Joseph Kurantsin-Mills

Plasma factor VII and thrombin–antithrombin III levels indicate increased tissue factor activity in sickle cell patients

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasmin in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin–antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D‐dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme‐linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0·70 ± 0·19 U/ml versus 1·16 ± 0·41 U/ml), whereas F(VII)z in the patients and controls were 0·47 ± 0·15 U/ml and 1·15 ± 0·33 U/ml respectively, P<0·001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 ± 44 pM versus 42 ± 2 pM) (P<0·001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12·85 ± 1·96 ng/ml versus 4·22 ± 0·37 ng/ml) (P<0·001). The D‐dimer levels were also higher in the HbSS than control plasmas (1029·6 ± 58·6 ng/ml versus 224·3 ± 27·6 g/ml) (P<0·001), with the patients’values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso‐occlusive processes associated with sickle cell painful episodes.

Marked alterations in circulating inflammatory cells during cardiomyopathy development in a magnesium-deficient rat model

Rodents fed on a Mg-deficient (Mg-D) diet develop cardiomyopathic lesions, as well as other types of cardiovascular dysfunction. In the rat, inflammatory cell infiltration of the myocardium begins to occur by week 1, and the lesions develop extensively in the third and fourth weeks on the Mg-D diet. Although the aetiologic mechanisms of Mg-D cardiomyopathy are unknown, we have previously reported that once plasma Mg is markedly reduced, one of the earliest molecular markers of the pathophysiological process is elevation of plasma substance P, calcitonin gene-related peptide and prostaglandin E2, followed by histamine and the inflammatory cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha). In order to evaluate the potential role of specific circulating inflammatory cell subpopulations in the mechanisms underlying pathophysiological changes observed in Mg-deficiency-induced cardiomyopathy, we analysed these cells by flow cytochemistry. Leucocyte subpopulation pools increased progressively in the Mg-D rats. Elevated circulating levels of neutrophils and lymphocytes appeared to contribute to both the acute (week 1-2) and chronic phases (week 3-4) of the inflammatory responses; monocytes, eosinophils, basophils and large unstained cells which are lymphoid in stained smears, on the other hand, increased significantly in the third and fourth weeks and thus contributed to the chronic inflammatory phase. Changes in the circulating leucocyte subpopulations paralleled the chronological progression of the cardiomyopathic lesions, particularly in weeks 3 and 4. Since a pronounced neutrophilia preceded leucocyte infiltration and deposition within the myocardial tissue, modifications of the microvascular barrier may be a prerequisite for cardiomyopathy in this model of neurogenic inflammation.

Flow dynamics of human sickle erythrocytes in the mesenteric microcirculation of the exchange-transfused rat

To analyze the microvascular rheology of sickle cells in an intact animal model, rats were isovolemically exchange transfused with human normal (hemoglobin AA) or sickle (hemoglobin SS) erythrocytes (blood group O) or autologous red cells under ambient conditions, and the effects of the heterologous or autologous cells on (a) hemodynamics and respiration, (b) blood gases, and (c) acid-base status of the recipients were determined. Exchange transfusion of rats with autologous red cells or hemoglobin AA or hemoglobin SS erythrocytes was associated with stable mean arterial blood pressure, central venous pressure, respiration rate, blood pH, pCO2, and pO2 during the experimental period, except for tachycardia among the group of rats that received HbSS cells. Arteriovenous oxygen content varied among the three groups of animals, but, nonetheless, suggested adequate tissue oxygen supply under the conditions of the study. Acid-base status also was similar in the three groups of rats. The exchange-transfused rats were utilized to investigate the flow dynamics of red cells in the mesenteric microcirculation by applying intravital microscopy. Time-averaged velocities of the autologous red cells in 16- to 30-microns (id) vessels ranged from 1.07 to 1.25 mm/sec in single unbranched arterioles with varying flux and wall shear rates. Time-averaged velocities of the HbAA cells in single 15- to 35-microns arterioles ranged from 1.16 to 1.24 mm/sec with wall shear rates similar to the estimates for the autologous cells. For both rat and human HbAA RBCs, the flow dynamics were indicative of normal shear-dependent and deformability characteristics of the cells under the flow conditions. Sickle cells exhibited time-averaged velocities of 0.384 to 0.452 mm/sec, lower wall shear rates in 10- to 35-microns single unbranched arterioles, and three times less volumetric flux. In some arterioles, sickle cells with high axial ratio and low deformability showed definite adhesion to the endothelial surface, residing at such sites for several seconds until dislodged by the force of flow. Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and high axial ratio obstructed flow and exhibited residence times of 2 to 88 sec, thereby causing stasis. These data illustrate the microvascular flow behavior of sickle cells and demonstrate the rheological disequilibrium state that can result as sickle cells course through successive segments of the microcirculation.

