Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Metastable polymerization of sickle hemoglobin in droplets

Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin beta chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolysing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37 degrees C, though the particular values depend on the details of the experiment. We propose that polymer growth stops because the growing ends reach the droplet edge, whereas new polymer formation is thwarted by long nucleation times, since the concentration of hemoglobin is lowered by depletion of monomers into the polymers that have formed. This finding suggests a new aspect to the pathophysiology of sickle cell disease; namely, that cells deoxygenated in the microcirculation are not merely undeformable, but will actively wedge themselves tightly against the walls of the microvasculature by a ratchet-like mechanism driven by the supersaturated solution.     

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.     

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.     

Nitric oxide reduces sickle hemoglobin polymerization: potential role of nitric oxide-induced charge alteration in depolymerization

We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P₅₀ values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO–HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.     

Polymerization in erythrocytes containing S and non-S hemoglobins

We analyzed the effects of protein and water nonideality and of erythrocyte heterogeneity on the polymerization of hemoglobin S in cells where there were significant amounts of non-S hemoglobins, sickle trait (AS), and SC disease. For AS erythrocytes, the calculated predicted results were in good agreement with measured polymer formation as previously reported (Noguchi C.T., D.A. Torchia, and A.N. Schnechter, 1981, J. Biol. Chem. 256:4168-4171). Throughout much of the physiologically relevant oxygen saturation region, polymer was not formed in AS erythrocytes. Measurements of polymer formation in SC erythrocytes as a function of oxygen saturation using 13C NMR are reported here and also are in good agreement with the calculated predicted results. As in sickle (SS) erythrocytes, polymer can be detected in SC erythrocytes in the region above 60% oxygen saturation. The increased polymer formation in SC erythrocytes as compared with AS erythrocytes can be explained in terms of hemoglobin composition and concentration in SC erythrocytes, with the concomitant increase in the proportion of dense cells. These findings provide a basis for understanding the pathophysiology of sickle cell and of SC disease, in contrast to benign sickle trait, in terms of intracellular polymer formation.     

Autologous immunoglobulin-G binding to erythrocytes subjected to cyclical deoxygenation in vitro

This study demonstrates that low-density metabolically replete HbSS erythrocytes suspended in heat-inactivated autologous plasma and subjected to 15 hr of cyclical deoxygenation (under nitrogen) bind significantly increased quantities of autologous IgG as compared with oxygenated paired samples. IgG binding to the erythrocyte surface was quantified by a nonequilibrium 125-iodinated protein A binding assay and by flow cytometry. Sickle cells deoxygenated 15 hr (37 degrees C) in the presence of 2 mM calcium bound 2.2 +/- 0.2 (mean +/- SD)-fold more IgG (p less than 0.01) than oxygenated paired samples. Sickle erythrocytes deoxygenated in 0.4 mM EDTA bound 1.7 +/- 0.3 (mean +/- SD)-fold more autologous IgG than oxygenated controls (p less than 0.05). Indirect immunofluorescence assays also demonstrated that the relative levels of autologous IgG bound to sickle cells after 15 hr cyclical deoxygenation in the presence or absence of calcium was increased as compared with IgG binding by oxygenated paired samples. After 3 hr of cyclical deoxygenation in the presence of 2 mM calcium sickle erythrocytes exhibited a 40-60% increase in IgG binding, as compared with 10-20% increased IgG binding by paired samples treated in EDTA. These findings demonstrate that repeated morphologic sickling will increase the IgG binding capacity of low-density sickle cells, and suggest that sickling-associated alterations of the cell surface will produce new binding sites recognized by autologous IgG. These studies also show that the sickling-induced increase in IgG binding may be slightly enhanced by the presence of extracellular calcium.     

Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation

We have examined the kinetics of changes in the deformability of deoxygenated sickle red blood cells when they are exposed to oxygen (O(2)) or carbon monoxide. A flow-channel laser diffraction technique, similar to ektacytometry, was used to assess sickle cell deformability after mixing deoxygenated cells with buffer that was partially or fully saturated with either O(2) or carbon monoxide. We found that the deformability of deoxygenated sickle cells did not regain its optimal value for several seconds after mixing. Among density-fractionated cells, the deformability of the densest fraction was poor and didn't change as a function of O(2) pressure. The deformability of cells from the light and middle fraction increased when exposed to O(2) but only reached maximum deformability when equilibrated with supraphysiological O(2) concentrations. Cells from the middle and lightest fraction took several seconds to regain maximum deformability. These data imply that persistence of sickle cell hemoglobin polymers during circulation in vivo is likely, due to slow and incomplete polymer melting, contributing to the pathophysiology of sickle cell disease.     

