Aggregation and disaggregation kinetics of human blood platelets: Part II. Shear-induced platelet aggregation.

A population balance equation (PBE) mathematical model for analyzing platelet aggregation kinetics was developed in Part I (Huang, P. Y., and J. D. Hellums. 1993. Biophys. J. 65: 334-343) of a set of three papers. In this paper, Part II, platelet aggregation and related reactions are studied in the uniform, known shear stress field of a rotational viscometer, and interpreted by means of the model. Experimental determinations are made of the platelet-aggregate particle size distributions as they evolve in time under the aggregating influence of shear stress. The PBE model is shown to give good agreement with experimental determinations when either a reversible (aggregation and disaggregation) or an irreversible (no disaggregation) form of the model is used. This finding suggests that for the experimental conditions studied disaggregation processes are of only secondary importance. During shear-induced platelet aggregation, only a small fraction of platelet collisions result in the binding together of the involved platelets. The modified collision efficiency is approximately zero for shear rates below 3000 s-1. It increases with shear rates above 3000 s-1 to about 0.01 for a shear rate of 8000 s-1. Addition of platelet chemical agonists yields order of magnitude increases in collision efficiency. The collision efficiency for shear-induced platelet aggregation is about an order of magnitude less at 37 degrees C than at 24 degrees C. The PBE model gives a much more accurate representation of aggregation kinetics than an earlier model based on a monodispersed particle size distribution.     

Aggregation and disaggregation kinetics of human blood platelets: Part I. Development and validation of a population balance method.

Hydrodynamic shear stress of sufficient intensity is known to cause platelet activation and aggregation and to alter the effects of biochemical platelet agonists and antagonists. In this work, a population balance equation (PBE) model is developed for analysis of platelet aggregation and disaggregation kinetics under the influence of a shear field. The model incorporates both aggregation and disaggregation by splitting and/or erosion mechanisms. This paper, the first of a series of three, deals with the formulation, simplification, and validation of the PBE and with the estimation of parameters involved in the PBE. These population parameters include collision efficiency, void fraction (related to the particle collision diameter), and the breakage rate coefficient. The platelet particle size distribution is determined experimentally, both initially and at some later times. The PBE can then be used to match satisfactorily the observed particle histograms, by appropriate choice of parameters of the model as functions of time, platelet size, and magnitude of physical or chemical stimuli. Besides providing information on adhesive forces and on the rates of aggregation and disaggregation, these parameters infer the physical properties of platelets and platelet aggregates. These properties are of potential value in increasing our understanding of the processes involved in thrombotic disease and/or therapy. A numerical procedure for solving the PBE is validated by application to simple cases for which analytical solutions are available. The model is applied to analysis of experiments, and parameter sensitivity studies are used to order the importance of the parameters and to reduce the complexity of the model. The simplified model is shown to give good agreement with experimental observations.     

Aggregation and disaggregation of red blood cells

The aggregation of red blood cells may be analyzed as an interaction of an adhesive surface energy and the elastic stored energy which results from deformation of the cell. The adhesive surface energy is the work required to separate a unit adhered area and is the resultant of adhesive forces due to the bridging molecules that bind the cells together and the electrostatic repulsion due to surface charge. The elastic strain energy in the case of the red blood is associated with the membrane elasticity only since the interior of the cell is liquid. The membrane elasticity is due both to bending stiffness and shear. The area expansion is small and may be neglected. These assumptions allow realistic computation of red cell shapes in rouleaux. The disaggregation of rouleaux requires an external force which must overcome the adhesive energy and also supply additional elastic energy of deformation. Depending on the geometry, the initial effect of elastic energy may tend to aid disaggregation. In a shear flow, the stresses on a suspended rouleau alternately tend to compress and to disaggregate the cells if they are free to rotate. This introduces a time dependence so that viscous effects due to the viscosity of the cell membrane, the cell cytoplasm and the external fluid may play a role in determining whether disaggregation proceeds to completion or not.     

Biosynthesis of major platelet proteins in human blood platelets

We studied de novo protein biosynthesis in platelets of normal adult donors and in newly formed platelets isolated from splenectomized patients with idiopathic thrombocytopenic purpura (ITP). After metabolic labelling of platelet proteins, performed with different radiolabelled amino acids or carbohydrates, a tenfold increase in incorporation of radioactivity into trichloroacetic-acid-precipitable material was obtained with ITP platelets compared to control platelets. Electron microscopic studies of ITP platelets revealed the presence of rough endoplasmic reticulum and polyribosomes, providing morphological evidence for protein synthesis. SDS-PAGE of radiolabelled ITP platelet proteins followed by autoradiography showed that [35S]methionine and [3H]leucine were incorporated into almost all Coomassie-blue-stained proteins whereas [3H]carbohydrates only labelled a few bands. Using crossed-immunoelectrophoresis we identified some of the labelled platelet compounds and demonstrated that major membrane glycoproteins (GPIb, IIb, IIIa) and alpha-granule proteins, such as fibrinogen, thrombospondin, albumin and von Willebrand factor, were synthesized in newly formed circulating platelets.     