Deformability of Normal and Sickle Erythrocytes in a Pressure-flow Filtration System

In order to measure resistance of erythrocyte suspensions to flow through nuclepore filters, a positive pressure cell filtration system has heen developed and applied to measure the deformability of erythrocytes from normal (AA) individuals and patients with sickle cell trait (AS), sickle cell anemia (SS) and hereditary spherocytosis (HS). The pressure flow filtration system permits manipulation of flow rate (Q), hematocrit, pO2, pH, filter pore size and other metabolic parameters. Erythrocyte deformability is defined in this system as the reciprocal of “relative resistance” (Rr) to flow of a given cell suspension. Resistance is derived as the quotient of the pressure generated in response to a specific flow rate; and Rr is the ratio of actual resistance due to the cell suspension to the resistance of the medium alone. Utilizing this system, the following observations have been made: 1. Increase in hematocrit produces an increase in Rr particularly at low flow rates with a direct relationship between hematocrit and resistance until the cell concentration exceeds the filter pore density, resulting in an exponential rise in the pressure generated by increase in Q 2. With 5 μ filters, at low Q and under ambient pO2, AA and AS cells show slight increase in Rr, whereas, SS and HS cells show almost 10-fold greater Rr; 3. Plots of Rr vs. Q simulate a standard viscometric plot and thus data derived from this positive pressure filtration system is comparable to data obtained from cone-plate viscometry; 4. Reduction of pO2 from 165 to 50 mm Hg produces no effect on Rr vs. Q for normal cells, moderate increase for AS cells and marked increase for SS cells; 5. Scanning electron microscopy of the Nucleopore filters fixed during filtration flow depicts cell deformation and pore occlusion by various cell suspensions providing a morphologic basis for the observed resistance-flow kinetics.

Plasma and urinary leukotrienes in sickle cell disease: possible role in the inflammatory process

Abstract. Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady‐state and age‐matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean ± SEM, ng ml‐1) were: LTB4, 8·95 ± 0·26; LTC4, 7·24 ± 0·21; LTD4, 11·42 ± 0·40; and LTE4, 14·51 ± 0·50. Corresponding values for HbSS patients were: LTB4, 6·15 ± 0·42; LTC4, 13·61 ± 1·45; LTD4, 6·44 ± 0·51 and LTE4, 4·97 ± 0·37. The differences were significant at P < 0·05. Urine levels (mean ± SEM, ng mmol‐1 creatinine), for controls were: LTB4, 10·60 ± 0·35; LTC4, 360·0 ± 9·82. Values for HbSS urine were: LTB4, 27·50 ± 3·33; LTC4, 356·0 ± 17·87; LTD4, 69·90 ± 14·51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.