Kinetics of water transport in sickle cells

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Mixed Exciton-Charge-Transfer States in Photosystem II: Stark Spectroscopy on Site-Directed Mutants

Sickle erythrocytes exhibit abnormal morphology and membrane mechanics under deoxygenated conditions due to the polymerization of hemoglobin S. We employed dissipative particle dynamics to extend a validated multiscale model of red blood cells (RBCs) to represent different sickle cell morphologies based on a simulated annealing procedure and experimental observations. We quantified cell distortion using asphericity and elliptical shape factors, and the results were consistent with a medical image analysis. We then studied the rheology and dynamics of sickle RBC suspensions under constant shear and in a tube. In shear flow, the transition from shear-thinning to shear-independent flow revealed a profound effect of cell membrane stiffening during deoxygenation, with granular RBC shapes leading to the greatest viscosity. In tube flow, the increase of flow resistance by granular RBCs was also greater than the resistance of blood flow with sickle-shape RBCs. However, no occlusion was observed in a straight tube under any conditions unless an adhesive dynamics model was explicitly incorporated into simulations that partially trapped sickle RBCs, which led to full occlusion in some cases.     

Dynamics of oxygen unloading from sickle erythrocytes.

The objective of this work is to theoretically model oxygen unloading in sickle red cells. This has been done by combining into a single model diffusive transport mechanisms, which have been well-studied for normal red cells, and the hemoglobin polymerization process, which has been previously been studied for deoxyhemoglobin-S solutions and sickle cells in near-equilibrium situations. The resulting model equations allow us to study the important processes of oxygen delivery and polymerization simultaneously. The equations have been solved numerically by a finite-difference technique. The oxygen unloading curve for sickle erythrocytes is biphasic in nature. The rate of unloading depends in a complicated way on (a) the kinetics of hemoglobin S polymerization, (b) the kinetics of hemoglobin deoxygenation, and (c) the diffusive transport of both free oxygen and oxy-hemoglobin. These processes interact. For example, the hemoglobin S polymer interferes with the transport of both free oxygen and unpolymerized oxy-hemoglobin, and this is accounted for in the model by diffusivities which depend on the polymer and solution hemoglobin concentration. Other parameters which influence the interaction of these processes are the concentration of 2,3-diphosphoglycerate and total hemoglobin concentration. By comparing our model predictions for oxygen unloading with simpler predictions based on equilibrium oxygen affinities, we conclude that the relative rate of oxygen unloading of cells with different physical properties cannot be correctly predicted from the equilibrium affinities. To describe the unloading process, a kinetic calculation of the sort we give here is required.     

Red blood cell magnetophoresis

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.     

Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking

Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.     

Sickling-induced binding of autologous IgG to erythrocytes heterozygous for sickle hemoglobin

This study reports that sickling-induced increased autoantibody binding can be demonstrated in varying degrees for deoxygenated S/beta-thalassemic (2-fold) and hemoglobin-SC (1.2-fold) erythrocytes as compared with oxygenated paired samples. In contrast, HbAS erythrocytes deoxygenated in autologous plasma exhibited less than 2% morphologic sickling and no increased IgG binding as compared with control samples. Sickling in the presence or absence of plasma increased the IgG binding capacity of S/beta-thalassemic erythrocytes, comparable to previous findings for HbSS erythrocytes, while increased IgG binding to HbSC erythrocytes was detected only after deoxygenation in plasma. It is concluded that specific IgG binding to deoxygenated S/beta-thalassemic RBCs results from subtle permanent sickling-induced alterations of the membrane surface, while IgG binding to HbSC erythrocytes sickled in plasma results from transitory membrane changes. These findings suggest that sickling in vivo will produce cumulative autoantibody binding to S/beta-thalassemic erythrocytes, a process which could lead to immune-mediated erythrocyte destruction. In contrast, comparatively small fractions of the autoantibody bound to HbSC erythrocytes in vivo would result from sickling-induced membrane alterations. These studies indicate that sickling-associated autoantibody binding in vivo will not occur for sickle cell trait (HbAS) erythrocytes protected by plasma.     

