Intracellular origin and ultrastructure of platelet-derived microparticles.

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. SUMMARY: Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays.

Heparin enhances uptake of platelet factor 4/heparin complexes by monocytes and macrophages.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, platelet factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. OBJECTIVE: To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. METHODS AND RESULTS: Using unlabeled and fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular-weight heparin and complexes composed of heparin and murine PF4, protamine or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show that cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. CONCLUSIONS: Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

BACKGROUND: ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. METHODS AND RESULTS: Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. CONCLUSION: ERp57 regulates thrombosis via multiple targets.

Platelet and monocyte antigenic complexes in the pathogenesis of heparin-induced thrombocytopenia (HIT).

Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder that occurs in a small subset of patients receiving heparin. Twenty-five per cent (or higher) of affected patients develop limb or life-threatening thrombosis. The effectiveness of therapy is incomplete and may be complicated by bleeding. HIT is caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAGs). However, antibodies with the same apparent specificity are found in many more patients without clinical disease and the reason why so few develop HIT is uncertain. We propose that HIT antibodies recognize cell surface PF4/GAG complexes on intravascular cells, including platelets and monocytes that are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications.

Platelet-delivered factor VIII provides limited resistance to anti-factor VIII inhibitors.

BACKGROUND: Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti-FVIII inhibitors. OBJECTIVE: We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIII(null) mouse in the presence of circulating inhibitors. METHODS: FVIII(null) mice that were transgenic for pFVIII (pFVIII/FVIII(null)) were compared with FVIII(null) mice receiving infused FVIII in a FeCl(3) carotid injury model in the presence of anti-FVIII inhibitors. RESULTS: After injury, pFVIII/FVIII(null) mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia. CONCLUSION: In FVIII(null) mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of approximately 6-fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail-clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail-vein exsanguination model to low levels of pFVIII.

High-dose intravenous immunoglobulins for lupus nephritis--a salvage immunomodulation.

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with a great diversity of clinical manifestations which is difficult to manage. IVIGs represent promising immunoregulatory agents with the ability to control SLE without subsequent predisposition to infectious complications. Despite the implied risk of developing renal failure due to IVIG, considerable beneficial effects on lupus nephritis are reported. In this review, the clinical and adverse effects, and mechanism of action, with special emphasis on modulation, of idiotypic network is discussed.

Adverse effects of intravenous immunoglobulin therapy in 56 patients with autoimmune diseases.

OBJECTIVE: To test the adverse effects and viral safety of intravenous immunoglobulin (IVIg) use in autoimmune diseases. METHODS: Fifty-six patients with various autoimmune diseases who were treated with one to six IVIg courses were evaluated for the presence of adverse effects following IVIg therapy and were screened before and after the treatment for the presence of serum human immunodeficiency virus antibodies, hepatitis C virus antibodies, and hepatitis B surface antigen. RESULTS: Among the 56 patients, 20 (36%) had at least one adverse effect following at least one of the treatment courses. These included headache, low-grade fever, chills, anemia, low-back pain, transient hypotension, nausea, intensified perspiration, and superficial and deep vein thromboses. Whereas the presence of adverse effect to IVIg was unrelated to either the clinical response to the treatment or to the nature of the autoimmune disease, the occurrence of an adverse effect in the first treatment course was significantly associated with a greater chance for an adverse effect in the subsequent courses. No transmission of any of the three viral agents examined could be detected. CONCLUSIONS: Although IVIg use in autoimmune diseases is associated with adverse effects in about one third of the patients, these effects are usually mild and transient. Patients who develop adverse effects during the first treatment course may be at increased risk of adverse effects during the subsequent IVIg courses.

Intravenous immunoglobulin treatment of lupus nephritis.

OBJECTIVE: To evaluate the clinical response of treatment-resistant membranous and membranoproliferative lupus nephritis to intravenous immunoglobulin (IVIg). METHODS: Seven lupus nephritis patients who failed to respond to at least prednisone and cyclophosphamide were studied. A kidney biopsy showing either membranous or membranoproliferative glomerulonephritis was available in six patients. They were treated with six courses (patients 1 and 2) or 1 or 2 courses (patients 3 through 7) of high-dose IVIg. For patients 3 through 7, the plasma levels of albumin, total cholesterol, urea, creatinine, dsDNA antibody titers, and daily proteinuria were measured just before the IVIg therapy, immediately on completion, and 6 months later. RESULTS: All seven patients had a beneficial response to IVIg. In patient 1, decrease in proteinuria was evident 2 weeks after IVIg was started, nephrotic syndrome gradually disappeared, and she had no proteinuria in 3 years' follow-up. Decline in proteinuria was evident in patient 2 after the 4th IVIg course, but proteinuria reached the pretreatment level 4 months after the therapy ended. In patients 3 through 7, the mean daily proteinuria before IVIg (5.3 +/- 2.1 g) decreased after 1 or 2 IVIg courses (3.3 +/- 1.4 g), and further decreased when measured 6 months later (2.1 +/- 1.3 g). Similarly, the plasma cholesterol level decreased while the plasma albumin level increased after IVIg. CONCLUSIONS: IVIg might be effective in treatment-resistant membranous or membranoproliferative lupus nephritis. Future studies should concentrate on determining the preferred treatment protocol of IVIg for the various classes of lupus nephritis.