Aggregation of intramembrane particles in erythrocyte membranes treated with diamide

Treatment of erythrocytes with diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) results in oxidation of sulfhydryl groups of the membrane, and cross-linking of membrane proteins into high molecular weight complexes. Concomitant freeze-etching studies show aggregation of intramembrane particles on the protoplasmic fracture face of erythrocyte ghost membranes treated with the oxidant. Furthermore, after a 3 h incubation of erythrocytes with 10 mM diamide at 37 degrees C, cellular energy levels declined to about 70% of control values. The data suggest that disulfide cross-linking of the major membrane proteins releases the apparent physical occlusion of the band 3 proteins within the interstices of the cytoskeletal shell. This results in the translational mobility of band 3 proteins which is reflected ultra-structurally in the freeze-etch images.

Indium-111 oxine labeled erythrocytes: Cellular distribution and efflux kinetics of the label

Indium-111 oxine label erythrocytes are useful in scintigraphic studies of splenic function because of the high yield of gamma-photons [172(90%) and 247(94%) keV] of indium-111. However, the effects of indium-111 oxine on the structural and functional integrity of erythrocytes which might influence their reticulo-endothelial (RE) sequestration are unknown. We examined the morphology of human and rat indium-111 labeled erythrocytes by SEM, the distribution of the label within the cell by analysis of the membrane and cytosol (hemoglobin solution) and the kinetics of efflux of indium-111 from erythrocytes incubated at 37 degrees C in plasma or physiological buffer. Indium-111 oxine labeled red cells retain their discocytic morphology and the cell indices, and density characteristics on phthalate ester are similar to those of the control cells. The efficiency of labeling may be as high as 97%. Human or rat erythrocyte membranes retain 33 and 41% of indium-111, and the cytosol contains 67 and 59%, respectively. About 98% of the indium-111 is bound to the membrane proteins and 1% to the lipid bilayer. Efflux of indium-111 from cells in autologous plasma showed a multiphasic release resulting in about 4-5% release of the label in 2 h and 11.5% in 20 h. Cells in PBS showed 1-5% release of the label during the incubation period. These findings suggest that indium-111 oxine labeling of erythrocytes does not grossly alter the structural and deformability integrity of the cells to induce selective RE sequestration, unless the cells have been damaged prior to or during the labeling procedure, or the spleen is hyperactive.

Irreversible erythrocyte volume expansion induced by tellurite

Tellurite (K2TeO3) has been suggested as a potential anti‐sickling compound because it causes a selective increase in the water content of RBC. To investigate the conditions underlying the increase in RBC volume due to tellurite, normal RBCs were incubated with the compound in a physiological medium and the cells washed with a 10‐fold volume of the medium. The washed cells were then incubated at 24°C for periods up to 4 h and the following parameters were determined: MCV, MCH, MCHC and supernatant haemoglobin concentration by standard methods, the density distribution profile by phthalate esters and cell morphology by scanning electron microscopy (SEM). The effect of hypertonic PBS on the tellurite‐treated cells was also tested. K2TeO3 induced concentration and time dependent increases in MCV and decreases in MCHC without any apparent change in MCH. The median density and the transitional 60% density range of the cell distribution profile respectively decreased and increased in proportion to [K2TeO3] and time. Hypertonic PBS did not inhibit or reverse the tellurite‐induced changes in MCV and MCHC, SEM and photovolumetric measurements demonstrated tellurite‐induced large vesicles ranging in size from 24 to 32 μ3. The proportion of these vesicles increased with time and K2TeO3 concentration. Since tellurite is an oxidant, these findings suggest that its influx into the red cell results in irreversible reactions that disrupt the ion and water regulatory properties of the membrane.

