Coronary artery bypass grafting in immune thrombocytopenic purpura.

BACKGROUND: Reports of patients with idiopathic thrombocytopenic purpura undergoing cardiac operations are scarce and no recommendations exist regarding their management. We report 3 patients with idiopathic thrombocytopenic purpura and severe coronary artery disease who underwent uncomplicated coronary bypass grafting. METHODS: The case history of each patient with idiopathic thrombocytopenic purpura who underwent coronary artery bypass grafting and the literature were reviewed. RESULTS: All 3 patients underwent uncomplicated coronary artery bypass grafting after preoperative treatment with intravenous immunoglobulin and intraoperative platelet transfusions if needed. Prophylactic splenectomy was not performed. There was no increased incidence of bleeding complications. CONCLUSIONS: Coronary artery bypass grafting can be safely performed in patients with idiopathic thrombocytopenic purpura using conventional conduits after pretreating with immunoglobulin G and avoiding splenectomy.

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes.

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H2O2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H2O2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N-ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N-ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N-ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N-ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N-Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N-ethylmaleimide showed that over 50% of N-ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N-ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H2O2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Antiglobulin serum mediated phagocytosis of normal senescent and oxidized RBC: role of anti-IgM immunoglobulins in phagocytic recognition.

Fresh human monocytes usually do not recognize normal RBCs; however, in our newly developed assay antiglobulin-opsonized normal RBCs were phagocytized. Both anti-IgG and anti-IgM fractions present in the antiglobulin serum were involved but the major opsonin was anti-IgM. The anti-IgM opsonized mainly senescent RBCs and therefore could be used to discriminate young from senescent RBCs. The antiglobulin serum and monospecific anti-IgM increased opsonization of in vitro oxidized and desialylated RBCs, whereas trypsin-treatment of RBCs decreased phagocytosis. The material removed by trypsin from the RBCs surface inhibited the antiglobulin and monospecific anti-IgM phagocytic assay supporting the view that membrane associated elements crossreacted with anti-IgM. These results suggest that both internal cellular events and external removal of sialic acid play a role in the emergence of non-IgG covered epitopes on the surface of senescent and oxidized erythrocytes.

Demonstration of haemoglobin associated with isolated, purified spectrin from senescent human red cells.

Employing a direct and sensitive radioimmunoassay (RIA) we have confirmed the presence of haemoglobin associated with isolated, purified spectrin from senescent red cells. Haemoglobin associated with spectrin occurs in the highest amount in cells with an MCHC greater than 36 g/dl and is approximately 3% of the total spectrin extract. Spectrin from the young cells had the least haemoglobin, while an intermediate amount was found in unfractionated, whole red cells. The RIA results were in close approximation with estimation of the haemoglobin-spectrin complex obtained by carefully integrating the Coomassie blue stain profiles from 4% SDS PAGE in densitometric scans from isolated spectrin.

Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

Modulation of mononuclear phagocyte cytotoxicity by alpha-tocopherol (vitamin E).

The in vitro effect of colloidal suspensions of alpha-tocopherol (alpha-T) on phorbolmyristate-acetate (PMA)-induced monocyte cytotoxicity and on antibody-dependent monocyte cytotoxicity (ADCC) was studied. We observed that 1) in the presence of alpha-T, the inhibition was twice as high in the PMA-induced assay than in ADCC; 2) monocytes preincubated with alpha-T were inhibitory in both assays but much less in ADCC, and 3) target erythrocytes preincubated with alpha-T decreased the cytotoxicity in the PMA-induced assay only. Since alpha-T preincubated monocytes showed a decreased release of H2O2 but not of O2-, we concluded that one of the mechanisms by which alpha-T decreased cytotoxicity could be decreased release of H2O2. Whereas the role of H2O2 was documented in the PMA-induced cytotoxicity, in ADCC non-oxidative injury seems more important. This is supported by 1) lesser inhibition of the assay with alpha-T preincubated monocytes; 2) lack of protection with alpha-T preincubated erythrocytes, and 3) mild inhibition with protease inhibitor.

Properties and characterization of vesicles released by young and old human red cells.

We have presently demonstrated morphologic differences between young and senescent red cells following 18 h of metabolic depletion in vitro. Young and old red cells both form echinocytes, whereas only young cells demonstrated myelin forms or microspheres. Furthermore, vesicles were released in greater quantities into the cell-free supernatant from young cells. Isolated vesicles from both young and old red cells contained lipids, intrinsic membrane proteins (especially band 3), and haemoglobin, and they were also enriched in acetylcholinesterase (AChE). Young cells produced more vesicles than old cells but the composition of the low density vesicles was similar except that haemoglobin-spectrin complex was found exclusively in vesicles from young cells. Oxidation of young red cells prior to metabolic depletion prevented both myelin formation and vesicle release.