Demonstration of lag phase in the sol-gel transformation of deoxygenated S hemoglobin without temperature alteration.

1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.     

Sickle hemoglobin polymer melting in high concentration phosphate buffer

Sickle cell hemoglobin (HbS) prepared in argon-saturated 1.8 M phosphate buffer was rapidly mixed with carbon monoxide (CO)-saturated buffer. The binding of CO to the sickle hemoglobin and the simultaneous melting of the hemoglobin polymers were monitored by transmission spectroscopy (optical absorption and turbidity). Changes in the absorption profile were interpreted as resulting from CO binding to deoxy-HbS while reduced scattering (turbidity) was attributed to melting (depolymerization) of the HbS polymer phase. Analysis of the data provides insight into the mechanism and kinetics of sickle hemoglobin polymer melting. Conversion of normal deoxygenated, adult hemoglobin (HbA) in high concentration phosphate buffer to the HbA-CO adduct was characterized by an average rate of 83 s-1. Under the same conditions, conversion of deoxy-HbS in the polymer phase to the HbS-CO adduct in the solution phase is characterized by an average rate of 5.8 s-1 via an intermediate species that grows in with a 36 s-1 rate. Spectral analysis of the intermediate species suggests that a significant amount of CO may bind to the polymer phase before the polymer melts.     

Spectrophotometric determination of H2O2-generating oxidases using oxyhemoglobin as oxygen donor and indicator

1. Spectrophotometric determination of oxygen uptake using oxyhemoglobin as oxygen donor and indicator was used for assay of H2O2-generating oxidases like monoamine oxidase and glucose oxidase. 2. In order to decompose H2O2 formed during the oxygen uptake, catalase and methanol (or ethanol) was added to the respiratory system. At pH values higher than 7.5 the oxydation of deoxygenated hemoglobin to methemoglobin was less than 3%. 2. Oxidases with low Km for oxygen can be assayed using the spectrophotometric method if suitable correction factors are introduced into the calculation of oxygen uptake. The correction factor represents the ratio of the rate of formation (or disappearance) of one of the reactants and the rate of oxyhemoglobin deoxygenation, measured under identical experimental conditions.     

Multiple nature of polymers of deoxyhemoglobin S prepared by different methods

Studies on the aggregation of deoxy-Hb S in concentrated phosphate buffer revealed the formation of three types of polymers, the difference depending on the method employed for polymerization: 1) random or linear polymers without birefringence, 2) helical polymers with birefringence, and 3) crystals. Random or linear polymers were formed when oversaturated deoxy-Hb S was polymerized by the so-called salting out or isothermal method. Helical polymers were formed when oversaturated deoxy-Hb S (120% of the solubility) was polymerized by the temperature jump method. Crystals were formed preferentially by agitation of the sample during the polymerization below 12 degrees C. The solubilities of deoxy-Hb S measured after preparation of these three types of polymers were different, as were the activation energies for the formation of the three polymers. When a mixture of deoxy- and CO-Hb S was crystallized, the crystalline phase did not contain CO-Hb S molecules. To study the relationship among these three types of polymers and red cell sickling, the morphology of erythrocytes was studied after deoxygenation by several different methods. When erythrocytes were prepared by deoxygenation with 2% sodium dithionite at 30 degrees C, a condition similar to that for the isothermal method, red cells did not form the typical sickle shape but rather an irregular shape. In contrast, with the same experiments carried out by using the temperature jump method, typical sickle-shaped cells were formed. These data suggest that the morphological difference may be attributed to the different types of polymers formed inside erythrocytes.     

Triclinic crystals associated with fibers of deoxygenated sickle hemoglobin.

Triclinic crystals have been found in capillaries that initially contained deoxygenated sickled erythrocytes, and in solutions of sickle hemoglobin that were stirred during deoxygenation. In both cases these crystals occur as a phase transition from fibers. They have been observed only as twins; the a-axis of one member is related to that of its twin by 180 degrees rotation about the b* direction. The volume of the triclinic crystal unit cell is half that of the monoclinic crystals that have also transformed from fibers. Analysis of X-ray diffraction data indicates that the two molecules in the triclinic unit cell that repeat at an interval of 64 A form double filaments similar to those found in the monoclinic crystals and in the fiber. The existence of the triclinic crystals which contain only one double filament per unit cell removes a postulated requirement that antipolar double filament pairs be the sole unit of the fiber architecture.