Sickle erythrocyte-endothelial interactions in microcirculation: the role of von Willebrand factor and implications for vasoocclusion

To determine the role of von Willebrand factor (vWF) in adhesion of sickle (SS) erythrocytes in microvascular flow conditions, we have perfused the ex vivo mesocecum vasculature of the rat with desmopressin, an analogue of vasopressin that causes the release of endothelial vWF. Analysis of vWF in the venous effluent of the isolated vasculature showed mainly the presence of extra-large molecular weight forms characteristic of endothelial vWF, which in the presence of desmopressin showed an average increase of 54%. Also, desmopressin induced a significant increase in adhesion of washed oxygenated (oxy) unseparated SS erythrocytes, accompanied by a persistent microvascular obstruction and a pronounced increase in the peripheral resistance (PRU). In contrast, infusion of SS deformable discocytes (SS2) in desmopressin-perfused vasculature resulted in a significant adhesion but not in persistent vasoocclusion, showing that SS2 discocytes alone are not sufficient for microvascular obstruction. Furthermore, SS4 erythrocytes (dense discocytes and irreversibly sickled erythrocytes) caused a persistent microvascular blockage and a significantly higher PRU than SS2 discocytes. However, the increase in PRU for SS4 erythrocytes following desmopressin treatment was 50% less compared with a corresponding increase for SS2 discocytes over the control values, which showed a smaller effect of desmopressin on the hemodynamic behavior of SS4 dense erythrocytes. Incubation of desmopressin-treated vasculature with anti-vWF antibodies resulted in a pronounced decrease in adhesion and significantly improved hemodynamic behavior of SS cells. Also, in untreated vasculature, similarly incubated with anti-vWF antibodies, there was almost complete inhibition of adhesion. Under the described perfusion conditions, antibodies to fibronectin and thrombospondin, as well as incubation of SS erythrocytes with anti-vWF antibodies did not affect adhesion. These results are compatible with a model for SS vasoocclusion in which extra- large vWF-mediated adhesion of deformable SS erythrocytes is the first step followed by an accelerated entrapment of dense SS erythrocytes.     

Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.     

A Medical Curriculum for all seasons - a dialogue

Ten years have now passed since the College of Medicine of the University of Malawi was opened. The College''s Curriculum is firmly based on community needs. We describe the genesis and implementation of the curriculum of the College of Medicine and how it has persisted over the past ten years. The challenges that have so far been met are outlined.     

Optic disc haemorrhages and vascular abnormalities in a glaucoma population

Purpose: To retrospectively examine the optic disc photographs of a glaucoma population for optic disc haemorrhages, vascular occlusions and vascular abnormalities. Methods: The optic disc photographs of 906 eyes of glaucoma and suspect glaucoma patients were examined. Optic disc photographs were taken annually, where possible, with the follow-up period varying between 1 and 14 years duration (mean, 2.89). Glaucoma patients are regularly reviewed every 4–6 months and glaucoma suspects every 1–2 years, depending on the ophthalmologist. Low-tension glaucoma patients were reviewed more frequently (mean, every 2.6 months). The results of the findings were compared to a control group of 39 subjects with a mean follow-up period of 7 years, using Fisher's exact test. Results: It was found that during the period under review, 7.4% (n= 67) of eyes had optic disc haemorrhages. The highest frequency of optic disc haemorrhages (37.5%) was found in the low tension glaucoma group (P= 0.0001) followed by 11.4% of primary open-angle glaucoma eyes (P= 0.03). In the normal group there were three eyes with optic disc haemorrhages and one with a disc collateral, which constitutes 5.1% vascular changes in this sub-group. Of the study eyes 2.8% had central retinal vein occlusions, 1.3% branch vein occlusion, 1.2% disc vessel abnormalities (loops) and 1.1% disc collaterals. Discrete nerve fibre layer haemorrhages and microaneurysms were found in 0.8% and 1.8% of eyes, respectively. Conclusions: A total of 16.8% of the eyes observed in this study had either disc haemorrhages or vascular changes. The underlying trend of vascular and haemorrhagic changes in glaucoma are demonstrated in this sample, which is in general agreement with previous studies. The high percentage of optic disc haemorrhages in low tension glaucoma is highlighted. The presence of microaneurysms and nerve fibre layer haemorrhages is interesting but of unknown significance.     

Comparison of thrombin and ristocetin in the interaction between von Willebrand factor and platelets

It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that low concentrations of thrombin also cause platelet membrane receptors to become available for FVIII/vWF. As a consequence, the suspicion has been raised that thrombin provides similar or equivalent activity in vivo to that observed for ristocetin under in vitro conditions. In this study, we quantitated the extent to which thrombin promotes the binding of FVII/vWF to platelets and determined whether or not this interaction initiates or complements platelet aggregation. With ristocetin present, the amount of 125I-FVIII/vWF that became platelet-bound correlated closely with the onset, rate, and extent of platelet aggregation. In contrast, at every thrombin concentration tested, the amount of 125I- FVIII/vWF that specifically bound to platelets was about 6% of that observed with ristocetin. Significantly, FVIII/vWF did not augment the rate of aggregation of platelets in response to thrombin or initiate platelet aggregation when subaggregating doses of thrombin were used. These observations indicate that the minimal association that occurs between FVIII/vWF and the platelet membrane in the presence of thrombin does not correlate with platelet aggregation and therefore is not analogous to the effects of ristocetin. Whether the low level of binding relates to another process, such as platelet-endothelial interactions, remains unknown.     

Platelet-derived growth factor promotes polymorphonuclear leukocyte activation

The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.     

Molecular and cellular effects of antisickling concentrations of alkylureas

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.     

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.     

Marrow transplantation from HLA-identical siblings for treatment of aplastic anemia: is exposure to marrow donor blood products 24 hours before high-dose cyclophosphamide needed for successful engraftment?

The present study in patients with aplastic anemia was undertaken to determine whether exposure of recipients to donor blood products 24 hr before preparation with cyclophosphamide (1) enhanced the rate of sustained engraftment of marrow from HLA-identical siblings as suggested by animal experiments, (2) increased the rejection rate, in particular in transfused patients who may already have been exposed to donor antigens by blood products, or (3) was of no relevance to the outcome of transplantation of marrow from HLA-identical siblings. One- hundred fifty-five patients were studied, of whom 78 received blood products from the marrow donor 24 hr before cyclophosphamide and 77 did not. A binary logistic regression analysis was applied to the data, simultaneously considering five previously known risk factors for rejection. Results showed that preceding transfusion of donor blood products had neither a significant beneficial nor detrimental effect on the incidence of sustained engraftment.