Influence of ionized calcium on thrombin-induced down regulation of GPIb/IX receptors on human platelets.

The influence of ionized calcium on the down-regulation of GPIb/IX receptors on human platelets was evaluated by flow cytometry using monoclonal antibodies. Addition of EDTA alone to a washed platelet suspension did not cause decreased monoclonal antibody binding to the cells. However, introduction of thrombin to the washed platelets containing EDTA resulted in a marked decrease in binding of monoclonal antibodies to the GPIb/IX receptors. If calcium at 1-3 mmol/L was added to buffered platelets instead of EDTA before thrombin, down-regulation was prevented or significantly reduced. Restoring calcium to EDTA platelets after the thrombin-stimulated down-regulation had been in progress for 1-3 minutes caused reversal of decreased antibody binding by GPIb/IX to near resting levels. Results demonstrate that extracellular calcium is a major factor regulating thrombin-induced down-regulation.

Physiology and function of platelets from patients with Alzheimer's disease.

The discovery that intact Alzheimer amyloid precursor protein is present in platelet granules, has created a great interest in the biochemistry, physiology and function of platelets of patients with Alzheimer disease (AD). In this study we monitored various biochemical and physiological parameters, such as serotonin and adenine nucleotide levels, membrane fluidity, agonist-mediated release of arachidonic acid, thromboxane formation, calcium mobilization, as well as irreversible aggregation and secretion of granule contents. Platelets of patients with AD responded poorly when stirred with weak or potent agonists on a platelet aggregometer. Although capable of agonist-mediated calcium mobilization and synthesis of thromboxanes, the aggregation response of platelets of patients with AD to thrombin and archidonate was considerably compromised. In view of the normal biochemistry and signal transduction capabilities, the compromised response of these cells to potent agonists like thrombin suggested an extrinsic defect. The present study has shown that a plasmatic factor is at least in part responsible for the functional abnormalities of AD platelets.

Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds.

Vol. 61, no. 6, p. 2338, Table 5, column 2, last line: "9" should read "97.9." [This corrects the article on p. 2335 in vol. 61.].

Influence of the calcium-sensitive fluorophore, Quin 2, on platelet function.

Recent investigations using Quin 2, a fluorophore used to monitor cytosolic free calcium shifts, have shown that strong agonists cause a dramatic dose-dependent increase in platelet fluorescence. However, weak agonists stimulated little or no increase in light emission of Quin 2-loaded platelets, suggesting that calcium flux is not involved in activation by these agents. The present study has sought an alternative explanation for the failure of weak stimuli to cause a rise in cytosolic free calcium in platelets containing Quin 2. Conditions used to prepare, wash, load, gel-filter, and evaluate the fluorophore-filled cells were examined for their compatibility with retention of sensitivity to activation by weak agonists. The technique used to measure shifts in cytosolic calcium with Quin 2 requires multiply washed, unstirred platelets. Under these conditions, platelets do not aggregate or secrete in response to weak agonists. Quin 2, at concentrations greater than 40 mumol/L, inhibits the response of platelets to strong agonists, and completely blocks their reaction to weak agonists. Quin 2 inhibition of platelet function appears related to high buffering capacity for free calcium, although other mechanisms cannot be ruled out. This suggestion is supported by the observation that Quin 2-induced blockade can be overcome by membrane modulation, which is a calcium-dependent process. However, since both agonists are weak, significant elevation in cytosolic calcium concurrent with functional restoration could not be demonstrated under the experimental conditions used for monitoring calcium. Thus, the conditions used to prepare platelets for Quin 2 evaluation and Quin 2 itself appear to be responsible for the failure of weak agonists to cause evidence of a calcium shift in fluorophore-loaded cells.

Defective DNA-receptor function in systemic lupus erythematosus and related diseases: evidence for an autoantibody influencing cell physiology.

The receptor for DNA was functionally defective in the majority of patients with systemic lupus erythematosus (SLE; 91% of 35 studied) and allied rheumatic disorders. This functional defect was manifest by impaired binding of exogenous DNA to the cell surface of peripheral blood mononuclear cells and an inability of cells to internalise and degrade DNA. The receptor defect was not constitutive, since it could be reversed by overnight incubation of cells; this process was sensitive to cycloheximide, suggesting a requirement for active receptor regeneration. The DNA-receptor defect could be induced in healthy controls, by incubating their cells with the serum of patients with SLE. The humoral factor inducing the defect was an autoantibody.

Measurement of ionized calcium in blood platelets with a new generation calcium indicator.

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.

Angiographic findings in mixed connective tissue disease. Correlation with fingernail capillary photomicroscopy and digital photoplethysmography findings.

