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.     

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.     

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.     

Effect of secondary flow on biological experiments in the cone-plate viscometer: methods for estimating collision frequency, wall shear stress and inter-particle interactions in non-linear flow.

We present a theoretical analysis of fluid flow and particle interactions in the cone-plate viscometer under conditions typically applied in biological studies. The analysis demonstrates that at higher shear rates, besides linear primary flow in the rotational direction, prominent non-linear secondary flow causes additional fluid circulation in the radial direction. Two parameters, the cone angle and Reynolds number, characterize flow in the viscometer over all ranges of shear rate. Our results indicate that secondary flow causes positional variations in: (i) the velocity gradient, (ii) the direction and magnitude of the wall shear stress at the plate surface, (iii) inter-particle collision frequency, (iv) magnitude and periodicity of normal and shear forces applied during particle-particle interactions, and (v) inter-particle attachment times. Thus, secondary flow may significantly influence cellular aggregation, platelet activation and endothelial cell mechanotransduction measurements. Besides cone-plate viscometers, this analysis methodology can also be extended to other experimental systems with complex non-linear flows.     

Elongational flow studies on DNA in aqueous solution and stress-induced scission of the double helix.

Elongational flow techniques are used to investigate the birefringent response and flow-induced molecular scission of monodisperse phage-DNA samples in aqueous solution. A 4-roll mill apparatus was used to characterize the solutions at low stain rates, epsilon less than or equal to 300 s-1, and the opposed jets apparatus used to study fracture of the DNA molecules at strain rates up to 15 x 10(3) s-1. The molecular weight values were measured before and after fracture in elongational flow using the high-resolution technique of pulsed field gel electrophoresis (PFGE). The birefringent response incorporates both rigid and flexible components. The birefringence is nonlocalized and rises gradually to a plateau value, similar to rigid-rod behavior. In addition a certain minimum value in the strain rate is necessary, an onset value epsilon 0, before the signal appears, indicating a flexible component. This behavior is consistent with a hinged-rod model and is similar to that observed for the protein collagen molecule at elevated temperature. We propose that this type of behavior is likely for multistrand rope-like macromolecules where localized separation or partial untwisting of the intertwined chains occurs, creating temporary hinges, in accordance with biochemical evidence for sequence-specific sites of flexibility. Results are presented on the entanglement effects at high concentrations. We have calculated rotational diffusion rates as a function of concentration and molecular weight. Using PFGE to measure the molecular weight profiles, our fracture studies at high strain rates demonstrate chain halving and quartering in accordance with the predictions of the thermally activated barrier to scission theory for single-chain polymers.     

Nonlinear flow affects hydrodynamic forces and neutrophil adhesion rates in cone-plate viscometers

We present a theoretical and experimental analysis of the effects of nonlinear flow in a cone-plate viscometer. The analysis predicts that flow in the viscometer is a function of two parameters, the Reynolds number and the cone angle. Nonlinear flow occurs at high shear rates and causes spatial variations in wall shear stress, collision frequency, interparticle forces and attachment times within the viscometer. We examined the effect of these features on cellular adhesion kinetics. Based on recent data (Taylor, A. D., S. Neelamegham, J. D. Hellums, et al. 1996. Biophys. J. 71:3488-3500), we modeled neutrophil homotypic aggregation as a process that is integrin-limited at low shear and selectin-limited at high shear. Our calculations suggest that selectin and integrin on-rates lie in the order of 10(-2)-10(-4)/s. They also indicate that secondary flow causes positional variations in adhesion efficiency in the viscometer, and that the overall efficiency is dependent not only on the shear rate, but also the sample volume and the cone angle. Experiments performed with isolated neutrophils confirmed these predictions. In these experiments, enhancing secondary flow by increasing the sample volume from 100 to 1000 microl at 1500/s for a 2 degrees cone caused up to an approximately 45% drop in adhesion efficiency. Our results suggest that secondary flow may significantly influence cellular aggregation, platelet activation, and endothelial cell mechanotransduction measurements made in the viscometer over the range of conditions applied in typical biological studies.     

Platelet lysis and aggregation in shear fields.

