Phosphatidylethanolamine is progressively exposed in RBCs during storage.

BACKGROUND: It is well established that as a blood unit ages, fewer of the unit's red blood cells (RBCs) remain in circulation post-transfusion. The mechanism for clearance is not well defined. Phosphatidylethanolamine (PE) is a phospholipid that is primarily found on the inner leaflet of healthy cells, and is an important ligand for phagocytosis of dead cells when exposed. OBJECTIVES: The objective of the present study was to measure the change in PE exposure in donor RBCs over increasing storage ages using the novel PE-specific probe, duramycin. METHODS: Five adsol (AS-1) preserved RBC units were sampled weekly for 6 weeks and were labelled with duramycin. The percentage of PE exposed on red cells in each sample was determined using flow cytometry. Surface phosphatidylserine (PS) was evaluated for comparison. RESULTS: We found that RBCs in AS-preserved donor units increasingly exposed PE, from less than 1% in freshly processed RBCs, to nearly 20% at 42 days of storage and correlated with increased relative vesiculation or microparticle concentration and release of cell-free haemoglobin. By comparison, only 5% of cells exposed PS at 42 days. CONCLUSION: We conclude that exposure of PE in the RBC outer membrane was higher than that of PS during 42 days of storage and correlated significantly with increased vesiculation and release of haemoglobin.

D-4F, an apoA-1 mimetic, decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma.

Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DWFKAFYDKVAEKFKEAFNH(2) (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)ß-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michael's adducts in lungs of OVA-sensitized mice. 4F decreased TGFß-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.

Bleeding disorders: A common cause of menorrhagia in adolescents.

OBJECTIVE: To determine the frequency of underlying bleeding disorders in adolescents with menorrhagia. STUDY DESIGN: We retrospectively reviewed the charts of all girls, aged 10 to 19 years, who presented to our children's hospital for inpatient or outpatient evaluation of menorrhagia between January 1990 and November 1998. RESULTS: At presentation, 9 of the 71 girls (13%) had thrombocytopenia (platelet count <150,000/microL; range, 5000-106,000/microL). The most common causes for thrombocytopenia were immune thrombocytopenic purpura (n = 5) and myelosuppression caused by chemotherapy (n = 2). Of 14 girls who underwent a more detailed hemostatic evaluation, 8 were given a diagnosis of a hereditary coagulation disorder: 6 had platelet function defects and 2 had type 1 von Willebrand disease. Excessive menstrual bleeding commonly results in anemia. One half of the total group had anemia (hemoglobin <12.0 g/dL). Seven girls (10%) had potentially life-threatening anemia (hemoglobin <5.0 g/dL). CONCLUSIONS: Acquired and congenital bleeding disorders are common causes of menorrhagia in adolescent girls. Severe anemia is a frequent complication of menorrhagia. We recommend that adolescents without thrombocytopenia who present with menorrhagia receive a comprehensive hemostatic evaluation, including testing for von Willebrand disease and platelet function defects.

Hydroxyurea therapy decreases the in vitro adhesion of sickle erythrocytes to thrombospondin and laminin.

The adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix probably contributes to the pathogenesis of vaso-occlusive disease. The chemotherapeutic agent hydroxyurea (HU) decreases the frequency of vaso-occlusive crises in patients with sickle cell disease. However, the exact mechanism(s) of HU's effect on vaso-occlusive crises is not fully understood. The goal of this study was to determine the effect of HU therapy on the adhesion of sickle erythrocytes to the subendothelial matrix proteins thrombospondin (TSP) and laminin under conditions of flow in vitro. Erythrocytes from patients with severe sickle cell disease on HU therapy (n = 14) had significantly less adhesion to TSP (687 +/- 92 erythrocytes/mm2, mean +/- SE) than untreated patients with severe disease (n = 18, 1176 +/- 117 erythrocytes/mm2, P = 0.003). In addition, there was significantly less adhesion of erythrocytes to immobilized laminin in patients treated with HU (1695 +/- 293 erythrocytes/mm2) than in the untreated patients (2590 +/- 296 erythrocytes/mm2, P = 0.02). Erythrocytes from an additional nine patients with severe sickle cell disease were studied both before and after initiation of HU therapy. Erythrocytes from these patients became less adhesive to both TSP (P = 0.001) and laminin (P = 0.01), a change that was sustained in most patients throughout the duration of the study (2 months to > 12 months). This study suggests that HU modulates the adhesive phenotype of sickle erythrocytes, an effect that may be in addition to, or independent of, other known effects of HU, such as an increase in fetal haemoglobin level.

Low-molecular-weight heparin in thrombotic disease in children and adolescents.