Thirteen patients with mixed connective tissue disease underwent hand angiography to assess the degree of vascular abnormalities and their correlation to nailfold capillary microscopy and digital photoplethysmography findings. Organic obstruction was found in 60% of ulnar arteries, 87% of superficial arches, 13% of deep arches, and 65% of digital arteries. Fingernail capillary abnormalities were seen in 90% of patients. Normal photoplethysmography results had a predictive value of 91% for identifying digits without bilateral occlusions. These findings indicate a hitherto unrecognized propensity for disease of both small and medium-sized vessels in patients with mixed connective tissue disease.

Anaphylactoid reaction to corticosteroid: case report and review of the literature.

It has been suggested that corticosteroids can cause allergic reactions including anaphylaxis. Thirty-five patients have been reported to have anaphylaxis-like reactions following exposure to hydrocortisone in topical and parenteral preparations. We describe a further case of a nonatopic woman who developed urticaria during intravenous infusion of hydrocortisone. A review of the literature reveals that IgE-mediated immediate hypersensitivity has not been definitely proven in any case. We conclude that the clinical manifestations occasionally experienced after receiving hydrocortisone are most likely pseudoallergic reactions.

Interactions of zinc and arachidonic acid.

To probe the interaction of zinc with polyunsaturated fatty acids we have studied the effect of zinc on the cooxygenation of ferrous iron and arachidonic acid. Zinc inhibited the process of cooxygenation in a concentration dependent fashion. Further evaluation of the interaction of zinc and arachidonic acid gave spectroscopic evidence that zinc, oxygen and arachidonic acid can form an unstable hydroperoxide-like complex similar to that postulated earlier for iron, oxygen and arachidonic acid. However, in the case of zinc the complex will not proceed further to form stable peroxides and the unstable complex falls apart to give zinc and arachidonic acid intact. The findings have implications for the role of zinc in enzyme reactions and for antioxidant reactions of zinc within the cell. The influence of zinc on platelet aggregation was also evaluated. Zinc was found to inhibit cell-cell aggregation. However, in contrast to the known ability of zinc to inhibit prostaglandin synthesis in a broken cell preparation, zinc did not inhibit prostaglandin or thromboxane synthesis in the intact platelet. Inhibition of platelet aggregation by zinc must result from some other action of this cation.

Lysophosphatidic acids. Influence on platelet aggregation and intracellular calcium flux.

Decanoyl-, palmitoyl-, and oleoyl-lysophosphatidic acid (LPA) were studied for their effects on platelet aggregation and intracellular calcium flux. Palmitoyl-LPA and oleoyl-LPA both caused a concentration-dependent aggregation of human blood platelets at concentrations of 12--300 microM. Aggregation by adenosine diphosphate (ADP) was enhanced at slightly lower concentrations. First-wave aggregation induced by these LPAs was not blocked by aspirin, indomethacin, or heparin, suggesting similarities to ADP aggregation. However, in washed platelets with a high calcium concentration, no serotonin secretion was observed, even though full aggregation occurred, suggesting that aggregation was not due to released ADP. This concept was supported by studies of platelets deficient in the storage pool of ADP and serotonin, which had a normal first-wave aggregation response to palmitoyl-LPA. Aggregation induced by palmitoyl LPA was inhibited by prostaglandin E1 (PGE1), theophylline, and ethylenediaminotetraacetate (EDTA), though in the presence of EDTA shape change occurred. Aggregation stimulated by palmitoyl-LPA or oleoyl-LPA was characterized by changes in the shape of the platelets with development of pseudopods and centralization of granules closely surrounded by contractile microfilaments and supporting microtubules. The addition of palmitoyl-LPA and oleoyl-LPA, but not decanoyl-LPA, caused the release of calcium from a platelet membrane fraction that contains elements of the intracellular calcium storage system and actively concentrates this cation in the presence of adenosine triphosphate (ATP) and magnesium. It is suggested that LPAs cause aggregation by stimulating the release of calcium intracellularly.

Vitamin E inhibits the release of calcium from a platelet fraction in vitro.

Vitamin E, an inhibitor of platelet aggregation, was evaluated for its effects on platelet intracellular calcium flux. These studies used a platelet membrane fraction containing membranes of the dense tubular system which actively sequesters calcium in the presence of ATP and magnesium. After these membrane vesicles have accumulated calcium, the cation can be released by addition of the calcium ionophore A23187. Vitamin E had no effect on uptake of calcium by the membrane vesicles, but showed a concentration dependent inhibition of the release of calcium induced by A23187. In similar or slightly higher concentrations than inhibited calcium release, vitamin E also inhibited platelet aggregation, internal contraction and secretion, but had no effect on prostaglandin and thromboxane synthesis and potentiated phospholipase A2 activity. It is suggested that vitamin E acts to inhibit platelet internal contraction and secretion by preventing efflux of calcium from the dense tubular system. The potentiation of phospholiplase A2 by vitamin E could be explained by a localized increase of calcium at the site of the phospholipase A2 on the inner side of the dense tubular system membrane proximal to the vitamin E block.

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.