiation than normal cells.

Increased platelet thromboxane A2/prostaglandin H2 receptors in patients with pregnancy induced hypertension.

Pregnancy induced hypertension (PIH) is associated with a variety of disturbances in the hemostatic system including alterations in platelet function, thrombocytopenia, and an increase in platelet turnover. The density of platelet Thromboxane A2 (TXA2)/Prostaglandin H2 (PGH2) receptors was determined in patients with PIH and normal pregnant women, using [125I]-PTA-OH, a TXA2/PGH2 receptor antagonist. The number of platelet TXA2/PGH2 receptors significantly increased (p < 0.008) from 1734 +/- 370 sites/platelet (n = 8) in normal pregnant women to 3703 +/- 846 sites/platelet (n = 9) in patients with severe PIH. The sensitivity of platelets to the TXA2 mimetic U46619 was significantly (p < 0.0005) increased in platelets obtained from severe PIH patients (EC50 = 150 +/- 10nM, n = 3) compared to controls (EC50 = 290 +/- 60 nM, n = 5). These results indicate that an increased number of TXA2/PGH2 receptors as well as increased sensitivity to TXA2/PGH2 mimetics occurs in PIH. Collectively, these results provide further support for the notion that TXA2 and its receptor may play an important role in the pathophysiology of PIH.

Radiation damage to the erythrocyte membrane: the effects of medium and cell concentrations.

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.

New combination of 5-fluorouracil and interferon-gamma effective against human myeloid leukemia in vitro.

The therapeutic potential of recombinant interferon gamma (IFN gamma) alone or in combination with two cytotoxic drugs - 5-fluorouracil (5-FU) and cytosine arabinoside (Ara-C) - was studied in vitro on two myeloid leukemia systems: HL60 promyelocytic cell line and chronic granulocytic leukemia (CGL) progenitor cells. When applied individually, IFN gamma and the drugs inhibited in a dose-dependent manner HL60 cell colony formation in semisolid culture. Moreover, IFN gamma or the cytotoxic drugs dose-dependently reduced the colony formation of CGL progenitor cell in agar. When added in combination, IFN gamma potentiated synergistically the inhibitory action of 5-FU in both systems. The most pronounced potentiation was detected at concentrations of 0.5 microgram/ml 5-FU and 50 U/ml IFN gamma. On the contrary, the antiproliferative effect of Ara-C was enhanced only subadditively when combined with IFN gamma. In view of the present findings, which are supported by new evidence from the literature, the use of 5-FU in leukemia should be reconsidered. The results further imply the potential value of combined treatment of 5-FU and IFN gamma in leukemia.

Non-steroidal antiestrogens induce apoptosis in HL60 and MOLT3 leukemic cells; involvement of reactive oxygen radicals and protein kinase C.

The antitumoral activity of non-steroidal antiestrogens on promyelocytic leukemia HL60 and T lymphoblastic MOLT3 cell lines was studied. Tamoxifen and its derivatives, clomiphene and nafoxidine, caused reduction of cell viability in a dose-dependent manner. These drugs showed differences in their potency following four days incubation, with nafoxidine being the most efficient inhibitor and tamoxifen the least active. Apoptosis was induced as assessed by the DNA ladder pattern and formation of pre G0/G1 population as detected by flow cytometry analysis of DNA. The effect of these drugs was abrogated by antioxidants: alpha-tocopherol was most effective in antagonizing the drugs' effect. N-acetyl L-cysteine reversed mainly the decrease in cell viability caused by the drugs, but was less active on induction of apoptosis. GF109203X, a protein kinase inhibitor, attenuated apoptosis induced by clomiphene in MOLT3 cells. The results suggest that the antileukemic activity of the antiestrogens is mediated by oxidative stress and protein kinase C (PKC) activation. Triphenylethylene antiestrogens and their derivatives may be used as antileukemic drugs which kill cells by apoptosis mediated by oxidative stress and activation of PKC.

TPK inhibitors differentially affect IFN-gamma activities.

