Two-step mobilization of arachidonic acid in platelet activation induced by low concentrations of TP 82, a monoclonal antibody against CD9 antigen.

Arachidonic acid mobilization in platelets activated by low concentrations (less than or equal to 1.6 micrograms/ml) of TP 82, a monoclonal antibody against CD9, appears to consist of two distinct phases. In the first phase, limited arachidonic acid release occurs concomitantly with a shape change induced by TP 82.

The role of apoE in inhibitory effects of apoE-rich HDL on platelet function.

Apolipoprotein E- (ApoE-) rich high-density lipoprotein (HDL), which was prepared from the bound fraction of normolipemic volunteers on heparin-Sepharose and from a hyperalphalipoproteinemic patient, potently inhibited aggregation of human platelets in a dose-dependent fashion. Dimyristoyl phosphatidylcholine liposome with apoE (apoE.DMPC) also inhibited platelet aggregation, and incubation of washed platelets with apoE.

Characteristics of a highly thermostable neutral protease produced fromBacillus stearothermophilus.

A highly thermostable neutral protease was found in culture filtrates ofBacillus stearothermophilus. The optimum reaction pH and temperature of this protease were 6.0 and 60°C, respectively, and 90% activity remained even after heat treatment at 90°C for 30 min.

Effects of various inhibitors on platelet activation induced by TP 82, a CD 9 monoclonal antibody.

TP 82, a monoclonal antibody against CD 9 antigen, induced human platelet activation at concentrations higher than 0.4 microgram/mL in terms of aggregation, release of intracellular granule contents, production of arachidonic acid metabolites, and elevation of the intracellular Ca2+ concentration. The effects of a competitive inhibitor of ADP, acetylsalicylic acid, EGTA, and GRGDSP which blocks fibrinogen binding to IIb/IIIa complex suggested that each of released ADP, thromboxane A2, extracellular Ca2+, and close cell contact acts together to potentiate platelet activation induced by TP 82.

Intracellular ionized calcium mobilization of CD 9 monoclonal antibody-activated human platelets.

Cytoplasmic Ca2+ mobilization in human platelets triggered by a CD 9 monoclonal antibody, TP82, was monitored by the Ca2(+)-sensitive photoprotein, aequorin and Ca2(+)-sensitive fluorophores, fura 2 and quin 2. Aequorin-indicated Ca2+ values were proportional to the concentration of TP82, which was in inverse proportion to the lag time before the onset of platelet aggregation and serotonin release. When fura 2 was used as a Ca2+ indicator, above a threshold concentration, the TP82-induced intracellular Ca2+ value remained unchanged even with increasing concentration.

Modification of Na+/H+ exchanger in human platelets by 1,2-dioctanoylglycerol, a protein kinase C activator.

Protein kinase C activation in human platelets has a modulatory role in maintaining intracellular pH (pHi), by adjusting pHi at a particular value (7.22). Changes in pHi induced by protein kinase C appeared to be dependent upon the difference between H+ efflux catalyzed by the Na+/H+ exchanger and H+ production.

Mastoparan, a wasp venom, activates platelets via pertussis toxin-sensitive GTP-binding proteins.

Mastoparan induced limited release of serotonin from intact human platelets, while neither intracellular calcium ion elevation nor arachidonic acid mobilization was observed. Cytolysis induced by mastoparan was negligible in the concentration range that induced serotonin release. In digitonin-permeabilized cells, mastoparan induced Ca(++)-independent release of serotonin and Ca(++)-dependent arachidonic acid release.

Ionomycin, a Ca++ ionophore, increases platelet volume independently of the Na+/H+ exchanger.

A Ca++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca++ concentrations, suggesting that the volume change occurs when the intracellular Ca++ reaches a certain level (greater than uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na+ with membrane-impermeable N-methyl-D-glucamine or Cs+, but not with Li+, K+, or Rb+.

The role of intracellular pH and Ca++ on arachidonic acid metabolism in human platelets.

High concentrations of ionomycin induced arachidonic acid metabolism, while its magnitude appeared to be poorly correlated with the increase in intracellular Ca++ induced by ionomycin. Intracellular pH elevation had no direct effect on arachidonic acid release. On the other hand, the elevation in intracellular pH potentiated arachidonic acid release induced by low concentrations of ionomycin, and appeared to increase the sensitivity to Ca++ of the arachidonic acid releasing mechanism.

Functional activity of protein S determined with use of protein C activated by venom activator.

A new screening procedure, an easy and specific assay for determining functional Protein S activity, has been developed, with use of Protein C activated by venom activator (Protac). Purified Protein C (100% amidolytic activity) was activated by venom activator (6 units/mL). To a mixture of 100 microL of Protein S-deficient plasma, 20 microL of sample plasma, 100 microL of cephalin (Actin), and 20 microL of activated Protein C we added 100 microL of 25 mmol/L CaCl2 solution and measured the clotting time with a KC 10 coagulometer.

