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The present study has evaluated the influence of high concentrations of cytochalasins B and E on the detergent-resistant actin levels in fully spread platelets by PAGE gel electrophoresis, and the effects of the two inhibitors of new actin filament assembly on translocation of fibrinogen gold (Fgn/Au) labelled GPIIb-IIIa receptors on the surface-activated cells. Concentrations of 10(-4) M and 10(-5) M cytochalasin B and E reduced detergent-resistant actin in fully spread platelets to levels present in resting discoid platelets in suspension. Despite reduction of actin filaments to levels in resting cells, cytochalasin B did not prevent translocation of Fgn/Au from platelet margins into channels of the open canalicular system (OCS). Similar concentrations of cytochalasin E completely blocked translocation of receptor-ligand complexes and produced a patching phenomenon not observed in previous studies. Rinsing of the spread cells to remove cytochalasin, followed by incubation of the treated platelets in Hank's buffered salt solution (HBSS) restored levels of detergent-resistant actin to those found in untreated, spread platelets. Resting grids of 10(-5) M cytochalasin E-treated platelets on drops of HBSS for 15 min restored their ability to clear FGN/Au linked to GPIIb-IIIa from exposed surfaces to the OCS, but 10(-4) M cytochalasin E-treated cells remained anergic after incubation on drops of HBSS. Thus a fully assembled cytoplasmic actin filament cytoskeleton does not appear to be essential for translocating receptor-ligand complexes on the platelet surface to the OCS, nor does its presence guarantee that the ability to clear GPIIb-IIIa receptors will be restored.

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Hemorheologic alterations induced by incremental treadmill exercise were examined in 5 Thoroughbreds. Blood viscosity; PCV; RBC filterability, density gradient profile, and shape; serum and RBC electrolyte concentrations; and plasma total solids and lactate concentrations were measured before exercise, at treadmill speeds of 9 and 13 m/s, and 10 minutes after exercise. Exercise was associated with significant (P < 0.05) increases in PCV, blood viscosity, and plasma total solids concentration. After adjustment of PCV to 40% by adding or removing each horse's own plasma, blood viscosity remained significantly greater in the sample obtained at 13 m/s, compared with that in samples taken at rest. Filterability of RBC was significantly decreased at 13 m/s, compared with values from other sampling times. During exercise, a significantly greater proportion of the RBC were less dense and were found in the upper layers of the RBC density gradient profile, compared with resting values. This change was associated with a significant increase in RBC mean cell volume. Rapid increases in serum sodium and potassium concentrations during exercise were accompanied by significant increases in RBC potassium and chloride concentrations. This study revealed a consistent pattern of hemorheologic alterations associated with exercise in Thoroughbreds, suggesting that multiple hemorheologic tests are needed to adequately define these complex alterations during exercise in horses.

Effects of echinocytosis on blood rheology and exercise performance were evaluated for 5 Thoroughbreds. Echinocytosis was induced by administration of furosemide (1 mg/kg of body weight, IM, q 12 h) for 4 days. Furosemide treatment resulted in decreases in serum sodium and serum chloride concentrations and in RBC chloride and potassium concentrations. Echinocytosis was associated with increased RBC density as determined by RBC density gradient centrifugation. However, samples containing echinocytes were more filterable than control samples, indicating that echinocytes were not rigid cells. Erythrocyte sedimentation rate was decreased in blood samples containing echinocytes, indicating that cell-to-cell interaction was reduced. Whole blood viscosity was not altered by presence of echinocytes. Echinocytes did not impair the capacity of horses to complete treadmill exercise tests, nor did they alter heart rate or blood gas variables. However, plasma lactate concentration was higher in samples obtained during exercise at a treadmill speed of 9 m/s. Echinocytosis was associated with higher postrace creatine kinase activity. These data indicate that echinocytes may be dense, but not rigid cells, which have decreased tendency to aggregate and do not increase whole blood viscosity. Therefore, echinocytes are unlikely to inhibit or obstruct microvascular blood flow.

