D. Del Principe

Hydrogen peroxide has a role in the aggregation of human platelets

The aggregation of platelets induced by soluble and particulate stimuli is enhanced by the addition of minute amounts of H2O2. Externally added catalase strongly inhibits the aggregation induced by particulate stimuli and by phorbol myristate acetate (PMA). The addition of aminotriazole to stimulated platelets causes a significant inhibition of intracellular catalase. This indicates the formation of H2O2 inside the platelets during activation. No effects were observed when the platelets were stimulated by the ionophore A23187.

Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin.

Human blood platelets produce oxidant species when stimulated by collagen and thrombin. The oxidative burst of platelets has been studied by cytofluorimetry taking advantage of the fluorogenic dye DCFH2-DA, which is taken up and deacetylated by platelets and then oxidized to the fluorescent derivative DCF. The oxidation of DCFH2 is induced by stimulation with collagen but not with thrombin and inhibited by external catalase. Catalase also inhibited the aggregation induced by collagen, but not that induced by thrombin. Aspirin and indomethacin inhibited the formation of the fluorochrome only when platelets were stimulated by thrombin. Externally added H2O2 increased the cytoplasmic calcium content as probed by the fluorescence of Indo-1. The present data suggest that collagen induces the production of H2O2, which in turn may stimulate the aggregation of platelets through a calcium mobilization. Instead the stimulation by thrombin does not require the intermediacy of H2O2.

Stimulated platelets release factor(s) affecting the in vitro response of human polymorphonuclear cells.

The metabolic and functional responses of human polymorphonuclear cells (PMNs) to thrombin‐activated platelet supernatants were studied. The incubation of PMNs with supernatants from stimulated platelets (SPS) caused a 50% decrease in both killing of Staphylococcus aureus and luminol‐enhanced chemiluminescence (CL) by PMNs stimulated by opsonized‐zymosan (OZ), Concanavalin A (Con A), or calcium ionophore A23187. The levels of PMN intracellular fluorescence measured by flow cytometry, using the fluorochrome dichlorofluorescin diacetate (DCF‐DA), were considerably less in the presence of SPS than in resting platelet supernatants (RPS). No influence of platelet supernatant on O2 consumption and O2 ‐ generation by OZ‐activated PMNs was observed. The incubation of PMNs with SPS caused a significant increase in the rate of chemotaxis and aggregation elicited by Con A, OZ, and phorbol myristate acetate (PMA). The supernatant from resting platelets did not show any of the above‐reported effects. Platelets previously degranulated by thrombin were unable to inhibit CL when activated with agonists. Studies on the differential release of the granules by platelets showed that the CL‐quenching activity paralleled the discharge of lysosomal content The release of myeloperoxidase (MPO) from PMNs elicited by OZ was reduced in the presence of SPS. The platelet supernatant did not affect the MPO activity if PMNs were lysed with Triton X‐100. The leakage of lactate dehydrogenase (LDH) from platelets was less than 3%, and no catalase or superoxide dismutase was released. This activity withstood lyophilization, but was destroyed by 10 min heating at 100°C or by treatment with proteolytic enzymes.

DISSEMINATED ARTHRITIS AND OSTEITIS BY CANDIDA ALBICANS IN A TWO MONTH OLD INFANT RECEIVING PARENTERAL NUTRITION

Abstract. The case of a two‐month‐old female infant, who after a severe diarrhoea treated with prolonged intravenous infusion in peripheral veins alternated with total parenteral feeding, developed a Candida albicans septicemia (accompanied by disseminated intravascular coagulation syndrome) is reported. The course of her disease was also complicated by multiple foci of osteoarthritis in both knees, in the left hip and in several long‐bones. Radiographically the foci of Candida osteitis appeared as fine erosion of the cortex and minute round areas of osteolysis in the spongiosa, surrounded by a rim of perifocal sclerosis. During the acute stage of Candida sepsis a transitory cellular immunodeficiency was present. Treatment of Candida infection by 5‐fluorocytosine was followed by complete recovery.

