Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. II. Effect of shear rate, donor sex, and ADP concentration.

The effect of shear rate on the adenosine diphosphate-induced aggregation of human platelets in Poiseuille flow was studied using the method described in part I (Bell, D.N., S. Spain, and H.L. Goldsmith. 1989. Biophys. J. 56:817-828). The rate and extent of aggregation in citrated platelet-rich plasma were measured over a range of mean transit time from 0.2 to 8.6 s and mean tube shear rate, G, from 41.9 to 1,920 s-1. At 0.2 microM ADP, changes in the single platelet concentration with time suggest that more than one type of platelet-platelet bond mediates platelet aggregation at physiological shear rates. At low G, a high initial rate of aggregation reflects the formation of a weak bond of high affinity, the strength of which diminishes with time. Here, the fraction of collisions yielding stable doublets, the collision efficiency, reached a maximum of 26%. The collision efficiency decreased with increasing G and was accompanied by a progressive delay in the onset of aggregation. However, the gradual expression of a more shear rate-resistant bond at high shear rates and long mean transit times produced a subsequent increase in collision efficiency and a corresponding increase in the rate of aggregation. Although the collision efficiencies here were less than 1%, the high collision frequencies were able to sustain a high rate of aggregation. At 0.2 microM ADP, aggregate size generally decreased with increasing G. At 1.0 microM ADP, aggregate size was still limited at high shear rates even though the rate of single platelet aggregation was much higher than at 0.2 microM ADP. Platelet aggregation was greater for female than for male donors, an effect related to differences in the hematocrit of donors before preparing platelet-rich plasma.     

Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations

Using a cone-on-plate mechanical spectrometer, we have measured the linear and non-linear rheological properties of cartilage proteoglycan solutions at concentrations similar to those found in situ. Solutions of bovine nasal cartilage proteoglycan subunits (22S) and aggregates (79S) were studied at concentrations ranging from 10 to 50 mg ml-1. We determined: (1) the complex viscoelastic shear modulus G (omega) under small amplitude (0.02 radians) oscillatory excitation at frequencies (omega) ranging from 1.0 to 20.0 Hz, (2) the non-linear shear rate (gamma) dependent apparent viscosity napp (gamma) in continuous shear, and (3) the non-linear shear rate dependent primary normal stress difference sigma 1 (gamma) in continuous shear. Both the apparent viscosity and normal stress difference were measured over four decades of shear rates ranging from 0.25 to 250 s-1. Analysis of the experimental results were performed using a variety of materially objective non-linear viscoelastic constitutive laws. We found that the non-linear, four-coefficient Oldroyd rate-type model was most effective for describing the measured flow characteristics of proteoglycan subunit and aggregate solutions. Values of the relaxation time lambda 1, retardation time lambda 2, zero shear viscosity no, and nonlinear viscosity parameter muo were computed for the aggregate and subunit solutions at all of the solute concentrations used. The four independent material coefficients showed marked dependence on the two different molecular conformations, i.e. aggregate or subunit, of proteoglycans in solution.     

Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique

Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate-osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits. In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock. Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.     

A simple method for bakers' yeast cell disruption using a three-phase fluidized bed equipped with an agitator

A three-phase fluidized bed equipped with a turbine agitator was utilized as a simple device for disrupting bakers' yeast cells (Saccharomyces cerevisiae). The degree of yeast cell disruption was evaluated based on the number of broken cells and its validity was confirmed by the total amount of crude soluble proteins released and by microscopic observation. It was found that the equipment could yield 90% of yeast cell disruption. With the presence of glass beads, the degree of cell disruption became higher as agitating speed is increased. The disruption enhancement would be attributed to the grinding effect resulting from the interaction between yeast cells and glass beads. One-thousand micrometers of glass beads yielded a higher degree of disruption than larger ones. An increase in liquid flow rate hindered the degree of disruption because of shorter contact time although the shear rates in the yeast suspension would become more rigorous.     

The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.     

Concentration-dependent, temperature-dependent non-Newtonian viscosity of lung surfactant dispersions