Acetyltransferase activity and production of platelet-activating factor by human neutrophils activated with various stimuli.

The acetyltransferase activity and biosynthesis of platelet-activating factor (PAF) were assessed in human neutrophils activated by 4 microM A23187, 20 ng/ml phorbol myristate acetate (PMA), and 10(-6) M n-formyl-methionyl-leucyl-phenylalanine (fMLP). All three agents elevated the acetyltransferase activity dose-dependently. There were no significant differences in the Km values for acetyl CoA between non-stimulated and stimulated cells.

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization.

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites.

Stimulative effects of TMB-8 and trifluoperazine on pancreatic hormone release.

The effects of 8-N-N-diethylamino octyl 3,4,5-trimethoxybenzoate (TMB-8) and trifluoperazine (TFP) on the early phase (10 min) of the release of pancreatic hormones from isolated rat islets were investigated. TMB-8 and TFP stimulated insulin, glucagon, and somatostatin release in a dose-dependent manner at a low glucose concentration (2.5 mM).

Anion channels contribute to the regulation of intracellular pH in human platelets.

The presence of extracellular bicarbonate potentiated platelet intracellular pH rises induced by thrombin. The effect was most remarkable in sodium-depleted buffers. This effect of bicarbonate was dose-dependent and was inhibited by anion channel blockers.

Exacerbation of toxic effects by endotoxin contamination of recombinant human tumor necrosis factor.

The toxic effects of endotoxin-free human recombinant tumor necrosis factor (rH-TNF), shown to contain less than 50 pg endotoxin/mg rH-TNF, were investigated and compared with those of rH-TNF and endotoxin coadministered at 4-400 ng endotoxin/mg rH-TNF in female Sprague-Dawley rats. The mean lethal dose of 5.9 mg/kg rH-TNF found for the endotoxin-free rH-TNF was far higher than that attributed to rH-TNF by other investigators.

Localization of granular components in ADP- and/or teleocidin-stimulated human blood platelets as revealed by immunocytochemical methods.

Morphological aspects of the platelet release reaction induced by adenosine 5'-diphosphate (ADP) and/or teleocidin (a tumor promoter) in the presence of aspirin were studied by transmission electron microscopy. Human platelet-rich plasma treated with reagents at 37 degrees C for 1-5 min was fixed with aldehyde and embedded in Epon or Lowicryl K4M. Addition of ADP (10 microM) resulted in the centralization of granules without granule release, while teleocidin (100 ng/ml) induced the swelling of the open canalicular system (OCS) and the release of alpha-granules without centralization of these granules.

Functional responses of aequorin-loaded human neutrophils. Comparison with fura-2-loaded cells.

Aequorin-loaded human neutrophils in response to chemotactic peptides and ionomycin showed a sharp rise in their intracellular Ca2+ concentration which decayed within 2 min. Depletion of extracellular Ca2+ suppressed only the ionomycin-induced increase. Fura-2-loaded cells also showed a sharp rise in the intracellular Ca2+ concentration in response to each stimulator, while the decline was extremely slow in the ionomycin-induced Ca2+ increase.

Amidolytic kinetic assay of protein C by selective spectrophotometry in a centrifugal analyzer.

This rapid, simple amidolytic assay of protein C activity in whole plasma involves activation by protein C activator from the venom of Agkistrodon contortrix contortrix (Protac) and use of a Cobas Fara spectrophotometer programmed for kinetic assay. Plasma is incubated with activator venom in the presence or absence of antibody to human protein C in the instrument, chromogenic substrate (S-2366) is added, and the absorbance is measured at 405 nm. The difference between the absorbance of the sample plasma with and without antibody to human protein C correlated well with protein C antigen as assayed by enzyme-linked immunosorbent assay (ELISA) and the Laurell rocket technique in normal subjects, patients being treated with warfarin, and patients with liver cirrhosis or disseminated intravascular coagulation.

Effect of recombinant DNA-produced tumor necrosis factor on various parameters of neutrophil function.

The effect of recombinant DNA-produced human tumor necrosis factor (TNF) on various parameters of neutrophil function was evaluated. TNF was a weak direct activator of oxygen radical production. It released the specific granule contents to a limited extent, but the azurophilic granule contents were retained even in the presence of cytochalasin B.

Potentiation of neutrophil function by recombinant DNA-produced interleukin 1a.

The effect of interleukin 1a (IL-1a) produced by E. coli-derived recombinant DNA was evaluated on various parameters of human neutrophil function. IL-1a alone stimulated neutrophil hydrogen peroxide production in a dose-dependent manner, but the rate was much lower than that of opsonized zymosan.