The present investigation has evaluated the influence of temperatures ranging from 37 degrees C to 45 degrees C for intervals of 30, 60, and 90 minutes on the biochemistry, morphology, and function of human platelets. Exposure to temperatures up to 43 degrees C for an hour did not significantly alter platelet morphology or physiologic response to aggregating agents. Samples of platelets heated at 43 degrees for 60 minutes lost their ability to aggregate in response to arachidonate, but sensitivity was restored by pretreatment with epinephrine. Platelets heated to 45 degrees C for 90 minutes were converted from discs to spheres and failed to aggregate in response to all agonists, whether or not they were pretreated with epinephrine. The platelets heated at 45 degrees C for 90 minutes could adhere to formvar or denuded subendothelium but were unable to extend pseudopods or to spread. They maintained normal levels of adenine nucleotides and serotonin but failed to secrete these products on stimulation. Studies with fibrinogen coupled to gold (Fgn/Au) revealed only a few particles bound to glycoprotein IIb-IIIa (GPIIb-IIIa) on platelets heated at 43 degrees C for 90 minutes. Ligands that did bind to GPIIb-IIIa were transported to the open canalicular system. Biochemical studies demonstrated normal synthesis of thromboxane B2 and calcium flux by platelets heated at 45 degrees C for 90 minutes. Polyacrylamide gels of platelets heated at 45 degrees C for 90 minutes showed an increase in talin incorporation into heated platelet cytoskeletons but no increase in filamentous actin. The findings indicate that impaired function of heated platelets is due to the influence of heat on cytoskeletal proteins important for pseudopod extension, shape change, expression of GPIIb-IIIa, or other surface membrane receptors and secretion, but that impaired function is not due to inhibition of biochemical systems involved in activation events.

Forty-one children were identified with autoimmune neutropenia of infancy and early childhood (absolute neutrophil count [ANC] less than 500/microliters and demonstrable serum antineutrophil antibodies). There were 21 boys and 20 girls; the median age at diagnosis was 11 months (range 5-38 months). No life-threatening infections occurred. There was a gradual upward trend in ANC in all patients over many months, with 87% having an ANC > 1000/microliters by 24 months from diagnosis. Among various clinical and laboratory parameters analyzed statistically, only younger age at diagnosis was associated with earlier neutrophil recovery. There was no association between degree or duration of neutropenia and sex, race, antibody reactivity, or presence of serious illness at diagnosis.

This study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100-insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension-activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent-resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.

The effects of furosemide and pentoxifylline on blood flow properties in horses were investigated. Hematologic and rheologic changes were examined in 4 horses before and 3 minutes after administration of epinephrine (1 mg, IV). The next day, hemorheologic changes were determined before and 3 hours after administration of furosemide (1 mg/kg of body weight, IM), and after administration of epinephrine at the sampling at 3 hours. Hematologic and rheologic changes were evaluated weekly in 3 horses given pentoxifylline (8.5 mg/kg, q 12 h, PO) for 28 days. In addition, hemorheologic responses to epinephrine were determined on days 0, 14, and 28 of pentoxifylline treatment. Neutrophil filtration studies were also performed 2 hours after IV administration of pentoxifylline (8.5 mg/kg). Postepinephrine values for PCV, RBC and WBC counts, and blood viscosity were greater than preepinephrine values. Erythrocyte sedimentation rates decreased after epinephrine, whereas RBC filterability did not change. Treatment with furosemide was associated with increases in mean RBC hemoglobin concentration and blood viscosity. Filterability of RBC did not change. Treatment with pentoxifyllie resulted in an increase in RBC filterability and erythrocyte sedimentation rate and a decrease in PCV; however, mean values for hematocrit and RBC count did not change. Treatment with pentoxifylline did not result in a change in resting blood viscosity, but markedly reduced the postepinephrine increase in blood viscosity. Neither IV nor orally administered pentoxifylline had an effect on neutrophil filtration. It was concluded that pentoxifylline has beneficial effects on RBC filterability and postepinephrine changes in blood viscosity, which may contribute to improvements of microcirculatory blood flow. In addition, furosemide may exacerbate exercise-associated hyperviscosity in horses.