The bulk shear viscosities of aqueous dispersions of lavaged calf lung surfactant (LS) and its chloroform:methanol extract (CLSE) were measured as a function of concentration, shear rate and temperature. At 10-mg phospholipid per milliliter, dispersions of LS and vortexed CLSE in 0.15 M NaCl (saline) had low viscosities near 1 cp over a range of shear rates from 225 to 1125 s(-1). Lung surfactant viscosity increased with phospholipid concentration and became strongly non-Newtonian with higher values at low shear rates. At 37 degrees C and 40 mg/ml, LS and vortexed CLSE in saline had viscosities of 38 and 34 cp (77 s(-1)) and 12 and 7 cp (770 s(-1)), respectively. Viscosity values for LS and CLSE were dependent on temperature and, at fixed shear, were lower at 23 degrees C than at 37 or 10 degrees C. Hysteresis was also present in viscosity measurements depending on whether shear rate was successively increased or decreased during study. Addition of 5 mM Ca(2+) at 37 degrees C markedly reduced CLSE viscosity at all shear rates and decreased LS viscosity at low shear rates. Dispersion by sonication rather than vortexing increased the viscosity of CLSE at fixed shear, while synthetic phospholipids dispersed by either method had low, relatively Newtonian viscosities. The complex viscous behavior of dispersions of LS and CLSE in saline results from their heterogeneous aggregated microstructure of phospholipids and apoproteins. Viscosity is influenced not only by the aggregate surface area under shear, but also by phospholipid-apoprotein interactions and aggregate structure/deformability. Similar complexities likely affect the viscosities of biologically-derived exogenous surfactant preparations administered to patients in clinical surfactant therapy.     

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.     

Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth

Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time.     

Effect of high shear on proteins

Shear is present in almost all bioprocesses and high shear is associated with processes involving agitation and emulsification. The purpose of this study is to investigate the effect of high shear and high shear rate on proteins. Two concentric cylinder-based shear systems were used. One was a closed concentric-cylinder shear device (CCSD) and the other was a homogenizer with a rotor/stator assembly. Mathematical modeling of these systems allowed calculation of the shear rate and shear. The CCSD generated low shear rates (a few hundred s(-1)), whereas the homogenizer could generate very high shear rates (> 10(5) s(-1)). High shear could be achieved in both systems by increasing the processing time. Recombinant human growth hormone (rhGH) and recombinant human deoxyribonuclease (rhDNase) were used as the model proteins in this study. It was found that neither high shear nor high shear rate had a significant effect on protein aggregation. However, a lower melting temperature and enthalpy were detected for highly sheared rhGH by using scanning microcalorimetry, presumably due to some changes in protein's conformation. Also, SDS-PAGE indicated the presence of low molecular-weight fragments, suggesting that peptide bond breakage occurred due to high shear. rhDNase was relatively more stable than rhGH under high shear. No conformational changes and protein fragments were observed. (c) 1996 John Wiley & Sons, Inc.     

Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow.

The concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), thrombin, and von Willebrand factor (vWF) released extracellularly from the platelet granules or produced metabolically on the platelet membrane during thrombus growth, were estimated using finite element simulation of blood flow over model thrombi of various shapes and dimensions. The wall fluxes of these platelet-activating agents were estimated for each model thrombus at three different wall shear rates (100 s-1, 800 s-1, and 1,500 s-1), employing experimental data on thrombus growth rates and sizes. For that purpose, whole human blood was perfused in a parallel-plate flow chamber coated with type l fibrillar human collagen, and the kinetic data collected and analyzed by an EPl-fluorescence video microscopy system and a digital image processor. It was found that thrombin concentrations were large enough to cause irreversible platelet aggregation. Although heparin significantly accelerated thrombin inhibition by antithrombin lll, the remaining thrombin levels were still significantly above the minimum threshold required for irreversible platelet aggregation. While ADP concentrations were large enough to cause irreversible platelet aggregation at low shear rates and for small aggregate sizes, TxA2 concentrations were only sufficient to induce platelet shape change over the entire range of wall shear rates and thrombi dimensions studied. Our results also indicated that the local concentration of vWF multimers released from the platelet alpha-granules could be sufficient to modulate platelet aggregation at low and intermediate wall shear rates (less than 1,000 s-1). The sizes of standing vortices formed adjacent to a growing aggregate and the embolizing stresses and the torque, acting at the aggregate surface, were also estimated in this simulation. It was found that standing vortices developed on both sides of the thrombus even at low wall shear rates. Their sizes increased with thrombus size and wall shear rate, and were largely dependent upon thrombus geometry. The experimental observation that platelet aggregation occurred predominantly in the spaces between adjacent thrombi, confirmed the numerical prediction that those standing vortices are regions of reduced fluid velocities and high concentrations of platelet-activating substances, capable of trapping and stimulating platelets for aggregation. The average shear stress and normal stress, as well as the torque, acting to detach the thrombus, increased with increasing wall shear rate. Both stresses were found to be nearly independent of thrombus size and only weekly dependent upon thrombus geometry. Although both stresses had similar values at low wall shear rates, the average shear stress became the predominant embolizing stress at high wall shear rates.     

