Inhibition of hemoglobin S polymerization by N-terminal band 3 peptides: new class of inhibitors: solubility studies.

Two synthetic peptides corresponding to the N-terminal amino acids (AA) of band 3 were designed to inhibit deoxyhemoglobin S (deoxy S) polymerization through two different mechanisms. Peptide I, an N:1-15AA fragment, was employed to bind to the 2,3-diphosphoglycerate (2,3-DPG) receptor locus of single deoxy S molecules with 5-7 AA extending internally and the remaining 10-8 AA projecting external to hemoglobin (Hb) S, thereby inhibiting polymerization by steric hindrance. Peptide II consisted of two N:1-8AA + K (lysine) sequences linked by a coupler through the lysine, and it was employed to bind to the 2,3-DPG loci of two deoxy S molecules, tethering them together to form "binary hemoglobin complexes" incapable of entering the polymer chains. Decreased polymerization would result from reduction in effective concentration of deoxy S. Binding of peptides to the 2,3-DPG receptor loci was demonstrated by a progressive rightward shift in the hemoglobin oxygen binding curves as a function of increasing peptide concentrations. Inhibition of deoxy S polymerization was studied by equilibrium solubility measurements of purified, stripped solutions of Hb S. Physiologically significant inhibition was demonstrated for both peptides with near-maximum increases in solubility achieved by Peptide II at 1:1 peptide:Hb S ratios. These peptides represent a new class of inhibitors of deoxy S polymerization.

Pyridoxine responsive hereditary sideroblastic erythropoiesis and iron overload: two microcytic subpopulations in the affected male, one normocytic and one microcytic subpopulation in the obligate female carrier.

Mild hepatic iron overload has been demonstrated by magnetic susceptibility measurements in a 22-year-old man with hereditary sideroblastic erythropoiesis despite hemoglobin levels in the normal range and a normal erythropoietin level. His grandfather's sideroblastic anemia has been found to be responsive to pyridoxine; his mother's hemoglobin has persisted in the normal range but red cell volume distribution analysis demonstrated two subpopulations; 30% with estimated geometric mean of 68 fl and 70% an estimated mean of 93 fl. Red cell distribution analysis of the grandson demonstrated two microcytic subpopulations; 46% with an estimated geometric mean of 45 fl and 54% an estimated mean of 70 fl. A therapeutic regimen is outlined to reduce to normal his iron stores and to prevent the future development of excessive iron overload.

Band 3 peptides inhibit deoxy S polymerization: viscosity studies.

We have previously obtained evidence that N-terminal band 3 peptides inhibited deoxyhemoglobin S (deoxy S) polymerization as determined by equilibrium solubility assays. An N:1-15AA fragment binds to the 2,3-diphosphoglycerate (2,3-DPG) receptor locus of deoxy S with five to seven amino acids (AA) extending internally, while ten to eight AA remained external to deoxy S and inhibited polymerization by steric hindrance. A true mirror-image peptide, corresponding to two N:1-8AA + lysine (K) linked by coupler, binds to the 2,3-DPG loci of two deoxy S molecules, tethering them together to form "binary complexes" incapable of entering the polymer chains. The reduction in the concentration of deoxy S available for extended chain formation decreased polymerization. We now report time:viscosity profiles of the sol-gel transformation of purified solutions of deoxy S with and without peptides and studies of the gel solidity at equilibrium. Samples with peptides had longer lag times than controls of similar deoxy S concentrations. The mirror-image peptide was a more effective inhibitor than the N:1-15AA peptide. When the mirror-image peptide was present in peptide:hemoglobin molar ratios of 0.25-1:1, the increases in lag time were equivalent to decreasing the deoxy S concentrations by 15-25%, comparable to projected major therapeutic effects. Gel solidity, determined by yield temperature, was less in the sample with mirror-image peptide compared to control. These results support the proposed mechanisms of inhibition of deoxy S polymerization by band 3 peptides.

Incidence of thrombosis during central venous catheterization of newborns: a prospective study.

Forty-two newborns were studied prospectively to determine the incidence of thrombosis due to central venous catheterization. Following Broviac catheter placement, the catheter tip, distal superior vena cava, and right atrium were evaluated by weekly two-dimensional echocardiograms. The presence of thrombosis was examined in relation to birth weight, gestational age, age and weight at the time of catheter placement, antithrombin III levels, and platelet counts. Six newborns (14%) were noted to have a thrombus by echocardiographic examination after the catheter had been in place for a median duration of 7 weeks. The infants with thrombus formation had significantly lower birth weights (887 +/- 231 v 1,409 +/- 766 g; P = .003) and gestational ages (27 +/- 2.4 v 30.3 +/- 4.3 weeks; P = .018) than those without thrombus. Their weights (757 +/- 203 v 1,832 +/- 1,098 g; P = .000) and ages (2.75 +/- 0.76 v 7.24 +/- 7.8 weeks; P = .002) at the time of catheter placement were also lower; the antithrombin III levels were lower at the time of catheter placement (0.32 +/- 0.08 v 0.06 +/- 0.31 U/mL; P = .001), but were normal for gestational and postnatal age. The presence of thrombosis was not related to the sex of the baby, the platelet count, or the duration of catheterization.