Mild thrombocytopenia is common in alcoholic individuals. Ethanol appears to impair platelet production primarily by affecting the maturing megakaryocyte compartment. We recently showed that guinea pigs have an unusually large number of elongated platelet forms even under conditions of steady-state thrombopoiesis, and a portion of their mature (stage IV) megakaryocytes yield extremely long extensions on micropipette aspiration. This study evaluates the effect of a moderately low level of ethanol consumption by guinea pigs on platelet size and form and megakaryocyte deformability. Adult Duncan Hartley guinea pigs took ethanol 2.5% (vol/vol) ad libitum for 4 weeks under environmentally controlled conditions, never reaching detectable blood ethanol levels (< 0.01%); the platelet count fell 16%. Elongated platelet forms constituted 29% of the cardiac puncture platelets of control animals, but only 3% of the cardiac puncture platelets of animals given ethanol; discocytic platelets of ethanol-treated animals were also significantly smaller. Individual stage III and IV megakaryocytes were aspirated into 5 microns diameter micropipettes by stepwise increment in pressure from 10 to 200 cm water. The extensions drawn from megakaryocytes of ethanol-exposed animals were significantly shorter than the extensions from control megakaryocytes. Extremely long extensions over 50 microns in length were drawn from 21% of the control megakaryocytes but less than 1% of ethanol-exposed megakaryocytes. Moderately low level ethanol consumption was associated with reduced platelet count and size, along with rigidity of mature megakaryocytes in guinea pigs. Few ethanol-exposed megakaryocytes yielded extremely long cell extensions, nor were elongated platelet forms prevalent in the circulation of animals given ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo.

Echinocytes have been incriminated in the pathogenesis of exertional diseases in horses. To evaluate the hypothesis that echinocytes are dehydrated erythrocytes, we decreased blood sodium and potassium concentrations in 4 horses by administering furosemide (1.0 mg/kg of body weight, q 12 h) for 2 days and we monitored CBC, serum and erythrocyte sodium and potassium concentrations, and echinocyte numbers. Serum sodium concentration decreased progressively over the 48 hours of furosemide administration, then returned to near baseline concentration at 168 hours. A statistically significant decrease (P < 0.05) in serum potassium concentration was observed at 24, 48, and 72 hours after initial furosemide administration, and remained less than the baseline value at the end of the study. Mean erythrocyte potassium concentration decreased rapidly and remained low at the end of the study. Minimal changes were observed in erythrocyte sodium concentration during the first 72 hours after furosemide administration, but the value was significantly (P < 0.05) increased at 168 hours. Type-I and type-II echinocyte numbers increased by 4 hours after furosemide administration and persisted throughout the study. Type-III echinocytes were not seen in baseline samples, but numbers increased only modestly after furosemide administration. Administration of epinephrine to well-hydrated horses increased echinocyte numbers only minimally, indicating that splenic contraction was not the likely cause for the furosemide-associated increase. To determine whether the decrease in erythrocyte potassium concentration and increase in sodium concentration was caused by furosemide acting directly on the erythrocyte membrane, we quantified erythrocyte potassium and sodium concentrations before and after incubation with furosemide in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematologic and rheologic changes were examined in 49 Thoroughbreds before and after competitive racing. Mean postrace values for RBC count, hemoglobin concentration, and PCV increased by 58 to 61%, whereas blood viscosity increased 2 to 3 times. Postrace echinocyte numbers were 162% greater than prerace values. Smaller, but statistically significant, changes were found for mean corpuscular hemoglobin concentration, red cell distribution width, plasma total protein concentration, total WBC count, neutrophil count, and lymphocyte count. Variables measured did not predict whether a horse was a bleeder not treated with furosemide, a bleeder treated with furosemide, or a nonbleeder.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.