A rotational viscometer was used to study the effects of shear stress on platelets in human platelet-rich plasma (PRP). For 5-min exposure times, shear stresses above 160 dynes/cm2 induced platelet lysis (as determined by release of platelet lactic dehydrogenase). For 30-s exposure times, shear stresses greater than 600 dynes/cm2 were required to induce platelet lysis. The platelet counts of sheared PRP were decreased to as low as one-fifth the original count due largely to shear-induced aggregation. The count is a minimum at intermediate stress levels (200-400 dynes/cm2). Higher stresses induce disaggregation as well as lysis. The diminution in the counts was partially reversed in 2 h incubation after cessation of shearing. Experiments were carried out with three different viscometer configurations so that the shear stress and the solid surface area access could be varied independently. Surface access was not a significant variable in the conditions of the experiments. Thus aggregation and lysis may be induced by stress effects alone as well as by solid surface effects. The results also show that the response of platelets to shear stress is strongly dependent on exposure time. Platelets are much less resistant to shear stress than red cells for relatively long exposure times. However, the converse is true for very short exposure times.     

Potential fluid mechanic pathways of platelet activation

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.     

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.     

Analysis of shear-induced platelet aggregation with population balance mathematics.

Suspensions of blood platelets aggregate and degranulate when subjected to a shearing flow of sufficient intensity. This work examines, by means of a population balance technique, the kinetics of platelet aggregation in a shear field. The particle collision efficiency, epsilon, and the particle void volume fraction, phi, are estimated from particle number density data. The collision efficiency represents the fraction of particle collisions that result in the binding together of the involved particles. We term epsilon and phi population balance properties because they refer to physical characteristics of platelets and aggregates that are pertinent to their aggregation behavior. Experiments focused on the dependence of epsilon on platelet concentration, shearing rate, and time in a controlled shear field. The collision efficiency is lower in dilute platelet suspensions. This finding supports an ADP-mediated mechanism for shear aggregation. The collision efficiency passes through a maximum with respect to shearing rate, suggesting a competition between the opposing effects of increasing platelet activation and increasing collision violence. The collision efficiency is highest during the first ten seconds in the shear field and declines significantly thereafter. Even at its maximum, however, epsilon for shear aggregation is small: only about one in every thousand particle collisions results in binding.     

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.     

Visualizing protein motion in Couette flow by all-atom molecular dynamics

In living cells, biomacromolecules are exposed to a highly crowded environment. The cytoplasm, the nucleus, and other organelles are highly viscous fluids that differ from dilute in vitro conditions. Viscosity, a measure of fluid internal friction, directly affects the forces that act on immersed macromolecules. Although active motion of this viscous fluid – cytoplasmic streaming – occurs in many plant and animal cells, the effect of fluid motion (flow) on biomolecules is rarely discussed. Recently NMR experiments that apply a shearing flow in situ have been used for protein studies. While these NMR experiments have succeeded in spectroscopically tracking protein aggregation in real time, they do not provide a visual picture of protein motion under shear. To fill this gap, here we have used molecular dynamics simulations to study the motion of three proteins of different size and shape in a simple shearing flow. The proteins exhibit a superposition of random diffusion and shear-flow-induced rotational motion. Random rotational diffusion dominates at lower shear stresses, whereas an active “rolling motion” along the axis of the applied flow occurs at higher shear stress. Even larger shear stresses perturb protein secondary structure elements resulting in local and global unfolding. Apart from shear-induced unfolding, our results imply that, in an ideal Couette flow field biomolecules undergo correlated motion, which should enhance the probability of inter-molecular interaction and aggregation. Connecting biomolecular simulation with experiments applying shear flow in situ appears to be a promising strategy to study protein alignment, deformation, and dynamics under shear.     

A novel mathematical model of activation and sensitization of platelets subjected to dynamic stress histories

Blood recirculating devices, such as ventricular assist devices and prosthetic heart valves, are burdened by thromboembolic complications requiring complex and lifelong anticoagulant therapy with its inherent hemorrhagic risks. Pathologic flow patterns occurring in such devices chronically activate platelets, and the optimization of their thrombogenic performance requires the development of flow-induced platelet activation models. However, existing models are based on empirical correlations using the well-established power law paradigm of constant levels of shear stress during certain exposure times as factors for mechanical platelet activation. These models are limited by their range of application and do not account for other relevant phenomena, such as loading rate dependence and platelet sensitization to high stress conditions, which characterize the dynamic flow conditions in devices. These limitations were addressed by developing a new class of phenomenological stress-induced platelet activation models that specifies the rate of platelet activation as a function of the entire stress history and results in a differential equation that can be directly integrated to calculate the cumulative levels of activation. The proposed model reverts to the power law under constant shear stress conditions and is able to describe experimental results in response to a diverse range of highly dynamic stress conditions found in blood recirculating devices. The model was tested in vitro under emulated device flow conditions and correlates well with experimental results. This new model provides a reliable and robust mathematical tool that can be incorporated into computational fluid dynamic studies in order to optimize design, with the goal of improving the thrombogenic performance of blood recirculating devices.     

