Red cell flexibility and platelet aggregation in patients with chronic obstructive vascular disease (COAD) and study of therapeutic approaches

The red blood cell flexibility was studied in the blood of twenty patients with severe peripheral vascular disease and twenty matched controls. Patients exhibited significantly less red cell flexibility than controls. In both groups there was an inverse relationship between age and red cell flexibility. No correlation was found between red cell flexibility and sex or smoking history. Pentoxifylline, a xanthine derivative which inhibits phosphodiesterases and platelet aggregation, was found to increase red cell flexibility. This effect of the drug was greater on red cells with imparied flexibility than on normal cells. Various prostaglandins by contrast were found to decrease red cell flexibility, this could be compensated for by pentoxifylline. Forty patients with COAD were treated intravenously with PgE1. Significant inhibition of platelet aggregation and clinical improvement was noticed. It is suggested that combinations of PgE1 and pentoxifylline should be explored in clinical studies.     

A counter-rotating "rheoscope chamber" for the study of the microrheology of blood cell aggregation by microscopic observation and microphotometry

An improved “rheoscope” chamber with counterrotating cone and plate, which is suitable for in vitro studies of the microrheology of blood cells, is described. The recent clarification (5, 10) of the flow-dependent changes of the transparency of whole blood supplied the basis for simultaneous objective recording of the photometric effects of red cell aggregation, red cell alignment (12), and cell agglutination (not shown). Similarly, the optical effects of platelet aggregation on the light scattering of PRP in dark-field illumination are measured photometrically. This novel techniques supplements traditional ones and has brought to light characteristics of pathological red cell aggregation at intermediate rates of shear that have evaded viscometric analysis. The physiology of platelets and their role in the natural history of blood coagulation in stasis, at low and at high flow can easily be studied.     

Stored red‐blood‐cells inhibit platelet function under physiologic flow

Background and Objectives The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined. Material and Methods This study looked at buffy coat‐derived platelet concentrates on day 2 of storage along with various stored red‐blood‐cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated ‘young’ and ‘old’ red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis. Results A statistically significant difference was observed between red‐blood‐cells stored for ≤20 days compared with those which have been stored for 21–60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P < 0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time‐points e.g. ‘young’ red cells from stored units were poorer at supporting platelet adhesion and aggregation than ‘young’ red cells from fresh units. Conclusion Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality.     

Filtration measurements to determine the effects of red cell rigidity and aggregation on resistance to flow

By driving samples of red cells suspended in native plasma and in buffered saline through the same filters at the same controlled flow rates, we have determined the percentages of flow resistance associated with red cell aggregation, with single red cells, and with plasma viscosity for 21 blood samples drawn from healthy normal subjects. The filtration system employed uses an in-line series of five porosity-matched 10-μm Swank filters through which blood samples are driven at steady flow rates which approximate conditions in the living microcirculation. We define the red cell contribution to resistance as the difference between the pressure drop for the suspension and for the suspending medium at the same flow rate. Resistance due to aggregation is then defined as the difference between the red cell contributions in plasma and in saline, since no aggregation occurs in the latter. The results indicate that, for normal blood passing through 10-μm pores at 37° and 45% hematocrit, the percentages of flow resistance due to red cell aggregation, to single red cells, and to plasma viscosity are approximately 42, 38, and 20%, respectively. Furthermore, these percentages are relatively independent of flow rate over the range studied. The pressure drop-flow rate curves were found to exhibit a characteristic S-shaped behavior with rather sudden changes in slope occurring in some cases, always at about the same flow rate.     

Detection of a thrombolysis-related reduction in red blood cell adhesiveness/aggregation using a simple slide test

Increased red cell aggregability might have unfavorable rheological effects in the microcirculation. It has been suggested that thrombolysis-related hypofibrinogenemia might be associated with a reduced erythrocyte adhesiveness/aggregation. We followed the reduction in erythrocyte adhesiveness/aggregation using a simple slide test and image analysis that measures the spaces that are formed between the aggregated erythrocytes. A significant (p = 0.01) reduction in the degree of erythrocyte adhesiveness/aggregation was noted in patients with acute myocardial infarction who received thrombolysis as opposed to individuals with Braunwald Class IIIB unstable angina who had normal fibrinogen concentrations. No change was found in the Westergren erythrocyte sedimentation rate, which is an indirect method to detect changes in aggregability of red blood cells. The present study shows the superiority of using a direct measurement of red blood cell adhesiveness/aggregation. This extremely rapid, cheap and almost bedside methodology to detect changes in erythrocyte adhesiveness/aggregation might be useful to detect changes of hemorheological relevance following thrombolysis.     