Immunization of low-density lipoprotein receptor deficient (LDL-RD) mice with heat shock protein 65 (HSP-65) promotes early atherosclerosis.

Heat shock proteins are a family of approximately 25 highly conserved proteins upregulated in response to various forms of stress. They play an active role in the development autoimmune diseases in animals, and have been incriminated in human autoimmune diseases (i.e. rheumatoid arthritis, multiple sclerosis). It has been previously shown, that an induced immune response against Heat shock protein 65 (HSP-65) results in atherosclerotic lesions in normocholesterolemic rabbits. We have supported these findings showing that C57BL/6 mice immunized with HSP-65 and fed a high-fat diet develop enhanced fatty streaks. To create a model that will eliminate the need for exogenous supplementation of a high-fat diet, we have immunized LDL receptor deficient (LDL-RD) mice with HSP-65 or with heat-killed Mycobacterium tuberculosis (Mt). Seven groups of LDL-RD mice (n=10), were immunized subcutaneously with different concentrations of HSP-65, Mt or bovine serum albumin (BSA). All mice were fed a normal chow-diet for 3 months. The mice immunized with the higher doses of Mt developed significantly larger fatty streaks when compared with their BSA immunized littermates. The size of the lesions in the aortic sinus were: 31,562+/-5,994 microm(2)in the 10 microg Mt and 52,777+/-5,245 microm(2)in the 100 microg Mt vs. 11, 500+/-3,750 microm(2)in the BSA group (P<0.05). In the HSP-65-immunized mice, only the group injected with the highest dose (5 microg, twice) developed significantly larger fatty streaks when compared with the BSA-immunized group (28,611+/-4,716 microm(2)vs. 11,500+/-3,750 microm(2)respectively, (P<0.05). The HSP-65-but not the Mt- or BSA-immunized mice developed high titers of anti HSP-65 antibodies, beginning 10 days after the immunization, which persisted until they were killed. Immunohistochemical staining showed CD3-positive lymphocytes in the aortic sinus of mice immunized with Mt or HSP-65, but not in the control group. Thus, we established a mouse model of HSP-65 immune mediated atherosclerosis devoid of high fat diet supplementation. This model will enable us to further study the role of the immune system in atherosclerosis, via HSP-65 and raise novel immunomodulatory therapeutic modalities.

[Changes in the capacity of T-lymphocytes for spontaneous recovery of selected differentiation antigens in relation to age]. / Zmena schopnosti spontánnej znovuobnovy vybraných diferenciacných antigénov T-lymfocytov v závislosti od veku.

BACKGROUND: During physiological ageing changes of the immune system take place at several levels. The objective of the submitted work was to compare the ability of spontaneous restoration of selected differentiation antigens on lymphocytes in the peripheral blood stream after previous trypsin treatment in a group of healthy elderly and adult subjects. METHODS AND RESULTS: Twenty-four adults were examined (19-59 years) and 36 elderly subjects (60-90 years). Isolated lymphocytes from the peripheral blood stream were treated with trypsin and then incubated in a cultivation medium. The authors investigated the capacity of restoration of differentiation antigens CD2, CD4, CD8 and CD45RA. Antigen CD2 was not restored in any of the investigated groups to original levels. However the difference between its expression on lymphocytes before trypsin treatment and on lymphocytes after 16-hour incubation was higher in the elderly subjects 16% (p < 0.001) than in the group of adults 7% (p < 0.01). Restoration of antigen CD4 was in both investigated groups almost equal. The number of CD8+ T-lymphocytes was in elderly people lower (p < 0.05), spontaneous restoration of antigen CD8 did not differ among the investigated groups and reached in both instances the baseline value. Antigen CD45RA was restored more slowly in elderly subjects, the difference between groups was at borderline of statistical significance (p < 0.0595). CONCLUSION: From the results ensues that during physiological ageing the ability of spontaneous restoration of antigens CD2 and CD45RA declines but not of antigens CD4 and CD8. So far there is no unequivocal explanation why this change occurs, it is probably conditioned by several factors. Investigation of these changes and an attempt to influence them can help to understand age-conditioned immunological dysregulation, its consequences and the possibility to influence them by treatment.

The hypothalamic-pituitary response in SLE. Regulation of prolactin, growth hormone and cortisol release.