The effect of various tyrosine protein kinase inhibitors on processes involved in the antiproliferative effect of interferon-gamma on WISH cells was studied. Following 24 hr treatment interferon-gamma inhibited thymidine incorporation into DNA and thymidine kinase activity, but no significant effect on cell number was observed. The isoflavonoid, genistein, which is a specific inhibitor of tyrosine protein kinase, reversed the inhibition in thymidine incorporation caused by the cytokine in a dose dependent manner. Prunetin, a member of the same group, did not significantly antagonize this effect. N alpha-tosyl-L-lysyl-chloromethane, a serine protease inhibitor which also serves as a tyrosine protein kinase inhibitor, partially reversed the effect of interferon-gamma at a concentration of 100 microM. The bioflavonoid, quercetin, a non-specific tyrosine protein kinase inhibitor, at a concentration of 30 microM completely abolished the action of interferon-gamma on thymidine incorporation. Genistein completely reversed the inhibition of thymidine kinase exerted by interferon, while quercetin had only a slight effect. However, the drugs could not antagonize the antiproliferative effect of interferon following 48 hr incubation, as measured by reduction of cell number. The results indicate that tyrosine protein kinase may play a role in the effects of interferon on thymidine metabolism and thymidine kinase activity. The differential effects of the inhibitors on thymidine metabolism and cell proliferation could support dissociation between the effect of interferon-gamma on these processes. Alternatively, this dissociation of effects could point to the limited use of inhibitors in clarifying modes of action as described.

Studies of the effect of auranofin, a new antiarthritic agent, on platelet aggregation.

Auranofin (AF), at a concentration of 10 micrograms/ml, was found to be a potent inhibitor of ADP-, epinephrine-, or collagen-induced platelet aggregation utilizing platelet-rich plasma obtained from human blood. In contrast, aurothioglucose was less effective than AF in inhibiting epinephrine- or collagen- induced platelet aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation. The inhibitory effect of AF was more evident on the second phase of aggregation and was a function of drug preincubation time. Compared to platelet-rich plasma, washed platelets were superior for detecting the inhibitory action of AF (greater than or equal to 0.1 microgram/ml) on ADP-induced platelet aggregation. This potent inhibitory action of AF on ADP-induced platelet aggregation was antagonized by dithioerythriol, a potent reducing agent. These results suggest that AF can inhibit both platelet release and aggregation mechanisms which may be relevant to its antiarthritic activity. Further studies are required to elucidate the cellular mechanism by which AF inhibits platelet aggregation.

Cannabinoids block release of serotonin from platelets induced by plasma from migraine patients.

The effects were assessed of delta'THC (the psychoactive component of cannabis) and CBD and DMHP-CBD (the non-psychomimetic components of marijuana derivatives) on 14C labelled serotonin release from normal platelets, when incubated with patient's plasma obtained during migraine attack. A statistically significant inhibitory effect (p greater than 0.005) of 14C serotonin release was found at 10(-5)M, 10(-6)M, 10(-7)M delta'THC concentrations. Plasma of migraine patients obtained in attack-free periods revealed no significant inhibitory effect on 14C serotonin release from normal platelets using the same delta'THC concentration. CBD and DMHP-CBD had no significant inhibitory effect on 14C serotonin release from normal platelets when tested either at migraine-free period plasma or plasma obtained during migraine attack.

Plasma thromboglobulin and platelet aggregation index in transient ischaemic attack: effect of aspirin and dipyridamole therapy.

Beta-thromboglobulin (beta TG) plasma levels and platelet aggregation index (PAI) were determined in 14 transient ischaemic attack (TIA) patients, before and two weeks after starting therapy with aspirin and dipyridamole. Thirty healthy men were the control group. Decrement in beta TG plasma levels (without statistical significance) was found in treated patients when compared to the period before treatment. It is noteworthy that both these levels were significantly higher than plasma beta TG levels of normal controls. A highly significant difference was found between PAI of patients before treatment compared with PAI of patients treated with aspirin and dipyridamole. PAI was higher and similar to PAI of controls in the treated patients. No correlation between these two tests was established. It is concluded that the beta TG test is efficient as an aid for diagnosis of TIA, while PAI is better tool for follow-up.

Plasma antithrombin III levels in pre-eclampsia and chronic hypertension.