PURPOSE: The use of unfractionated heparin (UFH) in children is problematic. In adults, subcutaneous low-molecular-weight heparin (LMWH) is as effective as UFH in the treatment of thrombosis. Because pediatric data are limited, the authors studied the use of enoxaparin in children. PATIENTS AND METHODS: Nineteen children (ages 18 days to 19 years; median age, 40 months) with indications for thrombosis treatment or prophylaxis were studied. Six patients (median age, 33 months), treated on a protocol that included pharmacokinetic studies, initially received enoxaparin 1 mg/kg subcutaneously every 12 hours; doses then were adjusted until target plasma levels of 0.5 to 1.2 anti-Xa U/mL were achieved. The records of 13 additional patients treated with enoxaparin off study were reviewed. RESULTS: In the first six patients, enoxaparin pharmacokinetics was found to be similar to that in adults; once targeted levels were achieved, these remained stable. Among all 19 subjects, 14 had treatment of active thrombosis and 5 underwent thrombosis prophylaxis. For treatment of thrombosis, enoxaparin 1 mg/kg initially was administered subcutaneously every 12 hours. Target anti-Xa levels were achieved with 0.55 to 1.5 mg/kg every 12 hours (mean, 0.98 mg/kg; median, 1.0 mg/kg) in 1 to 7 days (median, 1 day). All patients in the treatment group had clinical improvement within 2 to 5 days, and 12 had follow-up radiological studies that confirmed this. For prophylaxis, enoxaparin was given at 1 mg/kg subcutaneously every 24 hours. No new thrombi were clinically evident in this group. There was no major bleeding with enoxaparin; one patient had transient mild mucosal oozing. CONCLUSION: In this limited population, enoxaparin seems to be a safe, effective, and convenient alternative to UFH in children and adolescents. The adult therapeutic target range of 0.5 to 1.2 anti-Xa U/mL is readily achievable with a starting dose of 1 mg/kg every 12 hours in most children. Initial close monitoring with plasma anti-Xa activity should be done and doses adjusted to achieve target range, particularly in neonates. In the population of this study, enoxaparin seems as effective as UFH in the period immediately thrombotic episode. These results should be confirmed in the ongoing randomized trial comparing LMWH with UFH in children.

The carboxy-terminal cell-binding domain of thrombospondin is essential for sickle red blood cell adhesion.

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% +/- 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6. 7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.

Potential therapeutic approaches for the treatment of vaso-occlusion in sickle cell disease.

Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The pathogenesis of vaso-occlusion likely involves many complex steps related to both the primary event of deoxyhemoglobin S polymerization and the many resultant pathologic changes in both the sickle erythrocyte and the vascular endothelium. Several therapeutic approaches have been designed to reduce the extent of deoxyhemoglobin S polymerization. Although direct chemical inhibition of deoxyhemoglobin S polymerization has proven difficult, strategies to increase levels of fetal hemoglobin or reverse sickle erythrocyte dehydration have been more successful. Although gene therapy is actively being pursued, the ability to cure sickle cell disease is currently limited to bone marrow transplantation with its attendant toxicities and limitations. Because the pathophysiology of vaso-occlusion in sickle cell disease is complex, its treatment will likely be optimized using a multifactorial therapeutic approach.

Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

Mechanisms of stroke in sickle cell disease: sickle erythrocytes decrease cerebral blood flow in rats after nitric oxide synthase inhibition.

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.

Hydroxyurea therapy in children severely affected with sickle cell disease.

HYPOTHESIS: The major clinical manifestations of sickle cell disease (SCD) are hemolytic anemia, predisposition to infection, and recurrent vaso-occlusive episodes resulting in pain, organ dysfunction, or both. There has been no satisfactory treatment for children with recurrent severe painful episodes caused by SCD. Hydroxyurea is an antimetabolite drug shown in adults with SCD to increase fetal hemoglobin levels and reduce the symptoms of SCD. We hypothesized that hydroxyurea therapy in children with severe (defined as > or = 3 vaso-occlusive events per year) SCD could improve hematologic parameters and reduce vaso-occlusive events. OBJECTIVE: To assess the safety and efficacy of hydroxyurea for the treatment of severe SCD in children. STUDY DESIGN: After obtaining informed consent, we initiated hydroxyurea therapy at a dosage of 10 to 20 mg/kg per day in 15 patients with severe SCD (hemoglobin SS, hemoglobin SS-alpha thalassemia, or hemoglobin S-beta0-thalassemia). Doses were escalated as tolerated. Patients were monitored with bimonthly physical examinations and monthly laboratory measures. To assess the impact of hydroxyurea on clinical symptoms, we recorded the number of inpatient days for each patient during the period of treatment and compared it with the number of inpatient days for the 12- to 24-month period before institution of hydroxyurea therapy, using the subject as his or her own control subject. RESULTS: Thirteen patients received hydroxyurea for a median of 24 months (range, 6 to 39 months). The mean dose of hydroxyurea was 21.4 +/- 5.2 mg/kg. Treatment with hydroxyurea induced statistically significant increases in the total hemoglobin concentration, mean corpuscular volume, and percentage of hemoglobin F, and a decrease in the serum concentration of bilirubin. Toxic effects included three episodes of a reversible myelotoxic reaction, two of which required transfusion of packed erythrocytes. For the entire group, hospitalization decreased from a prehydroxyurea median of 3.9 +/- 2.0 days per month to 1.1 +/- 2.1 days per month of therapy (p = 0.09). For those subjects (n = 10) completing at least 1 year of treatment, the decrease in hospitalization from 4.1 +/- 2.2 to 1.0 +/- 1.7 days per month was significant (p = 0.03). CONCLUSIONS: In this pilot trial, hydroxyurea treatment of severe SCD in children was associated with improved hematologic parameters, acceptable toxic effects, and a trend to reduced hospitalization. Hydroxyurea appears to be a safe and potentially effective agent for the treatment of severe SCD in children. A prospective, controlled trial to investigate the efficacy of hydroxyurea in children is therefore warranted.

Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin.

Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well-characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases.

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor.

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.