Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton.

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.

Effects of furosemide and pentoxifylline on blood flow properties in horses.

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.

Alcohol consumption in the guinea pig is associated with reduced megakaryocyte deformability and platelet size.

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)

Effects of pentoxifylline on equine neutrophil function and flow properties.

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.

High frequency of elongated platelet forms in guinea pig blood: ultrastructure and resistance to micropipette aspiration.

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)

Micropipette aspiration of guinea pig megakaryocytes: absence of fragmentation and dependence on maturation stage.

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.

Comparison of bovine and human platelet deformability, using micropipette elastimetry.

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.

Epinephrine-induced reversal of aspirin effects on platelet deformability.

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.

Altered platelet deformability in patients with type IIa and type IV hyperlipoproteinemia.

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.

Aspirin treatment reduces platelet resistance to deformation.

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.

Isolation of microtubule coils from platelets after exposure to aggregating agents.

The discoid shape of human blood platelets is supported by a circumferential microtubule (MT) organized in many loops or coils. A recent study reported from the authors' laboratory demonstrated that significant numbers of MT rings could be isolated from resting platelets by simultaneous exposure to detergent and a small amount of fixative. This method has been used in the present investigation to determine the number of MT coils obtained from platelets after activation by ADP, thrombin, and the calcium ionophore, A23187. Concentrations of the agonists that caused shape change and internal transformation in parallel samples did not influence the frequency of MT rings present in activated samples after treatment with fixative and detergent. As many or more MT coils were present 5, 15, 30, 60, 90, and 120 seconds after addition of an agonist as from the control. Statistical analysis revealed no significant difference between the number of isolated coils from controls and activated platelets at any time during early activation. Immunofluorescence microscopic examination of platelets stained with a monoclonal antibody to tubulin at intervals of 5, 15, 30, 60, 90, and 120 seconds after activation on glass surfaces confirmed the suggestion that platelet MTs are resistant to disassembly during the early response to stimulation.

Micropipette aspiration of human platelets after exposure to aggregating agents.

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).

Micropipette aspiration of human platelets: influence of rewarming on deformability of chilled cells.

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)

Micropipette aspiration of human platelets: influence of microtubules and actin filaments on deformability.

The deformability of human platelets has been evaluated by micropipette aspiration. Control discoid platelets were about ten times as resistant to deformation in the micropipette as red blood cells. Under a constant negative pressure of 10 cm H2O, control platelets developed extension lengths of 0.74 +/- 0.1 micron. Prior treatment with vincristine, colchicine, or low temperature, all of which remove platelet microtubules, was associated with marked increases in lengths of aspirated segments. Taxol or heavy water, which stabilize microtubules, prevented the increased deformability caused by agents that dissolve microtubules. Cytochalasin B, an agent that inhibits assembly of actin microfilaments, also caused an increase in lengths of aspirated segments that could not be prevented by taxol. Vincristine and cytochalasin B, together, caused a greater increase in deformability than either agent alone. These results indicate important roles for microtubules and microfilaments in platelet deformability.

Morphometry of platelet internal contraction.

Blood platelets have a characteristic discoid shape supported by a circumferential band of microtubules. Following stimulation by aggregating agents or foreign surfaces, platelets lose their discoid form, extend pseudopods, and undergo a process of internal reorganization. Randomly dispersed cytoplasmic organelles become concentrated in cell centers within rings of microtubules and masses of microfilaments. Questions have been raised about this process and its contractile nature by studies demonstrating that platelet microtubules dissolve within seconds after activation and reassemble several minutes later in new locations. Earlier investigations showed that Taxol, a microtubule-stabilizing agent, did not inhibit platelet shape change, internal transformation, secretion, aggregation, or clot retraction. In the present study the diameters of microtubule coils in discoid platelets treated or not treated with Taxol and in platelets activated by thrombin, ADP, and a foreign surface were measured. The results of the study reveal no significant differences in diameters of microtuble rings in control or Taxol-treated cells. However, after activation by ADP, thrombin, or the grid surface, the diameter of coiled microtubules decreased by 30% or more. The results support the concept that internal transformation is a contractile event.

Micropipette aspiration of human blood platelets: a defect in Bernard-Soulier's syndrome.