Red blood cell aggregation and sedimentation: the role of the cell shape

The influence of erythrocyte shape changes on the sedimentation rate was studied in vitro and in vivo. In vitro the highest sedimentation rate was observed with a slight degree of stomatocytosis (morphological index-0.3; i.e. one red cell out of three being a stomatocyte I). With increasing degrees of stomatocytosis the sedimentation rate gradually decreased. Echinocytosis reduced the aggregation and sedimentation very drastically; the sedimentation rate was virtually zero when echinocytosis I or higher degrees were present. The influence of abnormal cell shapes occurring in vivo was studied in patients with an abnormal blood smear. It was found that a severely abnormal red cell morphology reduced the sedimentation rate in a standardized, fibrinogen-rich plasma to about half. These results indicate that the shape plays a crucial role in the aggregation and sedimentation of red cells and they may contribute to the understanding of the interaction of red cells with other cells such as endothelium.     

Effect of changes in erythrocyte lipid spectrum on erythrocyte functional parameters in patients with diabetes mellitus]

Studies of the red cell lipid spectrum in diabetics and of the effects of bioactive substances and reconstruction of the red cell membrane lipid component on these cells aggregation have shown increased levels of lysophosphatidylcholine, cholesterol, cholesterol to phospholipid ratio, and saturated fatty acids in the presence of phosphatidylcholine reduction. Fluctuations in the levels of lipids of other classes were also revealed. Studies of red cell aggregation have revealed that incubation of donors' and diabetics' red cells with hormones did not lead to essential changes in the aggregation induced by alcian blue. Addition of an aliquot of plasma enriched for platelets, glycolipids and alpha-tocopherol resulted in a marked reduction of the aggregation degree and size of the aggregate. Phosphatidylcholine, phosphatidylinositol, and cholesterol-enriched liposome reconstruction of the red cell lipid composition also resulted in reduction of the aggregation degree and of the size of the red cell aggregate. This has brought the authors to a conclusion on a close relationship between the red cell lipid composition and the aggregation capacity in diabetics and on the possibility of regulating the red cell aggregation activity with bioactive substances.     

Rheological abnormalities and thromboembolic complications in heart disease: spontaneous echo contrast and red cell aggregation

The role of abnormal rheological changes in the pathogenesis of thromboembolism has received much attention in recent years, especially in the field of cardiology. Such changes are sometimes seen in an echocardiogram as a smokelike haze known as spontaneous echo contrast (SEC). The presence and severity of SEC correlate with dilated cardiac chambers and the incidence of thromboembolic complications. It is caused by increased red cell aggregation and increased fibrinogen levels, both of which are known risk factors for thrombosis. Although not used clinically, measurements of red cell aggregation can be made in research settings. This can provide findings that give insight into factors causing increased red cell aggregation. A small series of patients with angina pectoris was studied with the Myrenne aggregometer for red cell aggregation. The results, which show correlation between the plasma fibrinogen and triglyceride levels, are presented. As yet, there are only a few therapeutic guidelines for the correction of abnormally high fibrinogen levels in patients at risk.     

