Hydrodynamic shear regulates the kinetics and receptor specificity of polymorphonuclear leukocyte-colon carcinoma cell adhesive interactions.

The ability of tumor cells to metastasize hematogenously is regulated by their interactions with polymorphonuclear leukocytes (PMNs). However, the mechanisms mediating PMN binding to tumor cells under physiological shear forces remain largely unknown. This study was designed to characterize the molecular interactions between PMNs and tumor cells as a function of the dynamic shear environment, using two human colon adenocarcinoma cell lines (LS174T and HCT-8) as models. PMN and colon carcinoma cell suspensions, labeled with distinct fluorophores, were sheared in a cone-and-plate rheometer in the presence of the PMN activator fMLP. The size distribution and cellular composition of formed aggregates were determined by flow cytometry. PMN binding to LS174T cells was maximal at 100 s(-1) and decreased with increasing shear. At low shear (100 s(-1)) PMN CD11b alone mediates PMN-LS174T heteroaggregation. However, L-selectin, CD11a, and CD11b are all required for PMN binding to sialyl Lewis(x)-bearing LS174T cells at high shear (800 s(-1)). In contrast, sialyl Lewis(x)-low HCT-8 cells fail to aggregate with PMNs at high shear conditions, despite extensive adhesive interactions at low shear. Taken together, our data suggest that PMN L-selectin initiates LS174T cell tethering at high shear by binding to sialylated moieties on the carcinoma cell surface, whereas the subsequent involvement of CD11a and CD11b converts these transient tethers into stable adhesion. This study demonstrates that the shear environment of the vasculature modulates the dynamics and molecular constituents mediating PMN-tumor cell adhesion.     

Selectin-carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

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.     

Selectin–carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

Aggregation and disaggregation kinetics of human blood platelets: Part III. The disaggregation under shear stress of platelet aggregates.

In the preceding two papers (1, 2), a population balance equation (PBE) mathematical model was developed, validated, and applied to the analysis of platelet aggregation kinetics under the influence of hydrodynamic shear stress. The present work involves the application of the model to the analysis of platelet reactions under shear stress in circumstances where disaggregation processes are of dominant importance: the disaggregation of aggregates formed in response to added agonists. Aggregation-disaggregation experiments were performed in the constant shear field of a rotational viscometer, and the evolution of the particle size distribution was determined by use of an electronic particle counter. The PBE model was used to simulate the experimental results. Exploratory calculations made it possible to reduce a rather complete, complex model to a more tractable form which retains the capability of simulating the experimental observations. For the experimental conditions studied, disaggregation by a splitting mechanism was found to be of dominant importance. The surface erosion mechanism can be neglected without significant impact on results. Physical reasoning confirmed by exploratory calculations showed that a discontinuous form of the breakage rate expression which incorporates a minimum friable particle size, gives significantly better results than a continuous expression. A simple step function void fraction parameter was found to be at least as successful as a more complicated, continuous function. The resulting simplified model has the potential of increasing our understanding of kinetics and mechanisms of platelet reactions, and of characterizing the state of platelet activity. Hence, it may be useful in efforts to understand thrombotic and hemostatic processes.     

Poliovirus aggregates and their survival in water.

Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes.     

Poliovirus aggregates and their survival in water.

Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes.     

Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.

Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA相似文献   

Importance of temporal flow gradients and integrin alphaIIbbeta3 mechanotransduction for shear activation of platelets

Disturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm(2)). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin alpha(IIb)beta(3)-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin alpha(IIb)beta(3) adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin alpha(IIb)beta(3) calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y(1) purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction.     

X chromosome effects and their interactions with mitochondrial effects

We report a simple and rapid method for detecting additive genetic variance due to X-linked loci in the absence of marker data for this chromosome. We examined the interaction of this method with an established method for detecting mitochondrial linkage (another source of sex-asymmetric genetic covariance). When applied to data from the Collaborative Study on the Genetics of Alcoholism, this method found evidence of X-chromosomal linkage for one continuous trait (ntth1) and one discrete trait (SPENT). Evidence of mitochondrial contribution was found for one discrete trait (CRAVING) and three continuous traits (ln(CIGPKYR), ecb21, and tth1). Results for ntth1 suggest that methods that do not also allow for male-female heterogeneity in environmental variance may be overly conservative in detection of X-chromosomal effects.     