Rheologic properties of mixed hemoglobin gels: deoxyhemoglobins S and A.

To obtain new information concerning the behaviors, and in turn the structures, of gels formed from mixtures of S and A hemoglobins, their physical properties have been characterized by stress relaxation with a rotational rheometer. The variables manipulated were (1) initial total hemoglobin concentration, (2) mole fraction of hemoglobin S present in the mixture, (3) hemoglobin A as intact tetramer only or as both tetramer and hybridized hemoglobin AS, (4) annealing time, (5) shear history, (6) temperature, and (7) temperature and time of annealing. Characteristics monitored to gain information about the effect of these variables on gel properties were (1) lag time, (2) polymer mass, (3) polymer fraction, (4) polymer composition, (5) equilibrium total hemoglobin activity, and (6) solidity/total or hemoglobin S polymer mass (or total or hemoglobin S fraction). As expected, mixed hemoglobin SA gels were less solid than those of pure S of similar initial hemoglobin concentrations because of lower polymer mass, and gel properties were influenced by shear history, annealing time, temperature, and temperature and time of annealing. However, when solidities were compared on the basis of similar quantities of gel present, mixed hemoglobin SA gels were found to be more solid than those of pure S as the mole fraction of hemoglobin S decreased in the initial mixture. This is explained by the predominant influence on gel properties of high hemoglobin activity incurred by the volume exclusion effect of the total hemoglobin concentration. The presence of hemoglobin A with hemoglobin S results in polymers and gels that differ from those found in pure hemoglobin S. Pathophysiologic implications of these findings for sickle cell disorders are proposed.

Rheologic properties of mixed hemoglobin gels: deoxyhemoglobins S and F.

To obtain new information concerning the behaviors--and in turn, the structures--of gels formed from mixtures of S and F hemoglobins, their rheologic properties have been characterized by stress relaxation. The variables manipulated were (1) initial total hemoglobin concentration; (2) mole fraction of hemoglobin S present in the mixture; (3) hemoglobin F as only intact tetramer or as both tetramers and hybridized hemoglobin; (4) hemoglobin F as predominantly G gamma, A gamma, or a mixture of gamma chain types; (5) annealing time; (6) shear history, (7) temperature; and (8) temperature and time of annealing. Characteristics monitored to gain information about the effect of these variables on gel properties were (1) lag time, (2) polymer mass, (3) polymer fraction, (4) polymer composition, (5) equilibrium total hemoglobin activity, and (6) solidity/total or hemoglobin S polymer mass (or total or hemoglobin S fraction). As expected, mixed hemoglobin S and F gels were less solid than pure hemoglobin S gels of similar initial total hemoglobin concentrations because of lower polymer mass, and gel properties were influenced by annealing time, shear history, temperature, and temperature and time of annealing. However, when solidities were compared on the basis of similar quantities of gel present, mixed hemoglobin S and F gels were found to be more solid than those of pure S as the mole fraction of hemoglobin S decreased in the initial mixture. This is explained by the predominant influence on gel properties of high hemoglobin activity incurred by the volume exclusion effect of the total hemoglobin concentration. Pathophysiologic implications of these findings for various sickle cell disorders are proposed.

Solidity of sickle hemoglobin gels: relevance to pathophysiology of sickling disorders.

The physical properties of hemoglobin (Hb) S gels formed under conditions relevant to in vivo conditions have been characterized using viscometry. Mixed Hb SF and Hb AS gels of hemoglobin concentrations and compositions found in patients with Hb S-HPFH (mild sickle-cell disease) and Hb AS (sickle-cell trait) have been compared to those of pure Hb S. The results may explain why red cells containing gels occlude the microcirculation and/or undergo hemolysis in patients with sickle-cell anemia, why patients with Hb S-HPFH are mildly affected with red-cell sickling, and why individuals with Hb AS have no clinical manifestations of the Hb abnormality.

Rheologic behavior of deoxyhemoglobin S gels.