Polyelectrolyte precipitation of proteins: II. Models of the particle size distributions

A population-balance model has been used to characterize continuous polyelectrolyte precipitation of egg white proteins. We have modeled the particle size distributions of aggregates formed under a range of mixing conditions. The models, accounting for aggregate growth (by both shear-driven and Brownian-like collisions), breakage (by hydrodynamic shear or aggregate-aggregate collisions), and birth (by the breakage of large aggregates), fit the data well. The kinetic constants show dependencies on shear rate and residence time that have not been previously theoretically predicted; these dependencies are due in part to aging effects on the aggregate. The model constants show a dominance of growth over breakage, supporting qualitative interpretations of the particle size distributions. A mechanism for growth-rate enhancement, caused by polymer extensions from the particle surfaces, produced improved model performance. A collisional breakage mechanism is supported.     

Release of protein from Bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill

Release of protein from a suspension of bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill has been studied. Protein release on disruption in the mill is a first-order rate process. The rate constant is dependent on at least six parameters. Increased disruption efficiency was obtained at higher agitator speeds, greater loading of bead attritive elements and lower rates of upward recycle of yeast suspension through the mill. An increase in temperature from 5 to 42°C was accompanied by a reduction in disruption efficiency of approximately 20%. With optimal values of the parameters examined the throughput of the mill is 5.32 kg/hr of soluble protein for 90% disruption.     

The influence of precipitation reactor configuration on the centrifugal recovery of isoelectric soya protein precipitate

The strength and rheological characteristics of isoelectric soya protein precipitate, prepared in continuous tubular and batch-stirred tank reactors, were determined in relation to the separation efficiency and sludge discharge characteristics of intermittent disk and scroll discharge centrifuges. The batch tank precipitate showed greater resistance to aggregate shear breakup than the tubular reactor precipitate. This difference was of significance to the separation efficiency of the scroll centrifuge only. The tubular reactor precipitate sludge showed greater resistance to shear deformation and resulted in drier sludge for a given scroll differential speed. some compressive dewatering occurred for both types of precipitate sludge but shear-induced dewatering is proposed as the major mechanism. This study illustrates the need to integrate the design of protein precipitate formation and centrifugal recovery operations.     

Polyelectrolyte precipitation of proteins: I. The effect of reactor conditions

Lysozyme was recovered from egg white by continuous precipitation with polyacrylic acid (molecular weight of 4 x 10(6)). Precipitator residence time and shear rate had significant effects on the size distribution of the precipitate, but no clear effects on the compositions. Precipitate mean size increased with higher shear, indicating growth phenomena predominating over breakage. Also, an enhancement of growth rate at small sizes was noted. The Camp number successfully characterized the interaction of shear rate and residence time on the particle size.     

Shear and time-dependent changes in Mac-1, LFA-1, and ICAM-3 binding regulate neutrophil homotypic adhesion

We examined the relative contributions of LFA-1, Mac-1, and ICAM-3 to homotypic neutrophil adhesion over the time course of formyl peptide stimulation at shear rates ranging from 100 to 800 s-1. Isolated human neutrophils were sheared in a cone-plate viscometer and the kinetics of aggregate formation was measured by flow cytometry. The efficiency of cell adhesion was computed by fitting the aggregate formation rates with a model based on two-body collision theory. Neutrophil homotypic adhesion kinetics varied with shear rate and was most efficient at 800 s-1, where approximately 40% of the collisions resulted in adhesion. A panel of blocking Abs to LFA-1, Mac-1, and ICAM-3 was added to assess the relative contributions of these molecules. We report that 1) LFA-1 binds ICAM-3 as its primary ligand supporting homotypic adhesion, although the possibility of other ligands was also detected. 2) Mac-1 binding to an unidentified ligand supports homotypic adhesion with an efficiency comparable to LFA-1 at low shear rates of approximately 100 s-1. Above 300 s-1, however, Mac-1 and not LFA-1 were the predominant molecules supporting cell adhesion. This is in contrast to neutrophil adhesion to ICAM-1-transfected cells, where LFA-1 binds with a higher avidity than Mac-1 to ICAM-1. 3) Following stimulation, the capacity of LFA-1 to support aggregate formation decreases with time at a rate approximately 3-fold faster than that of Mac-1. The results suggest that the relative contributions of beta2 integrins and ICAM-3 to neutrophil adhesion is regulated by the magnitude of fluid shear and time of stimulus over a range of blood flow conditions typical of the venular microcirculation.     

The Multistep Process of Homotypic Neutrophil Aggregation: A Review of the Molecules and Effects of Hydrodynamics

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and β₂-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400–800 s^-1), β90% of the single cells are recruited into aggregates ranging from doublets to groupings larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from β20% at 100s^-1 to a maximum level of β80% at 400 s^-1. This increase to peak adhesion efficiency was dependent on L-selectin and β₂-integrin, and was resistant to shear stress up to β7 dyn/cm². When L-selectin was blocked with antibody, β₂-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s^-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.     