Plasmodium falciparum clearance with artemisinin-based combination therapy (ACT) in patients with glucose-6-phosphate dehydrogenase deficiency in Mali

BackgroundArtemisinin-based combination therapy (ACT) is currently the most effective medicine for the treatment of uncomplicated malaria. Artemisinin has previously been shown to increase the clearance of Plasmodium falciparum in malaria patients with haemoglobin E trait, but it did not increase parasite inhibition in an in vitro study using haemoglobin AS erythrocytes. The current study describes the efficacy of artemisinin derivatives on P. falciparum clearance in patients with glucose-6-phosphate dehydrogenase deficiency (G6PD), a haemoglobin enzyme deficiency, not yet studied in the same context, but nonetheless is a common in malaria endemic areas, associated with host protection against uncomplicated and severe malaria. The impact of G6PD deficiency on parasite clearance with ACT treatment was compared between G6PD-deficient patients and G6PD-normal group.MethodsBlood samples from children and adults participants (1 to 70 years old) with uncomplicated P. falciparum malaria residing in Kambila, Mali were analysed. Study participants were randomly assigned to receive either artemether-lumefantrine (Coartem®) or artesunate plus mefloquine (Artequin™). A restriction-fragment length polymorphism analysis of PCR-amplified DNA samples was used to identify the (A-) allele of the gene mutation responsible for G6PD deficiency (G6PD*A-). 470 blood samples were thus analysed and of these, DNA was extracted from 315 samples using the QIAamp kit for PCR to identify the G6PD*A- gene.ResultsThe DNA amplified from 315 samples using PCR showed that G6PD*A- deficiency was present in 56 participants (17.8%). The distribution of the specific deficiency was 1%, 7% and, 9.8% respectively for homozygous, hemizygous, and heterozygous genotypes. Before treatment, the median parasitaemia and other baseline characteristics (mean haemoglobin, sex and age groups) between G6PD deficiency (hemizygous, heterozygous, and homozygous) and G6PD-normal participants were comparable (p > 0.05). After treatment, parasite clearance did not change significantly whether the participants were G6PD deficient or G6PD normal on day 1 (OR = 1.3; CI = 0.70-2.47; p > 0.05) and on day 2 (OR = 0.859; CI = 0.097-7.61; p > 0.05).ConclusionsThe presence of G6PD deficiency does not appear to significantly influence the clearance of P. falciparum in the treatment of uncomplicated malaria using ACT.

Sickle erythrocytes and platelets augment lung leukotriene synthesis with downregulation of anti-inflammatory proteins: relevance in the pathology of the acute chest syndrome

Initiation, progression, and resolution of vaso-occlusive pain episodes in sickle cell disease (SCD) have been recognized as reperfusion injury, which provokes an inflammatory response in the pulmonary circulation. Some 5-lipoxygenase (5-lox) metabolites are potent vasoconstrictors in the pulmonary circulation. We studied stimulation of production of the inflammatory eicosanoids leukotrienes (LTs) and prostaglandin E2 (PGE2) by isolated rat lungs perfused with sickle (HbSS) erythrocytes. Our hypothesis is that HbSS erythrocytes produce more LTs than normal (HbAA) erythrocytes, which can induce vaso-occlusive episodes in SCD patients. Lung perfusates were collected at specific time points and purified by high-pressure liquid chromatography, and LTC4 and PGE2 contents were measured by enzyme-linked immunosorbent assay (ELISA). Rat lung explants were also cultured with purified HbAA and HbSS peptides, and 5-lox, cyclooxygenase 1/2, and platelet-activating factor receptor (PAFR) proteins were measured by Western blotting, while prostacyclin and LTs produced by cultured lung explants were measured by ELISA. Lung weight gain and blood gas data were not different among the groups. HbSS-perfused lungs produced more LTC4 and PGE2 than HbAA-perfused lungs: 10.40 ± 0.62 versus 0.92 ± 0.2 ng/g dry lung weight (mean ± SEM; P = 0.0001) for LTC4. Inclusion of autologous platelets (platelet-rich plasma) elevated LTC4 production to 12.6 ± 0.96 and 7 ± 0.60 ng/g dry lung weight in HbSS and HbAA perfusates, respectively. HbSS lungs also expressed more 5-lox and PAFR. The data suggest that HbSS erythrocytes and activated platelets in patients pulmonary microcirculation will enhance the synthesis and release of the proinflammatory mediators LTC4 and PGE2, both of which may contribute to onset of the acute chest syndrome in SCD.