Previous reports have suggested that platelets from patients with Bernard-Soulier's syndrome (BSS) are not giant cells. Rather, they are normal-sized in suspension, but spread out on glass slides more readily than control cells, yielding the impression of being giant. The present study has used cell sizing techniques, electron microscopy, and micropipette aspiration to evaluate platelets from three patients with BSS. Cell sizing techniques revealed that BSS platelets were considerably larger than normal. The increased size was confirmed in electron microscopic studies of BSS platelets fixed in suspension. However, the BSS platelets did not contain increased amounts of internalized surface membrane considered to be the source of membrane necessary for excessive spreading. A possible explanation for increased spreading of BSS platelets was found in studies of their resistance to deformation in micropipettes. BSS platelets were much less resistant to deformation than normal cells or other abnormal platelets when aspirated under the same negative pressure. Their unusual deformability may explain the tendency of BSS platelets to spread more readily than normal cells on glass slides.

Variable deformability of irreversibly sickled erythrocytes.

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.

Evaluation of the role of diacyglycerol in calcium-induced erythrocyte shape change and rigidity.

The intrusion of micromolar amounts of calcium into the calcium-poor interior of the human erythrocyte initiates a series of cataclysmic changes in cellular metabolism and morphology. These include (1) cellular shrinkage (elevated mean cell hemoglobin concentration), (2) loss of water and potassium, (3) rapid and near-total hydrolysis of intracellular ATP, (4) conversion of cell shape from biconcave disc to echinocyte and spheroechinocyte, (5) greatly diminished cellular deformability and elasticity, and (6) the accumulation of DAG. It has been suggested that this latter of phenomenon may account for the changes in cell shape and, perhaps, membrane viscoelastic properties. In the present study we have tested the hypothesis that DAG accumulation is important in the evolution of calcium-induced erythrocyte damage. Our results indicate that (1) calcium-loaded lamb erythrocytes become misshapen and inelastic but do not produce detectable DAG; (2) human red cells suspended in potassium-rich buffer do accumulate DAG after exposure to calcium and A23187 but do not become echinocytic or inflexible; (3) human erythrocytes artificially loaded with DAG are morphologically and elastically normal; and (4) human erythrocytes which have "naturally" accumulated DAG after prolonged incubation in the presence of calcium will retain this DAG after energy repletion. Despite marked elevations of DAG, these cells resume normal morphology and deformability. In aggregate, our data fail to support any direct role for DAG accumulation in the occurrence of calcium-induced damage to human erythrocytes.

Concurrent effects of microsieve aspiration and the ionophore A23187 on the morphologic characteristics of the surface of normal erythrocytes.

Previous studies using the calcium ionophore A23187 have shown that pathologic changes in erythrocyte shape and deformability can be induced by modest increases in intracellular calcium with the concomitant depletion of cellular adenosine triphosphate. Efforts to modify the cellular response to such ionophore-induced calcium loading by means of environmental and chemical manipulation of the erythrocyte membrane have suggested that the state of the membrane prior to calcium uptake influences the response of the cell. The present investigation used the microsieve aspiration technic to induce membrane tension mechanically prior to calcium loading with A23187, and thereby to evaluate the response of the membrane to another form of stress. Erthrocytes subjected to conditions of membrane tension in the microsieve were found to resist completely the A23187-calcium-induced conversion to echinocytes or spheroechinocytes. These results support the hypothesis that the state of the membrane before calcium loading has a marked influence on the response of the cell. This may be related to the mechanism underlying the development of irreversibly sickled cells in patients with sickle cell anemia.

Inhibition of prostaglandin (PG) synthesis in sheep vesicular gland microsomes (SVGM) by nitroblue tetrazolium (NBT) and vitamin E (VE).

Previous studies have shown that NBT and VE together are potent inhibitors of platelet aggregation, secretion and PG synthesis. In this study, we evaluated the capacity of NBT to detect PG synthesis by SVGM. Aspirin pretreatment of SVGM decreased the amount of NBT reduced after addition of arachidonic acid, demonstrating that products generated by the cyclo-oxygenase were involved in NBT reduction. The influence of NBT and VE on PG synthesis by SVGM was then evaluated by measuring malondialdehyde (MDA) production. NBT or VE alone had no significant effect, but together these agents were as effective as aspirin in preventing MDA formation. The effect of NBT and VE on 14C-arachidonic acid conversion was followed by thin layer chromatography and radioscanning. Again, NBT or VE alone were ineffective, whereas the combination was as effective as aspirin in preventing conversion of arachidonic acid. We speculate NBT and VE together inhibit pg synthesis by scavenging a free radical species of arachidonic acid generated in the initial step of fatty acid peroxidation.