Red blood cell aggregation changes are depended on its initial value: Effect of long-term drug treatment and short-term cell incubation with drug

This study was designed to investigate whether the red cell aggregation depends on its initial level under drug therapy or cell incubation with bioactive chemical compounds. Sixty six subjects were enrolled onto this study, and sub-divided into two groups: the first group of patients (n = 36) with cerebral atherosclerosis received pentoxifylline therapy (400 mg, thrice daily) for 4 weeks. The patients of the second group were initially treated with Epoetin beta 10,000 units subcutaneously thrice a week, for 4 weeks. The second group - adult anemic patients (n = 30) with the confirmed diagnosis of solid cancer (Hb < 100 g/L). After 4 weeks of pentoxifylline treatment the red cell aggregation increased (p < 0.05) in the patients with initially low RBCA. On the other hand in the patients with initially high RBCA treatment with pentoxifylline reduced it markedly (p < 0.01). In vitro experiments with pentoxifylline RBC incubation resulted in a decrease of the initially high RBCA by 47% (p < 0.01), whereas in the sub-group with initially low RBCA it increased. It was observed that after 4 weeks of epoetin-beta treatment 75% the anemic patients with initially high RBCA had an aggregation lowering. The drop of aggregation was about 34% (p < 0.01). At the same time 25% of the study patients had a significant RBCA increase (p < 0.05) after treatment. The initially low red cell aggregation after incubation with epoetin-beta was markedly increased by 122% (p < 0.05). On the contrary initially high RBCA was reduced by 47% (p < 0.05). When forskolin (10 μM) was added to the RBC suspensions the RBCA was increased in sub-group of subjects with initially low aggregation and it was decreased in sub-group with initially high one. The similar RBCA changes were observed when RBC suspensions were incubated with vinpocetine, calcium ionophore (A23187), Phorbol 12-myristate 13-acetate (PMA) as a protein kinase C (PKC) stimulator. A major finding of this study is that the red cell aggregation effects of some drugs depend markedly on the initial, pre-treatment aggregation status of the patients. These results demonstrate that the different red blood cell aggregation responses to the biological stimuli depend strongly on the initial, pre-treatment status of the subject and the most probably it is connected with the crosstalk between the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism.     

Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia

Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells.     

Red blood cell aggregation in diabetes mellitus

The aim of this work is to study the red blood cell aggregation in diabetic patients. Investigations have been performed via rheometric technics on four various types of diabetics (diabetics with good metabolic control, diabetics with poor metabolic control, diabetics with a distal angiopathy, without clinical signs, diabetics with a distal angiopathy characterized by cutaneous trophic disorders). Results, compared with those of healthy subjects, show that the reversible phenomenon of erythrocyte aggregation is significantly modified in diabetic. Indeed, the red cell aggregation tendency is found to be increased in diabetics. This hyperaggregation becomes more important when the diabetes is characterized by a poor metabolic control or a distal angiopathy with or without cutaneous trophic disorders. Disturbances of red cell aggregation observed in this report are discussed as a result of plasma protein changes and may confirm the role of hemorheological properties in the pathogenesis of diabetic angiopathy.     

Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease

Background

Recent evidence suggests that red blood cell aggregation and the ratio of hematocrit to blood viscosity (HVR), an index of the oxygen transport potential of blood, might considerably modulate blood flow dynamics in the microcirculation. It thus seems likely that these factors could play a role in sickle cell disease.

Design and Methods

We compared red blood cell aggregation characteristics, blood viscosity and HVR at different shear rates between sickle cell anemia and sickle cell hemoglobin C disease (SCC) patients, sickle cell trait carriers (AS) and control individuals (AA).

Results

Blood viscosity determined at high shear rate was lower in sickle cell anemia (n=21) than in AA (n=52), AS (n=33) or SCC (n=21), and was markedly increased in both SCC and AS. Despite differences in blood viscosity, both sickle cell anemia and SCC had similar low HVR values compared to both AA and AS. Sickle cell anemia (n=21) and SCC (n=19) subjects had a lower red blood cell aggregation index and longer time for red blood cell aggregates formation than AA (n=16) and AS (n=15), and a 2 to 3 fold greater shear rate required to disperse red blood cell aggregates.

Conclusions

The low HVR levels found in sickle cell anemia and SCC indicates a comparable low oxygen transport potential of blood in both genotypes. Red blood cell aggregation properties are likely to be involved in the pathophysiology of sickle cell disease: the increased shear forces needed to disperse red blood cell aggregates may disturb blood flow, especially at the microcirculatory level, since red blood cell are only able to pass through narrow capillaries as single cells rather than as aggregates.     