Fluid shear stress enhances the sphingosine 1-phosphate responses in cell-cell interactions between platelets and endothelial cells

Fluid shear stress modulates the functional responses of platelets and vascular cells, and plays an important role in the pathogenesis of vascular disorders, including atherosclerosis and restenosis. Since shear stress induces activation of platelets, which abundantly store sphingosine 1-phosphate (Sph-1-P), and upregulates the mRNA expression of S1P(1), the most important Sph-1-P receptor expressed on the endothelial cells, we examined the effects of shear stress on the Sph-1-P-related responses involving these cells. Shear stress was found to induce Sph-1-P release from the platelets in a shear intensity- and time-dependent manner. Inhibitors of protein kinase C suppressed this mechanical force-induced Sph-1-P release, suggesting involvement of this kinase. On the other hand, in vascular endothelial cells, shear stress increased S1P(1) protein expression, as revealed by flow-cytometric analysis, and the responsiveness to Sph-1-P, which was assessed by monitoring the intracellular Ca(2+) concentration. These results indicate that shear stress enhances the Sph-1-P responses in cell-cell interactions between platelets and endothelial cells.     

Post-stenotic core flow behavior in pulsatile flow and its effects on wall shear stress

Arteries of several species, including man, tend to adjust their diameters such that the mean wall shear stress is in the range of 10-20 dynes cm-2. Additionally, intimal thickening in the human carotid bifurcation correlates well with the reciprocal of wall shear stress as determined in model studies. The correlation indicates that wherever the local mean wall shear stress exceeds approximately 10 dynes cm-2, the artery tends to be spared from intimal thickening. However, it is not known whether mean shear stress, i.e. the time-averaged value, or the instantaneous shear stress is the appropriate correlative variable. Each of these variables suggests different mechanisms for the reaction of the artery wall to its hemodynamic environment. It is therefore important to devise means by which the effects of mean shear and pulsatile shear can be separated in the study of atherogenesis. The present investigation examines the post-stenotic flow field in Plexiglas models under pulsatile conditions approximating those in the aortas of the cynomolgus monkey, an animal often employed in atherogenesis research. Behavior of the core flow and its effects on wall shear stress are studied for stenoses of 75 and 90% area reductions using laser velocimetry. The results show that the post-stenotic field contains regions in which the mean wall shear stress is low, but the pulsatile excursions are large.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

The effects of shear flow on protein structure and function

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function. These studies are reviewed and the implications to bioprocessing and pathophysiology of certain diseases are discussed.     

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.     

Cell interactions and histogenesis in embryonic neural aggregates

Mixtures of cell suspensions from the optic lobe of 3-day-old and 6-day-old chick embryos form aggregates which show many ‘rosettes’ resulting from the invagination of peripheral ‘placodes’. However, in aggregates formed exclusively by optic lobe cells of either 3-day-old or 6-day-old embryos no rosettes are observed. By means of radioautographic studies of the combined aggregates, it was shown that rosettes are formed almost exclusively by cells from 3-day-old optic lobe, while those of 6-day-old embryos are predominantly found at the periphery of the aggregates. Cells from 3-day old optic lobe also form rosettes when cultured in combination with wing cells. Similar observations were done in aggregates formed by mixtures of 3-day-old optic lobe and 6-day-old liver cells. The cells from the optic lobe of 3-day-old embryos forming the concave surface of invaginating placodes and rosettes in the ‘combined’ aggregates appear polarized and wedge- or ‘bottle’-shaped. They are aligned in a highly ordered way, and abundant zonula adhaerentes, usually parallel to the cell major axis, are found at the cell apex where bundles of longitudinally oriented microtubules are present. The 3-day-old optic lobe cell which form the convex external surface of the ‘pure’ aggregates have a similar shape and organization, but in this situation apical cell bulges with randomly oriented microtubules interdigitate with those from neighbouring cells, and are joined by zonula adhaerentes usually perpendicular to the longitudinal axis of the cell.