Elongated cylindric and beaded platelet forms are infrequently observed in the circulation, and have been termed "proplatelets," as presumed precursors to discoid platelets. Some 15% of the blood platelets obtained from the guinea pig by cardiac puncture were elongated forms (1/5 beaded and 4/5 cylindric) under conditions of steady state thrombopoiesis, thus providing an opportunity to contrast the structure and deformability of platelets of potential graded maturation. Ultrastructural studies were consistent with graded maturation. Longitudinal bundles of microtubules in cylindric platelets gave way to the appearance of basket-weave arrays in the nodal thickenings of beaded forms, and, finally, to circumferential coils in a portion of the discoid platelets. Individual platelets were aspirated into micropipettes of 0.8 microns internal diameter by application of incremental pressure up to a maximum pressure of 10 cm water. None of the guinea pig platelets fragmented into smaller forms even when aspiration was performed under physiologic environmental conditions. All the guinea pig platelets passed through the micropipette in aspiration studies performed under conditions minimizing platelet activation and adhesion. Elongated platelets aspirated at the very ends of the cells passed through the pipette at lower pressure than discoid forms, whereas elongated platelets aspirated along the length of the cell were more resistant to deformation and cell passage than were discoid platelets. Beaded forms were similar in deformability to cylindric platelets at low aspiration pressure but were more resistant to deformation at high pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors have identified six Southeast Asian patients ranging in age from 14 to 21 years with hemoglobin E-beta(0) thalassemia and a coagulopathy involving von Willebrand factor (vWF). These patients had normal or only slightly decreased plasma clotting factor levels. The activated partial thromboplastin time was prolonged in four of the patients. The abnormal feature common to all patients was a qualitative loss of high molecular weight multimers of vWF by crossed immunoelectrophoresis (vWF:CIE). Plasma vWF antigen concentration (vWF:Ag) and ristocetin cofactor activity (vWF:RCo) also were decreased and bleeding time prolonged in three patients. Epistaxis was present in two. No family history of increased bleeding tendency was present in any patient. Coagulation parameters and vWF:CIE were normal in two first-degree relatives without this hemoglobinopathy. vWF abnormalities and clinical manifestations were greatest in those patients with the most severe anemia and hepatosplenomegaly. These six patients appear to have an acquired abnormality of vWF, although they lack the clinical characteristics of acquired von Willebrand disease. While the etiology of this abnormality is unclear, the authors speculate that proteolysis of vWF secondary to extramedullary hematopoiesis or loss through high cardiac output shear stress in these anemic patients may be involved.

Patients with a metabolic block in the conversion of THCA to cholic acid develop cirrhosis and hemolysis. Tauro-THCA has been shown to distort hepatic architecture and cause hemolysis in bile-fistula rats. In this study, the critical micellular concentration of tauro-THCA was found to be one fourth of that measured for the primary human bile salt, taurocholate. In short-term incubations with intact red cells, tauro-THCA was more effective than taurocholate in removing red cell membrane lipid, inducing morphological red cell sphering, and decreasing functional cellular membrane surface area. These detergent biological membrane effects were most apparent at a concentration above the critical micellar concentration, with the membrane toxicity of the two bile salts roughly paralleling their differences in critical micellar concentration. The lower critical micellar concentration, greater hydrophobicity, and enhanced surface-active properties of tauro-THCA are speculated on as possible factors contributing to the bile salt's toxicity in vivo.

Platelets from subjects with hyperlipoproteinemia (HLP) differ from normal platelets in lipid composition and function depending upon the phenotypic classification of the HLP. The present study has evaluated the deformability of platelets from human subjects with type IIa and type IV HLP. Platelets suspended in autologous plasma diluted 30-fold with buffer were aspirated into micropipettes 0.7-0.8 microns in diameter by step-wise increment in tension, and the resulting extension lengths were recorded. Platelets from type IIa subjects could not be aspirated as far into the micropipettes as normal platelets. However, less tension was required to reach maximum cell extension than with normal platelets, and the initial extension lengths and slopes of the stress responses were the same as the control. In contrast, platelets from subjects with type IV HLP showed a generalized increase in deformability. The initial cell extensions aspirated from type IV platelets were longer than normal, and larger maximum cell extensions were achieved at lower tensions than control platelets. The type IV platelets were also mechanically fragile and fragmented at lower tensions than control or type IIa platelets. The variance in platelet deformability between subjects of the same phenotype was not directly correlated to plasma lipid or lipoprotein concentrations. This study confirms alterations in the structural organization of platelets from subjects with type IIa and type IV HLP.

A perfluorochemical blood substitute emulsified with Pluronic F-68 has been shown to improve the filterability of deoxygenated sickle red cells. We questioned whether some of the effect was independent of oxygen loading and studied the influence of Fluosol DA (Green Cross, Osaka, Japan) and Pluronic on the rheology and adhesion of sickle red cells saturated with oxygen and carbon monoxide. A 5-vol/vol% concentration of Fluosol or equivalent concentration of Pluronic was equally effective at improving the filtration of washed sickle cells through 5-micron-diameter pores at wall shear stresses approximating 1,000 dyne/cm2. The same concentration of Pluronic reduced the extensional static rigidity of irreversibly sickled cells (ISC) by 25% and also abolished the adherence of gravity-sedimented sickle cells to endothelial monolayers in the presence of saline or plasma. The inhibition of adherence was not reversible by washing and was accomplished with equal ease by isolated treatment of sickle cells or endothelium. Pluronic had no effect on the rheology or adhesion of normal adult red cells. Neither Fluosol nor Pluronic changed sickle or normal cell shape, mean cell volume, mean cell density, or cell density distribution. A lubricating effect of Pluronic on cell surfaces could explain all of the rheological observations and offers another direction of inquiry in the search for therapy for sickle cell disease.