Red cell aggregation as a factor influencing margination and adhesion of leukocytes and platelets

Leukocytes and platelets must adhere to the wall of blood vessels to carry out their protective functions. Rheological factors influencing these processes are the delivery of the cells to the wall, referred to as margination, and the local shear rates and stresses at the wall. Margination requires leukocytes and platelets to be excluded from the central flow of the much more numerous red blood cells. This exclusion may be influenced by red cell aggregation. Red cell aggregation also influences development of plug flow in small vessels, which in turn modifies the wall shear rate and stress from those expected in ideal Poiseuille flow. Promotion of aggregation by added agents such as high molecular weight dextrans or by reduction in shear rate, increases margination of leukocytes and efficiency of attachment to the vessel wall. Interestingly, however, fewer studies exist for platelets, and these suggest that margination is actually promoted by increasing shear rate. Direct studies of the effects of red cell aggregation on platelets are required, but it appears that aggregation has different effects on delivery of platelets compared to leukocytes. These differences may represent adaptations for efficient adhesion of leukocytes and platelets in different regions of the circulation.     

Red cell aggregation during normal pregnancy

Red cell aggregation (RCA) is responsible for the increase in whole blood viscosity at lower shear rates. RCA depends on the concentrations of red cells and plasma proteins with a high molecular weight and a large and asymmetrical spatial structure such as fibrinogen, immunoglobulin M and alpha 2-macroglobulin. During normal pregnancy, changes occur in all these concentrations. In a prospective study these changes and their influence on the resulting RCA were investigated in 24 healthy women with normal pregnancies. RCA was determined by light reflection measurement (syllectometry). RCA considerably increased during normal pregnancy in spite of the physiological haemodilution. The aggregation half time, used as a measure for RCA, decreased from an average non-pregnant value of 5.6 s to 3.3 s at 37 weeks. Multiple regression analysis showed that the increase in RCA could be mainly attributed to the raised fibrinogen concentration. However, at 37 weeks other factors, in addition to fibrinogen, contribute significantly to the increase in RCA.     

The effect of diuretics on red blood cell microrheological parameters in female hypertensive patients

This study was designed to examine changes of hemorheological parameters and red cell aggregation particularly in essential arterial hypertension subjects receiving antihypertensive diuretic therapy. Fifty six female subjects were enrolled in this study. Thirty seven subjects (group I) were treated for four weeks with Hydrochlorothiaszide (25 mg/day); Nineteen patients (group II) were infused with dose of furosemide 40 mg i.v. Both prior to and following drug treatment for four weeks and four hours after furosemide infusion hemorheological measurements included plasma viscosity; hematocrit, total plasma protein, red cell rigidity index (Tk) and RBC aggregation indices. In addition to this protocol the erythrocytes of patients of group II were incubated with furosemide (0.03 mM; for 30 min at 37 degrees C) to study a direct furosemide effect on red cell aggregation. Treatment and infusion with each of the two drugs significantly (p<0.05) reduced blood pressure in both groups. However, the hemorheological effects of hydrochlorothiaszide therapy were not significant. The effect of furosemide infusion and red cell incubation with it led to significant RBCA elevation. These results thus suggest that the rheologic effects of saluretic diuretics therapy were not significant. Single furosemide infusion and using it in vitro resulted in strong effect of red cell aggregation increase.     

Red cell vacuoles: their size and distribution under normal conditions and after splenectomy

The frequency of occurrence of vacuoles in red blood cells was studied by transmission electron microscopy. Small vacuoles were found in about 13% of the cell sections, and they had a mean diameter of 130 +/- 72 nm (mean +/- SD). It can be estimated that there were about 20 small vacuoles per erythrocyte. The frequency of vacuoles was similar in density-separated cell fractions. In splenectomized patients, the small vacuoles were 4 times more frequent; there was again no difference in vacuole density between top and bottom fractions of density-separated red blood cells. The bottom fraction of red blood cells from splenectomized patients, however, had a high incidence of large vacuoles (greater than 300 nm in diameter) and clustering of small vacuoles. These large vacuoles were probably the result of aggregation and fusion of small vacuoles, and their size allowed detection by light microscopy. Hence, the well-known "pocked" or "pitted" red blood cells of splenectomized individuals were more frequent in the bottom fraction. We conclude that small vacuoles occur normally in erythrocytes, that they tend to cluster and fuse during cell aging, and that the spleen is capable of removing these structures when they reach a certain size.     