Increased sensitivity of isolated alpha subunits of normal human hemoglobin to oxidative damage and crosslinkage with spectrin.

After peroxidation, alpha and beta subunits of normal human hemoglobin demonstrated a significant differential reactivity in their ability to form methemoglobin subunits and irreducible crosslinkages with spectrin. The alpha subunits formed crosslinks with spectrin in the absence of exogenous hydrogen peroxide, whereas both the beta subunit and the intact hemoglobin molecule required a minimum of 40 and 4 microM peroxide, respectively, in order to form these crosslinks. Changes in the amount of methemoglobin occurred at much higher concentrations of hydrogen peroxide for the beta hemoglobin subunit (100 microM H2O2) than for the alpha subunit (0.1 microM H2O2). A possible explanation for the existing reactivity between each of the two hemoglobin subunits and spectrin is considered and discussed. In our notation, alpha subunit = alpha SH and the beta subunit = beta SH.

Evaluation of methods using adherence to substrate and density gradient for the isolation of human monocytes.

A new method for monocyte isolation based on cell adherence to gelatin-coated plastic and dislodgement of the adhering monocytes at low temperature was compared with 5 other methods based on cell adherence to substrate and density gradient separation. All methods produced yields of monocytes ranging approximately between 50-70% with about the same degree of purity (less than 90%) except for the method using Percoll density gradient centrifugation where the purity of monocytes was about 80%. When lidocaine at different concentrations was used for cell dislodgement or Percoll density gradient for separation, phagocytosis, Fc receptor function and cytotoxicity were adversely affected, unlike in methods using EDTA or low temperature for dislodgement of the adherent cells. In monocyte chemotaxis assays the rate of migration was affected but not the number of migrating cells for all the isolation procedures investigated. Cell spreading function was apparently well maintained only when gelatin coated plastic was used for adherence and low temperature for cell dislodgement. These data indicate that the newly described method, similar to methods using EDTA for cell dislodgement, yielded relatively intact monocytes but unlike the latter method with better preserved cell spreading. Thus, this method can be considered for standardization to obtain pure monocyte populations from peripheral blood which then can be submitted for comprehensive biochemical and physiologic studies.

Characteristics of peripheral blood monocytes in hereditary xerocytosis and spherocytosis.

Peripheral blood monocytes isolated from patients with congenital hemolytic anemia, hereditary xerocytosis and spherocytosis, demonstrated in vivo engulfment of red cell and platelet fragments. In addition, morphometric studies performed on these monocytes showed an increase in cytoplasmic/nuclear ratio as well as lysosome and phagosome volumes. The production of carbon dioxide from glucose-1-14C in abnormal monocytes was increased (15-80%) but the intracellular values of beta-glucuronidase and esterase activity were similar to control monocytes. Monocyte locomotion assessed in the presence of chemotactic stimuli was found significantly increased (73 +/- 12 monocytes/oil immersion fields vs. 46 +/- 5 for control monocytes). We concluded that the monocytes in hemolytic anemias associated with increased in vitro red cell fragmentation have some features resembling the 'stimulated' monocytes and that this alteration may be due to red blood cell fragment ingestion.

Decreased monocyte function in patients with Hodgkin's disease.

Monocyte function in nine untreated and nine treated patients with Hodgkin's disease in different stages was studied simultaneously with normal controls. Monocyte chemotactic responses were decreased in 6 of the 14 patients with advanced disease regardless of previous therapy. None of our patients with stage II disease had abnormal results. Decreased monocyte chemotactic responses correlated with the presence of cutaneous anergy. In addition, decreased chemotaxis was associated with diminished monocyte bactericidal activity. This suggests that decreased bactericidal activity may be related to abnormal migration and possible diminished ingestion. The data support the hypothesis that depressed monocyte function may contribute to the increased susceptibility to infections of patients with Hodgkin's disease and it may be an additional factor favoring tumor dissemination in the advanced stages of the disease.

Monoclonal macroglobulinemia with osteolytic lesions: a case report and review of the literature.

A patient with monoclonal macroglobulinemia and osteolytic lesions was studied and 24 previously reported closely related cases were reviewed. Cases studied by ultracentrifugation without an immunoelectrophoresis were not considered. Patients presenting with a primary increase in monoclonal IgM molecules may be divided into three groups: typical Waldenstrom's macroglobulinemia, macroglobulinemia with osteolytic lesions resembling multiple myeloma, and a group displaying features of both diseases. At present, cell morphology is not helpful in separating these groups, and a bone survey is recommended. The authors underscore the clinical importance of the early recognition of these entities since procedures such as plasmapheresis for the management of hyperviscosity syndrome, mobilization and local radiotherapy to symptomatic lytic lesions may be necessary for optimal management.