Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.     

Regulation of reverse cholesterol transport - a comprehensive appraisal of available animal studies

ABSTRACT: Plasma levels of high density lipoprotein (HDL) cholesterol are strongly inversely correlated to the risk of atherosclerotic cardiovascular disease. A major recognized functional property of HDL particles is to elicit cholesterol efflux and consequently mediate reverse cholesterol transport (RCT). The recent introduction of a surrogate method aiming at determining specifically RCT from the macrophage compartment has facilitated research on the different components and pathways relevant for RCT. The current review provides a comprehensive overview of studies carried out on macrophage-specific RCT including a quick reference guide of available data. Knowledge and insights gained on the regulation of the RCT pathway are summarized. A discussion of methodological issues as well as of the respective relevance of specific pathways for RCT is also included.     

Immunological and Antiviral Responses After Therapeutic DNA Immunization in Chronic Hepatitis B Patients Efficiently Treated by Analogues

A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ–producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ–producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4⁺ T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.     

An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.     

The inhibitory effects of exogenous arachidonic acid on rabbit platelet aggregation and the release reaction

Although arachidonic acid causes rabbit platelet aggregation and the release of granule contents in suspensions of washed platelets when used in concentrations of approximately 50-300 microM, higher concentrations (500 microM) cause neither aggregation nor release. Suspensions of platelets from rabbits wee exposed to arachidonic acid (250 microM) for 15 min, allowed to recover in the presence of PGE1 for 30 min, washed, and resuspended; in some experiments, the platelets were treated with aspirin before being exposed to arachidonic acid. Aggregation of platelets pretreated with arachidonic acid was inhibited in response to ADP; this effect was greater with the non-aspirin- treated platelets and persisted for at least 4 hr after resuspension. The association of 125I-fibrinogen with the platelets as a result of ADP stimulation was also inhibited. Aggregation and release of granule contents in response to collagen and low concentrations of thrombin was inhibited, but the inhibition could be overcome by higher concentrations. Thrombin induced further release of granule contents from platelets exposed to arachidonic acid without pretreatment with aspirin. Platelets that had been exposed to arachidonic acid, either with or without pretreatment with aspirin, did not aggregate or undergo further release upon stimulation with arachidonic acid after they were washed and resuspended. Inhibition of the lipoxygenase pathway with eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) did not affect the inhibition caused by arachidonic acid, so it is unlikely that a product of this pathway is responsible for the inhibition. Mixing experiments indicated that the pretreated platelets did not form a thromboxane-A2-like activity, and that they were unresponsive to aggregation and release induced by products formed from arachidonic acid. Experiments with 3H-arachidonic acid showed that after 45 min of incubation with platelets, only 1.1% of the 3H-arachidonic acid remained as free arachidonic acid in the platelets. Although cyclic-AMP was slightly increased 1 min after the addition of arachidonic acid, the cyclic-AMP concentration was the same as that of control platelets after the platelets were washed and resuspended, indicating that increased cyclic-AMP is not likely to be responsible for the persistent inhibitory effect. Thus, the inhibitory effect of pretreatment with arachidonic acid is a general effect on responses to a variety of aggregating agents that act through different mechanisms, and the inhibition is not related to thromboxane-A2 formation. The possibility of membrane perturbation resulting in the unavailability of receptors may explain the persistent inhibitory effect, but the responsible reactions have not been identified.     

Abnormal clonogenic potential of T cells from multiple myeloma patients

Peripheral blood lymphocytes (PBLs) from multiple myeloma patients are defective in both proportion and absolute numbers of OKT4+ cells and have a normal proportion but reduced absolute number of OKT8+ cells. To assess the functional capabilities of the T cells in myeloma patients, we cloned the T cells in PBLs using limiting dilution conditions in which 100% of OKT4+ and OKT8+ T cells in normal PBLs are able to form a clone. In contrast, the OKT8+ cells from PBLs of five of seven multiple myeloma patients were severely compromised in their clonogenic potential; only 7% to 25% of OKT8+ T cells appeared to give rise to a clone. Clonogenic potential of the OKT4+ cells in patients was more nearly normal. Analysis of two multiple myeloma patients with abnormally low numbers of T cells in PBLs revealed the existence of abnormalities in the progenitors of T cell clones. In both patients, two to three times as many T cell clones were observed as would have been expected based on the number of PBLs cultured at limiting dilution, indicating that OKT4-8- cells in PBLs are capable of giving rise to OKT4+ and, at lower frequency, to OKT8+ clonal progeny in vitro. We conclude that purely quantitative assessment of T cell subsets should be interpreted with caution, since proportionately normal numbers of OKT8+ cells in patient PBLs are seriously compromised in their ability to give rise to clonal progeny in vitro, and since there appears to be a OKT4-8- population of T cells in PBLs that are committed to become OKT4+ or OKT8+ T cells, but are unable to do so in vivo.     

Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats

Fibrinogen is a cofactor in the aggregation of human platelets, and is required for ADP-induced aggregation of washed platelets; however, exogenous fibrinogen is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Because with human platelets the cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), inhibits aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets, its effects on rabbit and rat platelets were studied to investigate the differences in the fibrinogen requirements of platelets from the three species. RGDS (50 mumol/L) caused greater than 80% inhibition of thrombin- induced or (ADP + fibrinogen)-induced aggregation of human platelets, but only 3% to 9% inhibition of the aggregation of rabbit or rat platelets, regardless of whether fibrinogen was added. RGDS inhibited the binding of 125I-fibrinogen to ADP-stimulated human platelets by 80% to 90%, but by only 15% to 27% in the case of rabbit or rat platelets. The differences were due to the species of platelets, since human and rabbit fibrinogens gave similar results. In addition, RGDS failed to displace fibrinogen from the surface of rabbit platelets that had been stimulated with ADP. Thus, there are species differences in the ability of the cell adhesion peptide, RGDS, to block the platelet fibrinogen receptor, even within the mammalian species.     

Thrombocytopoietic properties of oncostatin M

Oncostatin M (OM) is a 28-kD glycoprotein that exhibits a panoply of biologic effects. Based on histologic observations of increased splenic megakaryocytes in nude mice implanted with an OM-secreting cell line, the thrombocytopoietic properties of OM in mice were investigated in culture and in vivo. Alone, OM did not induce megakaryocytic colony formation, but in combination with murine interleukin-3 (IL-3), OM markedly enhanced colony formation. The effects of OM on colony formation were similar to those of IL-6. OM alone augmented acetylcholinesterase in short-term marrow cultures. In normal mice, the administration of OM augmented platelet counts without increasing other circulating blood cell counts. The increment in counts exceeded that observed with IL-6. The kinetics of the OM response suggested that maximal increases in platelets occurred 3 days after the cessation of OM administration, irrespective of the duration of administration. In irradiated mice, OM administration accelerated platelet recovery and prevented the decrease in red blood cells observed in irradiated control animals. The data show that OM behaves as a megakaryocytic maturation factor in vitro and augments platelet production in vivo. Based on these animal data, OM may have potential clinical utility as a thrombocytopoietic agent.