The relationship of altered deformability of irreversibly sickled cells (ISC) to their morphological and physiologic characteristics was evaluated in this study. ISC obtained by differential centrifugation and density gradient sedimentation were separated and enriched into hard and soft populations on the basis of their ability to pass through Nuclepore filters with an average pore size of 3.0 mu. Measurement of deformability by micropipette and Nuclepore elastimetry documented marked differences of erythrocyte rigidity in the two populations. Yet ISC morphology, intracellular viscosity determinants (mean cell hemoglobin concentration, density profile gradient, hemoglobin composition), and surface area geometry (mean cell volume, osmotic fragility) were similar in populations of hard and soft ISC. The similarity in intracellular viscosity and surface area geometry suggests that an intrinsic membrane alteration is responsible for the difference in deformability of filtration separated populations of ISC. Hard ISC may be more important to the pathophysiology of sickle cell disease than soft ISC, though ISC counts on peripheral blood smears would not be of help in distinguishing the two populations.

Concentrated adult erythrocyte suspensions were subjected to low amplitude oscillatory shear in a Weissenberg rheogoniometer equipped with a cone-and-plate assembly. The dynamic viscoelastic properties of the suspension were measured over a broad range of frequency by a numerical solution that accounted for fluid inertia. Variation of shear amplitude and cell volume percent, and comparison of buffered saline, plasma, and dextran as suspending media showed that the cellular elements had undergone small bending and shearing deformations. Studies of normal adult erythrocytes, hypotonically swollen cells, temperature-altered cells, and erythrocyte ghosts suggested that the method was evaluating membrane material properties. The normal membrane was found to exhibit a shear rate dependent elastic modulus that increased by more than a factor of 20 over a frequency range from 0.0076 Hz to 60 Hz. The membrane viscosity showed a substantial drop with frequency indicative of a frequency thinning phenomenon. At high frequency of deformation the viscous response of normal erythrocytes was no longer indicative of a membrane property due to the dominant influence of the internal hemoglobin solution. The studies generally supported the ability of the method to quantify relative membrane material properties and detect changes in membrane structure.

The present study has evaluated the influence of epinephrine on the resistance of platelets to aspiration into micropipettes, and the effect of epinephrine on the altered deformability of aspirin treated platelets. Unlike other platelet agonists previously studied, epinephrine stimulation did not alter platelet deformability at a concentration capable of causing platelet aggregation. Aspirin caused a dramatic decrease in the resistance of platelets to aspiration. Pretreatment of platelets with epinephrine prevented aspirin from altering platelet deformability, and exposure of platelets to epinephrine after treatment with aspirin reversed the increased deformability produced by the drug. Blockade of alpha-2 adrenergic receptors with yohimbine or clonidine prevented epinephrine antagonism of the mechanical effects of aspirin. The studies provide further evidence of the novel antagonism between epinephrine and aspirin on platelet structure and function.

We evaluated the deformability of bovine platelets and contrasted the effects of pharmacologic and thermal perturbations on cytoskeletal structure of human and bovine platelets. Platelets were aspirated into micropipettes (0.7 to 0.8 micron in diameter) by stepwise increments in tension. The resulting lengths of the cell extensions were recorded. The cell extensions aspirated from bovine platelets were shorter than the extensions drawn from human platelets. Disassembly of the circumferential microtubule coil allowed human platelets to pass through the pipette, but the same treatments only slightly increased the deformability of bovine platelets. Alteration of the actin filament cytoskeleton caused increased mechanical fragility of human platelets. In contrast, even the combined use of microtubule and actin filament-disrupting agents only modestly increased the deformability of bovine platelets and did not cause premature fragmentation of the cells. Unusual cytoskeletal structure, absence of an open canalicular system, and disparity in granule size may all contribute to the variance in deformability between the platelets of the 2 species. Reduced cell deformability may impair bovine platelet surface interactions by diminishing the ease of cell spreading and formation of areas of contact between the platelet and other cell surfaces.