Isoelectric precipitation of soy protein. II. Kinetics of protein aggregate growth and breakage

Protein aggregate growth and breakage in agitated suspensions are modeled. The model includes growth of particles by a turbulent collision mechanism and breakage by a hydrodynamic shear mechanism. In the model, breakage results in the splitting of the particles into several small fragments. The model parameters are a growth rate constant and a breakage rate constant. Aggregate size distributions were measured with a Coulter counter and the data interpreted using a population balance that governs the steady-state particle size distribution in a continuous stirred tank reactor. Effects of changes in the operating variables pH, concentration, mean residence time, ionic strength, and mixing power input on the model kinetic parameters are investigated.     

NASCA report 2: Longitudinal study of relationship of exposure to space radiation and risk of lens opacity

The NASA Study of Cataract in Astronauts (NASCA) was designed to measure the impact of exposure to space radiation on progression rates of cortical, nuclear, and posterior subcapsular cataract in U.S. astronauts who have flown in space and comparison groups of astronauts who had not flown in space, and subjects with a history of military aviation. We present our analyses of 5 years of data with an average of 3.8 exams per subject. All subjects had digital lens images with the Nidek EAS 1000 Lens Imaging System. Because of high variability and skewness of opacity measures, nonparametric methods were used to test for association between rates of opacification and space radiation exposure. First, median regression was used to collapse longitudinal data into robust estimates of progression rates (opacity severity compare to time for each eye of each subject). To quantify and test for a radiation effect, median regression with the dependent variable being the maximum of the two slopes (OD and OS) per subject was then used, adjusting for the confounding variables of age, nutritional, and sun-exposure histories. Median regression showed evidence of an association between the rate of cortical progression in the worse eye with radiation dose and age. The estimated median progression rate from space radiation being 0.25 ± 0.13% lens area/Sv/year (P = 0.062). We found no relationship between radiation exposure and progression of aggregate area of posterior subcapsular cataract or nuclear progression rates. However, longer follow-up may be needed to further understand any impact of space radiation on progression rates for posterior subcapsular cataracts and nuclear cataracts, and to characterize changes to visual acuity.     

Effects of shear rate on rouleau formation in simple shear flow

A kinetic equation for rouleau formation in a simple shear flow is derived, based on several assumptions. These are (a) colliding rouleaux stick to one another with a certain probability to form a single rouleau; (b) simultaneous collisions between more than two rouleaux are negligible; (c) rouleaux are broken by a viscous force exerted by the suspending fluid on the surfaces of rouleaux; (d) when a rouleau is broken by viscous forces, only two fragments are formed. Based on a simple mathematical model, collision rate, sticking probability and degradation rate are obtained as functions of applied shear rate. From the solution of the kinetic equation, the average size of rouleaux is obtained as a function of time with shear rate as a parameter. It is shown that the average size of rouleaux increases monotonically with increasing time and tends to an equilibrium size. The average size of rouleaux in a dynamical equilibrium decreases monotonically with increasing shear rate and tends to one cell as shear rate approaches infinity. It is also found that the initial rate of rouleau formation increases with increasing shear rate at very low shear rate, but this trend is reversed at higher shear rates. The theoretical results are compared quantitatively with experimental data.     

Substrate controlled development of anaerobic acidifying aggregates at different shear rates in a gas lift reactor.

The influence of liquid shear rates on the development of acidifying mixed-culture aggregates was studied in a gas-lift reactor. The glucose concentration was kept at a constant and relatively high level by operating the reactor in pH-auxostat mode. Size, strength, and wet density of aggregates cultivated at different superficial gas velocities (Ug) were investigated. Image analysis showed that the Sauter mean diameter (Ds) decreased with increasing Ug. A stirred tank was used to characterize the surface detachment rate (Rd) under non-growth conditions. An exponential decrease was observed in Rd with the applied Ug during cultivation, i.e., aggregates became stronger. The increased strength coincided with an increase in aggregate wet density. Size classified aggregates showed an increase in Rd with the square of the aggregate diameter (Dp), however, this contribution was much smaller than the effect of adaptation. Experiments in a similar gas-lift reactor under dynamic conditions without adaptation, showed that Rd increased exponentially with increasing Ug. So, two important contributions to Rd can be distinguished: adaptation, which induces stronger aggregates, and aggregate size, which makes them less susceptible to hydrodynamic shear. A general expression for Rd was derived, which depends on Dp and Ug. Combining this equation with the surface biomass growth rate (Rg) allowed for the estimation of the maximal diameter (Dmax) aggregates can reach at any Ug, and it was found that the estimated and measured Dmax were in good agreement.