The physical properties of deoxyhemoglobin S gels formed from solutions at concentrations and temperatures approaching those in vivo have been characterized by stress relaxation using a rotational rheometer. Gels were annealed in the rheometer and then subjected to a constant shear strain; thereafter the stress sustained was followed with time. Gels with solid-like behavior held stress indefinitely, and were characterized by yield temperature (the temperature at which stress decreased). Gels with less solid behavior were unable to hold target stress, and were characterized by yield stress (maximum stress attained) and equilibrium stress (final stress held). The samples were ultracentrifuged to calculate pellet and polymer masses. The solidity of the gels, as measured by yield temperature or yield stress, was related to the initial hemoglobin concentration, pellet and polymer masses, shear history, temperature, and the temperature and time of annealing. Solidity increased significantly with time when gels were annealed at 37 degrees C, whereas, when annealed at 25 degrees C, no or minimal increases in solidity were noted. Studies suggest that polymerization occurs rapidly and is completed early in or before the gel annealing period and that the increase in solidity with time of annealing is mainly due to factors other than polymer mass, i.e. alignment, increasing bond strength, water loss. The chemical activity of deoxyhemoglobin S did not affect the solidity of the formed gels. When the resultant polymer masses were comparable, gels formed from samples with albumin present (higher initial total protein concentration, but lower initial deoxyhemoglobin S concentration), had the same behavior as gels formed from solutions with higher initial hemoglobin S concentration. These findings demonstrate that gel annealing conditions must be standardized when comparing the rheologic behaviors of deoxyhemoglobin S gels and indicate that the gel's physical properties (influenced by polymer mass, shear history, annealing time) must be considered in understanding pathophysiology of sickling disorders.

Viscosity studies of deoxyhemoglobin S: evidence for formation of microaggregates during the lag phase.

To look for evidence for the formation of deoxy S microaggregates during the lag phase, the kinetics of gelation of purified deoxy S in dilute phosphate buffer were continuously monitored by a Wells-Brookfield cone and plate microviscometer. Three separate manipulations were performed during the lag phase. (1) The rotating cone of the viscometer was stopped for varying intervals and then restarted. (2) Samples were withdrawn from runs in mid-to-late lag phase ("poised") and reintroduced early in the lag phases of other runs with appropriate volume, temperature, and concentration adjustments. (3) Deoxy S was partially replaced in mid-lag phase by solutions with equivalent volumes, temperatures, and concentrations of deoxy A, F, or C or deoxygenated albumin. The results support the conclusions that (1) irreversible microaggregates are formed early in the lag phase, (2) their formation is favored by stirring, and (3) they are stable if the shearing is stopped for intervals up to six times the total lag times of control runs. Replacement of deoxy S by an equivalent concentration of deoxygenated nonsickle protein during the lag phase does not change the lag time, whereas mixtures of sickle hemoglobin and other proteins made before the reaction is initiated by temperature jump have markedly prolonged lag times. This suggests that once microaggregates of deoxy S are formed, the excluded volume effect of deoxygenated nonsickle hemoglobins or albumin supports polymerization at the same rate as sickle hemoglobin. These results are consistent with the proposed equilibrium nucleation/irreversible growth model of nucleation.

Magnetic-susceptibility measurement of human iron stores.

We made direct noninvasive magnetic measurements of hepatic iron stores with a specially designed superconducting quantum-interference-device (SQUID) susceptometer in 20 normal subjects and in 110 patients with liver disease, iron deficiency, hereditary hemochromatosis, or transfusional iron overload. Magnetic in vivo measurements of liver non-heme iron were closely correlated with chemical in vitro measurements in liver-biopsy specimens (r = 0.98, P less than 10(-5) up to 115 mumol per gram of liver tissue (wet weight) or more. Magnetically determined storage-iron concentrations were below 6.0 mumol per gram in iron-deficient patients and normal men and premenopausal women, but they were raised (9.7 to 31.4 mumol) in 12 of 67 patients with liver disease and were greatly increased (22.9 to 117.7 mumol) in patients with untreated hereditary hemochromatosis or transfusional iron overload. Magnetic measurements of iron stores provide a new quantitative technique for early detection of hereditary hemochromatosis and for rapid evaluation of treatment regimens for transfusional iron overload.

Polycythemia vera in childhood: case report and review of the literature.

A patient presented at 5 years of age with polycythemia vera. He subsequently developed splenic infarctions and died at 20 years of age following cerebral hemorrhage and infarctions. Two months before his death, he developed hypertension and had biochemical evidence of primary hyperparathyroidism and possibly pheochromocytoma. Only nine reported childhood cases of polycythemia vera fulfill the criteria of the Polycythemia Vera Study Group. These cases are summarized and the complications discussed. Although none have progressed to myeloid metaplasia or acute leukemia, these patients are at risk of developing thrombo-hemorrhagic complications; available evidence indicates that they should be managed to keep the hematocrit between 40 and 45%.

Delineation of peripheral bone infarcts in a child with a rare hemoglobinopathy (SOArab) and purpura fulminans: case report.

A 27-month-old patient with SOArab hemoglobinopathy complicated by purpura fulminants was studied with 99mTc-pyrophosphate. The study showed an absence of radiotracer in the bones of both feet and the distal portions of both hands, along with an increased concentration of radiotracer proximal to the regions of absent radiotracer. Subsequent amputation of the distal portions of all four extremities was necessary because of dry gangrene. The amputation site in each extremity (selected on clinical grounds only) corresponded closely to the junction of absent and increased bone radiotracer, suggesting that bone imaging may be used as an aid in determining the extent of nonviable tissue in similar patients.