Increase in band 3 density and aggregation in hereditary spherocytosis

PURPOSE: Red cells in hereditary spherocytosis are characterized by a reduced surface area/volume ratio. The mechanisms leading to the loss of membrane material and subsequent elimination of the cells have still not been clarified. It was the aim of the present study to analyze band 3 distribution in the red cell membrane and its putative role in red cell elimination. METHODS/RESULTS: Immunogold histochemistry was performed to detect band 3 in red cell membranes. Band 3 density and distribution were visualized by electron microscopy. Unsplenectomized spherocytosis patients (n = 12) showed reduced band 3 density and aggregation compared to controls (n = 15) (density: 1.2 +/- 0.1 gold particles/microm circumference of red cell membrane vs 1.5 +/- 0.07 gold particles/microm, x +/- SEM; P < 0.05; aggregation: 0.26 +/- 0.02 aggregates/microm vs 0.3 +/- 0.02 aggregates/microm). By contrast, band 3 density and aggregation were increased in spherocytosis patients who had undergone splenectomy (density: 2.8 +/- 0.1 gold particles/microm vs 2.0 +/- 0.1 gold particles/microm; P < 0.05; aggregation: 1.5 +/- 0.1 aggregates/microm vs 0.5 +/- 0.03 aggregates/microm; P < 0.01). Artificial ageing of red cells from healthy controls (n = 6) led to a significant increase in band 3 aggregation (2.06 +/- 0.2 aggregates/microm vs 0.33 +/- 0.1 aggregates/microm; P(Wilcoxon) < 0.01) but no change in band 3 density. In hereditary spherocytosis (n = 6), both band 3 density and aggregation increased significantly after artificial ageing of the red cells. The elevated band 3 aggregation was associated with a stimulated erythrophagocytosis in vitro. CONCLUSION: Band 3 aggregation characterizes the red cells in hereditary spherocytosis. It may be the cause of selective splenic phagocytosis of both spherocytes and senescent erythrocytes.     

Approach to the assessment of platelet function: comparison between optical-based platelet-rich plasma and impedance-based whole blood platelet aggregation methods.

Platelet aggregation studies play an important role in the assessment of hereditary and acquired platelet function defects. The first aggregation test introduced into laboratory practice used platelet-rich plasma (PRP) where aggregation was detected by an optical method. The assessment of platelet function using whole blood (WB) aggregation by an impedance method followed up nearly 20 years later. The WB impedance aggregation assay appears to be superior to the optical method because it 1) evaluates platelets in a physiologic milieu in the presence of red and white blood cells, which are known to modulate platelet function; 2) is faster; 3) has higher sensitivity; and 4) does not require centrifugation, thus avoiding injury to platelets and loss of giant thrombocytes. These two assays were compared. Clearly, the WB impedance aggregation methodology has many advantages over the optical PRP assay for the assessment of the hyperactive platelet syndrome and the effects of anti-platelet drugs.     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

A method for on-line measurements of red cell velocity in microvessels using computerized frame-by-frame analysis of television images

We have used a television signal as the input to a digital computer system to measure red cell velocity in capillaries in a sartorius muscle in frogs. In principle, the velocity was measured in terms of the displacement of red cells and plasma gaps along the capillary (represented here by the shift of an optical density waveform) that occurred in a fixed time interval. Instead of using the conventional spatial cross-correlation of the waveforms, the displacement was determined by means of the absolute differences between the waveforms. The quality of the velocity measurement depended on the optical contrast between the cells and plasma gaps and an index of this quality was determined. The velocity in the capillaries of the frog muscle did not exceed 0.7 mm/sec, but it was demonstrated that, in theory, the velocity could be measured over a range of 0–21 mm/sec. This method is simple, allows further computer analysis of the velocity data and is very useful for studies of the microcirculation.