It has been suggested that neuroendocrine regulation plays an important role in the pathogenesis and activation of autoimmune diseases. The aim of this investigation was to clarify the hypothalamic-pituitary response to a well-defined stimulus under standardised conditions in patients with SLE. Plasma concentrations of prolactin (PRL), growth hormone (GH) and cortisol were determined in venous blood drawn through an indwelling cannula during insulin-induced hypoglycaemia (0.1 U/kg b.w., i.v.) in ten patients and in 12 age-, gender- and weight-matched healthy subjects. Basal PRL concentrations were higher in patients vs healthy controls (12 vs 6 ng/ml, P < 0.01), though still within the physiological range. Insulin-induced plasma PRL and GH were significantly increased both in patients and healthy subjects; however, the increments or areas under the curves were not different in the two groups. Plasma cortisol response showed moderate attenuation in patients. Sensitivity of pituitary lactotrothrops to thyrotropin-releasing hormone (TRH) administration (200 microg, i.v.) was the same in patients and control subjects. In SLE patients with low activity of the disease the sensitivity of pituitary PRL release to TRH administration remained unchanged. The hypothalamic response to stress stimulus (hypoglycaemia) was comparable in patients and healthy subjects.

Relationship between endocrine, immune, and clinical variables in patients with systemic lupus erythematosus.

OBJECTIVE: To assess the frequency of increased plasma prolactin (PRL) in patients with systemic lupus erythematosus (SLE) and to evaluate its relationship to other hormonal and immune variables. METHODS: Thirty-five patients with SLE with various levels of disease activity were studied. Plasma PRL, cortisol, growth hormone (GH) were determined by radioimmunoassay and interleukin 6 (IL-6) by ELISA: SLE activity was evaluated using the European Consensus Lupus Activity Measurement (ECLAM). RESULTS: Increased plasma PRL concentration (> 20 ng/ml) was recorded in 11 patients (31%). No correlation was found between plasma PRL and GH, IL-6, cortisol, or C-reactive protein, nor was any significant correlation observed between plasma PRL and the ECLAM score. Patients with hyperprolactinemia were, however, found to have been treated with higher doses of prednisone therapy than patients with normal plasma PRL. Further analysis of the relationship of plasma PRL and therapy showed that patients with SLE selected by the attending physician for prednisone therapy in doses > or = 10 mg/day were more frequently hyperprolactinemic. CONCLUSION: Our findings that patients with SLE with a more active form of the disease and who are less responsive to therapy had increased plasma PRL levels more frequently may be indicative of a potential relationship of hyperprolactinemia to severity of disease.

Effect of domperidone-induced hyperprolactinemia on selected immune parameters in healthy women.

Domperidone, anti-emetic drug, given to healthy female volunteers, induced an elevation of plasma prolactin (PRL) concentration with the peak in 1-4 h. The release of prolactin had a transient stimulating effect on theophylline sensitive T lymphocytes and on concanavalin A induced mitogenic activity, suggesting an enhanced activity of T suppressor lymphocytes. The relative number of CD4+ lymphocytes decreased markedly one hour after domperidone administration and returned to normal values within 2 h (that means 3 h after taking the drug). The number of lymphocytes positive for dipeptidyl peptidase IV exhibited similar transient increase and normalization of activity. No change was observed in the number of CD8+ lymphocytes. The production of interferon by leukocytes treated with Newcastle disease virus was found to be significantly increased 2 h after domperidone administration. The results suggest that prolactin can selectively stimulate some functions of cellular immunity as well as the release of cytokines (IFN). The present study may contribute to the understanding of the role of the immune system in endogenous hyperprolactinemia.

[New findings on the immunologic diagnosis of systemic lupus erythematosus]. / Nové poznatky v imunologickej diagnostike systémového lupus erythematosus.

The authors review recent findings pertaining to the pathogenesis of systemic lupus erythematosus. It was revealed that knowledge of impaired humoral and cellular immunity is of great practical importance and substantially improves the prognosis of the disease.

[Determination of NK cell activity on flow cytometry]. / Stanovenie NK bunkovej aktivity na prietokovom cytometri.

The authors assessed the activity of NK cells on a flow cytometer using 5-carboxyfluorescein diacetate as fluorescent substrate. They examined 25 healthy subjects aged 23-52 years. In NK cellular activity the normal distribution applies and the value of NK cellular activity for the whole group as regards the ratio of effector cells to target cells was 25:1, 23.46 +/- 15.84 percentage. The authors did not detect any significant differences in the activity of NK cells in relation to age and sex. They revealed statistically significant correlations of NK activity and the number of CD16+ and CD57+ cells as well as CD57+ CD3- cells. The optimal ratio of effector and target cells for assessment of NK activity was 25:1. Assessment of NK activity on a flow cytometer can be considered an equivalent substitute of the classical method, using radiochromium.