Recent studies using micropipette elastimetry have shown that the circumferential microtubule supporting the discoid form of resting platelets has a direct influence on the resistance of the cell to deformation. However, the findings did not resolve whether the mere presence of microtubules, their organization into coils, or location under the cell wall was responsible for resistance to aspiration into micropipettes. In the present study platelets were cooled to 2 degrees to 4 degrees C to remove microtubules completely. The chilled cells were then rewarmed to 37 degrees C, and the influence of microtubule reassembly on resistance to deformation in micropipettes measured at intervals up to 1 hour. Chilled platelets without microtubules were aspirated more than twice as far as control platelets at all negative pressures from 1 to 10 cm H2O (tensions 4 to 41 X 10(-2) dynes/cm). At negative tensions beyond 32.5 X 10(-2) dynes/cm (8 cm H2O), the aspirated lengths of control platelets plateaued until the cells finally fragmented. Aspirated segments of chilled platelets continued to increase in length on exposure to greater negative pressure. Twenty minutes after rewarming at 37 degrees C, chilled cells began to return toward normal resistance to aspiration when only 6% had recovered discoid shape. The range of deformability at this time was narrow, indicating that partial recovery was not caused by development of two cell populations, one with and one without microtubules. The return toward normal resistance continued at 30 minutes when 75% of platelets were discoid, and was identical to that in control platelets after 1 hour at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The characteristics of irreversibly sickled cells (ISC) are reviewed in relationship to their potential significance in the initiation of sickle cell vaso-occlusion. Although ISC are implicated in the shortened red cell survival of sickle cell anemia, there is no direct correlation to the frequency or severity of sickle crisis. ISC are heterogeneous in their physical properties suggesting the possibility that a subpopulation may be disproportionately important in causing vaso-occlusion. Study of their deformability heterogeneity of ISC showed that filtration-separated hard ISC are similar in morphology and intracellular composition to soft ISC, and would not be distinguished on blood smear.

Erythrocyte fractions of varying density were isolated by discontinuous density gradient centrifugation of washed erythrocytes of five subjects (three adults and two cord bloods). Free and total carnitine concentrations were determined in each gradient fraction to compare the carnitine content of less dense with more dense erythrocytes. Erythrocyte, leukocyte, and reticulocyte counts and hemoglobin were measured on all fractions of each gradient. The density gradient studies showed that the highest proportion of reticulocytes were associated with the least dense gradient fractions of all five subjects. Linear regression analyses revealed significant positive correlations (r = 0.94 to 0.99, P less than 0.02 to P less than 0.001) between the number of reticulocytes per fraction and the total or free carnitine concentrations per fraction for all subjects. No correlation was found between free or total carnitine and hemoglobin, number of erythrocytes, or number of leukocytes per fraction. It appears that erythrocyte carnitine is localized in circulating reticulocytes which have mitochondria and carnitine-dependent fatty acid metabolism.

Although most apparent in permanently misshapen irreversibly sickled erythrocytes (ISC), biochemical and structural alterations are present in the majority of sickle cell membranes. The relationship of membrane rigidity to cell shape and its dependence upon the internal hemoglobin cytosol are not clarified. We therefore examined the frequency dependent viscoelasticity of oxygenated, packed sickle red cell and ghost suspensions and hemoglobin solutions prepared from density gradient separated ISC and reversibly sickled cell (RSC) fractions. Low amplitude, oscillatory shear was applied in a Weissenberg cone and plate viscometer and the resultant viscoelastic signals provided a dynamic viscosity (eta') and elastic storage modulus (G') which varied with frequency of deformation. The viscoelastic response of the cell and ghost suspensions reflected the material properties of the membrane over most of the frequency range tested. Sickle erythrocyte, red ghost, and white ghost suspensions demonstrated greater viscocoelasticity than comparable normal suspensions. The viscoelastic magnitude of ISC was several-fold greater than normal, with little variation of viscoelasticity with frequency. RSC samples which were characterized by normal shape, size, and internal hemoglobin concentration were also significantly harder than normal, although similar in frequency dependence. Red ghosts prepared from ISC manifested 80% of the viscoelasticity of intact ISC despite diminution of the internal hemoglobin concentration by 90%. Under conditions of low amplitude shear, the behavior of the RSC membrane is compatible with a cytoskeleton possessing an increased number of molecular associations. The mechanical stability of the ISC membrane is related to a substantial, intrinsic reorganization of the cytoskeleton.

