No influence of echinocytic shape transformation of erythrocytes on phagocytosis by autologous monocytes in vitro.

The influence of echinocytosis induced by ATP depletion on erythrophagocytosis was analysed. Monocytes were isolated from peripheral blood of healthy, rhesus-positive volunteers and cultured for a total time of 6 h at 37 degrees C with 5% CO2. Erythrocytes from the same donor, either fresh (discocytes) or incubated at 37 degrees C for 48 h (spheroechinocytes) were added. Phagocytosis was quantified by measuring the chemiluminescence of the ingested heme. The rate of phagocytosis was 0.03 +/- 0.15 discocytes per phagocytic cell and 0.01 +/- 0.08 spheroechinocytes per phagocytic cell. Autologous serum did not affect this rate. The addition of anti-RhD immunoglobulins, however, increased phagocytosis drastically for discocytes and spheroechinocytes (4.22 +/- 2.09 and 5.24 +/- 2.58 erythrocytes per phagocytic cell, respectively). It is concluded that spheroechinocytosis induced by metabolic depletion, certainly one of the most severe forms of erythrocyte shape abnormalities, does not stimulate monocytes to remove these cells.

Quantitative and qualitative analysis of platelet GPIb and von Willebrand factor in liver cirrhosis.

Numerous abnormalities of plasmatic coagulation and platelet function may contribute to the bleeding in liver cirrhosis with a defective platelet-von Willebrand factor interaction being a potential mechanism. To analyze GPIb and von Willebrand factor in cirrhosis, we quantified the number of GPIb molecules on the platelet surface by flow cytometry, assessed the total (and indirectly the internal) pool of GPIb by ELISA and measured the circulating amount of glycocalicin in plasma as a measure of proteolytic activity and platelet turnover. Von Willebrand factor was characterized by ELISA, by its ristocetin-cofactor activity and by multimer analysis. Botrocetin-induced agglutination was used for functional analysis. The data from 8 well-characterized cirrhosis patients indicate that total GPIb is insignificantly increased to 46,000 +/- 5,000 molecules/P (normal: 39,500 +/- 2,000 [SEM]), surface-GPIb is normal with some variability and that the glycocalicin levels are 2-3 times higher than would be expected from the platelet count (= 100 +/- 5 x 10(9)/l). Von Willebrand factor antigen levels and activity were 400-500% of normal with a 22% reduction of the high molecular weight multimers. A significant hyperagglutination response to botrocetin was observed with platelets from both patients and controls using patient plasma as a source of von Willebrand factor. In conclusion, a hyperresponsiveness rather than a defective platelet-von Willebrand factor interaction can be observed in cirrhosis which may compensate for other hemostatic problems and appears to be mediated primarily by increased levels of von Willebrand factor.

Erythrocyte deformability has no influence on the rate of erythrophagocytosis in vitro by autologous human monocytes/macrophages.

Erythrocytes with decreased deformability are known to be rapidly removed from the circulation by splenic macrophages. The exact mechanism is, however, not well understood. We have analysed the phagocytosis of less-deformable erythrocytes by macrophages in vitro. Human monocytes/macrophages were isolated from peripheral blood and cultured for a total time of 6 h at 37 degrees C with 5% CO2. Autologous erythrocytes of the rhesus positive donor were rigidified by heat treatment (47 degrees C for 1 h). The change in erythrocyte deformability was assessed with a filter aspiration technique; the membrane elastic modulus was found to be increased about 2.5-fold. For controls, untreated erythrocytes and erythrocytes incubated with anti-RhD-antibodies were prepared. The rate of phagocytosis during 2 h at 37 degrees C and 5% CO2 was 0.74 +/- 0.59 (erythrocytes per monocyte/macrophage) for controls, 3.58 +/- 2.72 for anti-RhD-loaded erythrocytes and 0.82 +/- 0.74 for heat-treated erythrocytes, respectively. We conclude that decreased erythrocytes deformability does not cause an increased rate of phagocytosis by monocytes/macrophages compared to normally deformable erythrocytes in our in vitro model. This suggests that the preferential removal of rigid cells in vivo is probably not a specific process, but is due to the increased splenic transit time of rigid erythrocytes and hence longer interaction time between erythrocytes and phagocytes.