Plasma levels of antithrombin III were tested during pregnancy in a control group of normal patients and in a study group that included patients with moderate and severe pre-eclampsia and chronic hypertension. The control group showed mean antithrombin III activity of 97.9 +/- 20.9%, the severe pre-eclamptic patients 22.33 +/- 18.22%, the moderate pre-eclamptic patients 56.0 +/- 7.56%, and the chronic hypertensive patients 77.5 +/- 6.69%. The difference between normal pregnancy and moderate pre-eclampsia was significant at P less than 0.002, normal pregnancy and severe pre-eclampsia P less than 0.002, moderate and severe pre-eclampsia P less than 0.002, chronic hypertension and normal pregnancy P less than 0.1, and chronic hypertension and severe pre-eclampsia P less than 0.002. All the severe pre-eclamptic patients and 2 out of 6 of the moderate pre-eclamptic women were below 55.7% (mean - 2S.D.) of normal antithrombin III activity. Patients with heavy proteinuria had depressed antithrombin III activity. However, chronic hypertensive pregnancies, although rather a small group, had almost normal values of plasma antithrombin III activity. The plasma antithrombin III value may thus help to distinguish between chronic hypertension and severe pre-eclamptic disease.

Acquired chromosome instability in the elderly--the effect of diepoxybutane.

Aging and Alzheimer's disease (AD) have been the subject of many studies. It has been suggested that chromosomal alterations may be involved in the etiology and/or pathogenesis of ageing and AD. The purpose of the present study was to examine the effect of diepoxybutane (DEB) on lymphocyte chromosomal instability in the elderly. We examined lymphocytes cytogenetically with, as well as, without DEB treatment, in a group of 12 elderly (range of age 72-96 years), nine of them suffering from AD type. Without DEB treatment six of the donors expressed chromosomal instability in at least 6% of the analyzed cells. After treatment with DEB, lymphocytes showed an increase in the chromosomal instability in up to 20% of the analyzed in eight donors. The sex chromosomes were the main chromosomes involved in the acquired chromosomal abnormalities. It is not clear from this study whether this chromosomal instability is related to the AD. The significance of the involvement of sex chromosomes either in ageing or in AD, as well as, the question whether the chromosomal instability is the cause of or part of ageing processes, has to be addressed.

Effect of activators and inhibitors on human blood platelets: study by freeze-fracturing technique and electron microscopy.

Samples of normal human platelet-rich plasma were activated by ADP (0.5 microM and 20 microM) or by cold treatment (4 degrees C). The effects of incubation in EDTA, colchicine or cytochalazin B were also studied. Samples were prepared by the freeze-fracturing technique and examined by electron microscopy. Faces of fractured membranes were examined for aggregation of particles, for pore-openings in the membrane, and for the general morphological appearance of the membrane-fractured faces. No aggregation of particles was found in platelet-rich plasma preparations treated with ADP, EDTA, colchicine or cytochalazin B, nor did the pore openings reveal any changes following these treatments. We conclude that platelet activation and an increase in microviscosity of the platelet membrane is not due to platelet aggregates in the membrane.

Effects of ethanol, CBD and delta 'THC on proliferation of K-562 cells.

The effect of ethanol and cannabinoids on proliferation of the leukaemic cell line K-562 is described. The effects on the cells were assessed by counts of viable cells and 3H thymidine incorporation. delta 'THC was found to attenuate the proliferation of K-562 cells, the effect being prominent at 10(-4)M. CBD was effective only at high concentrations or after prolonged incubation time. Both drugs were dissolved in 0.3% ethanol solution; ethanol itself caused a biphasic effect, namely a mild increase in K562 proliferation at low concentrations and a marked attenuation at higher concentrations.

Specific impairment of ADP-induced platelet aggregation by cannabinoids.

The structure-activity relationship of the inhibition of ADP-induced platelet aggregation by cannabinoids was studied. A marked specificity was revealed: derivatives in which the two asymmetric centers have a configuration opposite to that of the natural cannabinoids and have a dimethylheptyl (DMH) side-chain were most inhibitory. It is concluded that the mode of inhibition includes a specific interaction of the cannabinoids with some membrane proteins, possibly including the receptor for ADP.