Platelet release has been alternatively viewed as a fragmentation of platelet territories demarcated within the cytoplasm of mature megakaryocytes or as a later event involving segmentation of proplatelet pseudopodia extended from the cell. The mechanical constraints on platelet release were evaluated by measuring the resistance of guinea pig megakaryocytes to aspiration into micropipettes of similar diameter to the width of naturally forming proplatelet projections. Application of increasing negative pressure to the surface of the cells resulted in progressively longer extensions being drawn into the pipette until maximal extension lengths were reached. None of the passively aspirated cytoplasmic extensions fragmented off the cells even at the highest aspiration pressure under physiologic study conditions. The longest extensions were aspirated from megakaryocytes of the most advanced maturation stage, and a proportion of the mature cells yielded very long extensions over 50 mu and up to 150 mu in length. Surprisingly, the ease of aspiration did not correlate to cell size during any stage of maturation. The mechanical behavior of guinea pig megakaryocytes indicates a large availability of surface for extension in mature cells ideal for active proplatelet projection. The lack of mechanical fragility suggests that platelet release is a very late maturational event not yet initiated in the "mature" megakaryocytes available for study from marrow harvests.

Splenectomized patients with hemoglobin (Hb) Köln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Köln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Köln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Köln RBC membranes and MDA-reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Köln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Köln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Köln RBCs. We conclude that Hb Köln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.

Pulmonary alveolar macrophages (PAM) obtained from healthy human cigarette smokers generated greater pressure in filtration through small cylindrical pores than PAM from nonsmoking controls. A well standardized hamster smoking model was employed to study the structural basis for the impaired PAM filtration accompanying cigarette smoke exposure. Hamsters also demonstrated increased PAM filtration pressure through cylindrical pores less than half a cell diameter in size after inhalation of cigarette smoke, but not after exposure to vapor phase smoke that had been strained through a particle filter. Cigarette smoker PAM were equally filterable as control through larger diameter channels, and showed comparable improvements in filterability with cytochalasin B treatment. Altered PAM filtration was associated with the formation of characteristic cytoplasmic smoker inclusions, an increase in cell size, and loss of redundant, ruffled surface membrane. The study of smoker and control PAM separated into different sizes on the basis of variations in cell density suggested that discrepancies in cell size were insufficient to explain the filtration disparity. A loss of availability of surface membrane for deformation appeared to be the most important factor responsible for the impaired filtration.

The present investigation has evaluated the influence of aspirin, its constituents, and other nonsteroidal anti-inflammatory agents on the resistance of human platelets to aspiration into micropipettes. Aspirin increased the length of platelet extensions into the micropipette over the entire negative tension range of 0.04 to 0.40 dynes/cm after exposure to the drug in vitro or after ingestion of the agent. Other cyclooxygenase inhibitors, ibuprofen and indomethacin, did not increase platelet deformability. The influence of aspirin was mimicked to some degree by high concentrations of salicylic acid, but acetylation of platelets with acetic anhydride had little influence on platelet deformability. Incubation of platelets with both salicylic acid and acetic anhydride had no more effect than salicylic acid alone. Benzoic acid, chemically similar to salicylic acid, had a minimal effect. The studies demonstrate that aspirin makes platelets more deformable, while components of the drug or other nonsteroidal antiinflammatory agents and cyclooxygenase inhibitors do not have the same influence on resistance to deformation.

The present study examined the influence of activation on platelet deformability. Aspiration of cells after exposure to thrombin, adenosine 5' -diphosphate, or the calcium ionophore A23187 at concentrations producing shape change and stickiness revealed significant changes from control cells. At the lowest negative pressure, 4 X 10(-2) dynes/cm (-1 cm H2O), there were no differences in lengths of membrane segments aspirated from control and activated platelets. Each subsequent increase in negative pressure up to 35 X 10(-2) dynes/cm (-7.5 cm H2O) resulted in significantly longer aspirated segments on activated cells compared to control cells. Greater negative pressures did not cause further increases in lengths of membrane segments drawn into the pipette. Thus, activation, which results in constriction of the circumferential microtubule, makes more membrane available for aspiration as negative pressure is increased. In both control and activated platelets, the microtubule coils served as a barrier to further lengthening of aspirated membrane segments as negative pressure was increased beyond 35 X 10(-2) dynes (-7.5 cm H2O).