Aggregation of human blood platelets in the presence of the pyridoindole stobadine

The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadine for 30 s inhibited stimulated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 micromol/l (adrenaline), 200 micromol/l (ADP), and 1,000 micromol/l (PMA). Aggregation induced with thrombin or Ca2+-ionophore A23187 was not changed in the presence of stobadine even in the concentration of 1,000 micromol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 micromol/l) or prolonged onset of its second phase (10 micromol/l). The presented experiments showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity.     

Aggregation kinetics of human mesenchymal stem cells under wave motion

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three‐dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.     

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.     

The effects of some salicylate analogues on human blood platelets. 2. The role of platelet acetylation in the inhibition of platelet aggregation

Aspirin, 2,3-diacetoxybenzoic acid, 2,6-diacetoxybenzoic acid and 2-propoxybenzoic acid were incubated in human platelet-rich plasma at 37°C for 5 and 10 min and the effects upon collagen induced platelet aggregation and the uptake by platelets of radioactive acetate and propionate groups from 14C-labelled analogues were studied to determine if a correlation existed between acylation of the platelet and inhibition of aggregation. Inhibition of aggregation and the uptake of radioactive label were both concentration-dependent and both increased with the time of incubation. Potency re inhibitors of aggregation was, in decreasing order, aspirin, 2,propoxybenzoic acid, 2,3-diacetoxybenzoic acid and 2,6-diacetoxybenzoic acid. Uptake of radioactive label however, was greatest with aspirin, intermediate with 2,3- and 2,6-diacetoxybenzoic acid, and lowest with 2-propoxybenzoic acid. Platelets exposed to a metabolic inhibitor (oligomycin, 10^?5M for 15 min) showed reduced uptake of labelled acetate and propionate and the degree of uptake did not correlate with the degree of inhibitory activity of the analogues on platelet aggregation. Platelet fragments produced by sonification did not take up radioactive label and chloroform: methanol extraction removed about 50% of the label from intact platelets. The results do not support the hypothesis that acetylation of platelets by aspirin is solely responsible for its inhibitory effects on aggregation but do not conflict with the suggestion that acetylation of platelets may be responsible for the persistent $\text{in}$$\text{vivo}$ effects of aspirin.     

The dynamics of shear disaggregation of red blood cells in a flow channel

Red blood cell (RBC) rouleaux were formed in a flow channel in the presence of 2 g/dl dextran (molecular weight 76,000). The partial separation of RBC rouleau doublets adhering to the floor of the flow channel in response to small oscillatory shear stresses was observed experimentally. Theoretical analyses on displacement and drag force were performed to determine whether the motion of the cell involves membrane rotation (i.e., rolling) or sliding. From the experimental data and the results of theoretical analyses, it is concluded that, under the conditions of the experiments, the RBCs in a doublet separate from each other by rolling, rather than sliding of the sheared cell.     

Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.     

The effect of colchicine on human blood platelets under conditions of short-term incubation.

The effects of colchicine on ADP-induced aggregation and on the phosphorylation of tubulin-like protein from human blood platelets were studied. Colchicine at 2mM concentration completely inhibits ADP-induced aggregation after 8min incubation. Under the same inhibitory conditions, phosphorylation of tubulin-like materials in intact platelets was also impaired whereas the endogenous kinase activity of tubulin, isolated through polymerization--depolymerization cycles, was not affected. It was also shown that, under conditions of maximal inhibition of both aggregation and tubulin phosphorylation, colchicine does not penetrate into the cells. The results obtained suggest that the effect of colchicine on platelet aggregation might be mainly, although not exclusively, due to a non-specific effect of the alkaloid on the plasma membrane, rather than to a direct action of the drug on the microtubular protein subunits.     

Aggregation efficiency of activated normal or fixed platelets in a simple shear field: effect of shear and fibrinogen occupancy.

Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.     

Secretome of human endothelial cells under shear stress

Endothelial cells are exposed to different types of shear stress which triggers the secretion of subsets of proteins. In this study, we analyzed the secretome of endothelial cells under static, laminar, and oscillatory flow. To differentiate between endogenously expressed and added proteins, isolated human umbilical vein endothelial cells were labeled with l-Lysine-(13)C(6),(15)N(2) and l-Arginine-(13)C(6),(15)N(4). Shear stress was applied for 24 h using a cone-and-plate viscometer. Proteins from the supernatants were isolated, trypsinized, and finally analyzed using LC-MS/MS (LTQ). Under static control condition 395 proteins could be identified, of which 78 proteins were assigned to the secretome according to Swiss-Prot database. Under laminar shear stress conditions, 327 proteins (83 secreted) and under oscillatory shear stress 507 proteins (79 secreted) were measured. We were able to identify 6 proteins specific for control conditions, 8 proteins specific for laminar shear stress, and 5 proteins specific for oscillatory shear stress. In addition, we identified flow-specific secretion patterns like the increased secretion of cell adhesion proteins and of proteins involved in protein binding. In conclusion, the identification of shear stress specific secreted proteins (101 under different flow conditions) emphasizes the role of endothelial cells in modulating the plasma composition according to the physiological requirements.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.