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.     

Velocity profiles in annular perfusion chamber measured by laser-Doppler velocimetry

p6e fluid flow in the annular perfusion chamber of Baumgartner developed to study platelet vessel wall interaction was examined with laser-Doppler velocimetry. A laminar and stable flow with a Reynolds number of less than or equal to 50 was measured at flow rates up to 3 ml s-1. No turbulence was found. The wall shear rate directly determined from measured velocity profiles agreed well with theory. The experiments underlined the necessity to work with vessels of uniform thickness and a smooth surface.     

"Shear induced platelet activation"--a critical reappraisal

Platelets were found to be stimulated by high shear exposure for 5 minutes. Using a new technique, shear stresses up to 255 N/m2, acting for pathophysiological relevant intervals of milliseconds did not elicit active release of beta-TG, beyond the amount explained by LDH-monitored passive lysis. Neither the plasma level of ionized calcium (citrate vs heparin anticoagulation), nor a potent platelet inhibiting agent like PgI2 (20 nM) did significantly alter platelet responses to short acting high shear stress. Ultrastructural signs of activation could largely be suppressed by adding ADP-scavengers. Direct "shear induced platelet activation" appears rather unlikely and mostly reducible to secondary biochemical activation by mediators, probably adenine nucleotides, from a small percentage of passively shear-destroyed platelets. The extent of this secondary activation is largely a matter of experimental conditions.     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). 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 stable adhesion.     

Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation

BACKGROUND: Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation. MATERIALS AND METHODS: Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry. RESULTS: The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation. CONCLUSIONS: These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.     

剪切应力诱导血小板聚集

剪切应力诱导血小板聚集（shear-induced platelet aggregation, SIPA）是指在高剪切流场诱导下血小板表面的膜糖蛋白（GPⅠb/Ⅸ/Ⅴ和GPⅡb/Ⅲa）与血浆中的von Willebrand因子（vWF）相结合,介导血小板的活化、黏附和聚集,是动脉血栓的重要成因.SIPA还需要Ca2+,ADP/ATP等生化因素的参与,因而SIPA现象是生化因素和力学因素偶合作用的结果.细胞外Ca2+是高剪切应力诱导血小板发生聚集的必需条件,Ca2+的跨膜内流引起细胞骨架结构的改变和GPⅡb/Ⅲa的活化.近来对ADP/ATP位于血小板膜上的P2受体的研究表明,P2受体与细胞内Ca2+协同作用通过多种生化途径调控血小板的活化过程在SIPA的信号传导中起着关键的作用.从力学环境与生化反应的偶合关系入手研究SIPA现象的触发机制,深入研究SIPA现象中的信号转导通路是今后的研究热点之一.     

Epinephrine and shear stress synergistically induce platelet aggregation via a mechanism that partially bypasses vWF-Gp Ib interactions

Elevated shear stress levels in pathologically stenosed vessels induce platelet activation and aggregation, and may play a role in the pathogenesis of arterial disease. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This study was designed to examine the synergistic interaction of shear stress and epinephrine in the activation of platelets. Platelets (in PRP) sheared at 60 dyn/cm² showed little or no aggregation unless pretreated with epinephrine. Pretreatment with 250 nM epinephrine followed by shear at 60 dyn/cm² induced >60% platelet aggregation. The specific α₂-adrenergic receptor antagonist yohimbine inhibited the synergistic aggregation, as did the ADP scavenging system phosphocreatine/creatine phosphokinase, indicating a three-way synergism with ADP. Chemical or monoclonal antibody blockade of von Willebrand factor (vWF) interactions with either platelet glycoprotein (Gp) Ib or Gp IIb/IIIa completely inhibited platelet aggregation induced by activating levels of shear stress alone. However, the combination of epinephrine and shear stress induced platelet aggregation that was blocked by 10E5, a monoclonal antibody that inhibits vWF binding to Gp IIb/IIIa, but not by aurin tricarboxylic acid or the monoclonal antibody 6D1, both of which inhibit vWF binding to Gp Ib. Synergistic platelet aggregation in response to epinephrine and shear stress was observed in washed platelets, platelet-rich plasma and whole blood in vitro, and also ex vivo following exercise to elevate endogenous levels of catecholamines. These results indicate that epinephrine synergizes with shear stress to induce platelet aggregation. This synergistic response requires functional Gp IIb/IIIa complexes, but is at least partially independent of vWF-Gp Ib interactions.