Hemoglobin (Hb) North Shore (beta 134 val leads to glu) is a mutant hemoglobin that is associated with the phenotype of mild heterozygous beta-thalassemia. Heterozygotes are characterized low normal hemoglobin levels or mild anemia, microcytosis, increased HbA2, and 34%-38% Hb North Shore. The mechanism of the anemia and microcytosis associated with Hb North Shore was explored by studies of hemolysate thermal instability, peripheral blood globin biosynthesis, and whole blood oxygen affinity. Hb North Shore was mildly heat unstable in comparison to normal adult hemolysate. Pulse labeling of reticulocytes with 3H-leucine showed an alpha/beta ratio of 1.35 (normal 1.0). The beta North Shore/alpha ratio was 0.22-0.27, which was less than expected on the basis of gene dosage and less than that seen for most beta-chain variants. The beta A/alpha ratio was 0.50, as would be expected. The beta North Shore/alpha ratio was 0.26 after a 15-min pulse and did not decrease during 120 min of chase. These findings suggest that suboptimal synthesis rather than posttranslational degradation is responsible for the thalassemic phenotype associated with this variant hemoglobin. These observations parallel the findings in heterozygous HbE. It is not presently known whether the thalassemia phenotype is conferred by the structural mutation itself or by another mutation cis to the beta North Shore gene.

Rabbit PAM deformability was evaluated by positive-pressure filtration through Nucleopore membranes of well-specified pore diameter. The PAM filtration method was standardized and was influenced by apparatus variations (pore size, flow rate, cell concentration), environment (temperature, pH, divalent cations, protein concentration), and differences in PAM cell volume. The influence of phagocyte function on filtration deformability was evaluated by exposing PAMs to pharmacologic and physiologic agents with somewhat exclusive influences on phagocyte physiology. Agents that interact with microfilament contractile protein (N-ethylmaleimide, cytochalasin B) altered deformability profoundly, but no effect was observed with agents interacting with microtubules (vinblastine, colchicine). Agents that cause general PAM activation (phorbol myristate acetate) or stimulate chemotaxis (F-Met-Leu-Phe) increased deformability. On the contrary, PAM deformability was not changed by phagocytosis of Staphylococcus aureus or latex beads. Pharmacologic agents that alter PAM adhesion (aspirin, indomethacin, physiologic dose hydrocortisone) or inhibit glycolysis (2-deoxyglucose) had no influence on filtration deformability. Filtration PAM deformability reflects passive whole cell rigidity, which appears to be determined by the state of polymerization of the actin-myosin microfilament complex.

A new method is presented to describe the low shear rate behavior of blood. We observed the response of a thin layer of sedimenting blood to a graded shear stress in a wedge-shaped chamber. The method allows quantitation of the degree of phase separation between red cells and plasma, and extracts the yield stress of the cell phase as a function of hematocrit. Our studies showed that the behavior of normal human blood underwent a transition from a solid-like gel to a Casson fluid. This transition began at the Casson predicted yield stress. The viscoelastic properties of blood were examined at shear stresses below the yield stress. The measured Young's elastic moduli were in good agreement with published data. The yield stress of blood showed a linear dependence on hematocrit up to 60%, and increased more rapidly at higher hematocrit.

The yield stress is a sensitive index of blood fluidity at low shear. Seven healthy adults were studied at hematocrits varying between 40 and 80% and fibrinogen concentrations from 0.0 to 0.935 g/dl. Multivariable analysis was used to determine the functional dependence of yield stress on hematocrit and fibrinogen level. The major findings from this analysis include a decreasing effect of fibrinogen at high concentrations (saturation effect), a relative insensitivity of yield stress to fibrinogen at low concentration (threshold effect), and a strong interaction between the effects of hematocrit and fibrinogen concentration on yield stress. Our results give the normal range of yield stress for a given value of fibrinogen and hematocrit and can be used to predict the effect of reductions in hematocrit or fibrinogen on the yield stress of normal blood.