Selective defect of a T helper subpopulation in severe combined immunodeficiency

The case of an infant female aged 1 month with severe infections, failure to thrive, and skin erythroderma is reported. Immunological studies demonstrated a severe combined immunodeficiency (SCID) with B cells and normal serum IgM levels. Studies of T-cell subpopulations showed a defect of a subset of T helper cells. Possible pathogenetic mechanisms are discussed.

Photodynamic Damage Induced by Bilirubin on Human Platelets: Possible Relevance to Newborn Pathology

Several reports have appeared showing the possibility of bilirubin-sensitized photodamage. We have extended these observations to platelets. In the presence of 300 microM bilirubin the in vitro irradiation of isolated platelets or platelet-rich plasmas with visible light induced significant lysis as determined by the release of lactate dehydrogenase (LDH). The extent of LDH release was a function of irradiation time, being about 20% after 2 h of irradiation. A loss membrane-bound ATPase activity was also observed at earlier times, indicating that membrane damage was preliminary to the lytic effect. The release of beta-thromboglobulin, induced by close cell-to-cell contact, was lower in bilirubin- and light-treated platelets with respect to controls. Our results suggest that bilirubin may act as a photodynamic agent producing some damage on human blood platelets.

A surface NAD-glycohydrolase of human platelets may influence their aggregation

Human platelets may hydrolyze externally added NAD+ yielding ADPR and nicotinamide. The extent of hydrolysis is significantly higher when the platelets are stimulated. The presence of external NAD+ strongly inhibits the aggregation induced by every agonist used. It seems that adenosine or ADPR itself generated by NAD+ hydrolysis may be responsible for the inhibition of aggregation. Evidence is given that some of the NAD+ hydrolysis product is taken up by stimulated platelets.

Interaction of terbium with human platelets

The effect of terbium on platelets has been studied by aggregation experiments and by fluorescence measurements. TbCl3 does not substitute for CaCl2 in the aggregation of platelets induced by ADP, but it may even inhibit, probably by a competition mechanism. It was impossible to observe a sensitized emission of Tb3+ in the presence of platelets. Instead the lanthanide, like Ca2+, significantly increases the aggregation of platelets induced by A23187. The fluorescence yield of this compound is greater in the presence of platelets than in buffer alone. Energy transfer appears to take place from the aromatic amino acids of the platelet membrane to the bound ionophore.

Malonyldialdehyde formation, oxygen consumption, fatty acid composition in newborn platelets stimulated by thrombin.

The release reaction, the formation of malonyldialdehyde (MDA), the pattern of oxygen consumption, and the variation in fatty acid composition after addition of thrombin (1.67 U/ml) have been investigated in newborn platelets, comparing the obtained data with analogous values showed by adult platelets assumed as normal controls. Newborn platelets showed a release reaction 20% lower than that of controls. MDA formation, even in the presence of NEM and the burst in oxygen consumption, resulted to be similar in newborn and adult platelets (p greater than 0.3); the burst was also similar after the addition of thrombin (10 U/ml) in the presence of antimycin and aspirin. The ratio between formed MDA and oxygen consumption was 1:10 in adult platelets while it was 1:15 in those of newborns. The study of fatty acid composition demonstrated that in newborn platelets at rest, arachidonic acid is significantly in a higher concentration than in controls and that it decreases after stimulation with thrombin. It is concluded that the pathway of prostaglandins is normally stimulated by thrombin in newborn platelets.

Effect of Terbium on Thrombin and Antithrombin-Thrombin Interaction

Interactions between thrombin and antithrombin are influenced by calcium ions.’ In fact, it has been reported that CaC!, potentiates the heparin-modulated antithrombin I11 inhibition of thrombin. A similar potentiation of calcium on thrombin inactivation by antithrombin may occur in the absence of heparin. Thus, calcium appears to play a complex role in the interactions involving thrombin, heparin, antithrombin, and fibrinogen. The mechanisms of these effects are poorly understood. Because lanthanide ions show chemical properties that make them suitable probes for the interactions of Caz+ with biological systems, we investigated the effect of one of them, namely Tb3+, on thrombin and on thrombin-antithrombin interactions.