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Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.
A cone and plate viscometer was modified to permit the continuous study of platelet response during shear stress exposure times on the order of one second to 180 seconds. Platelets may be stimulated by uniform, controlled shear stress alone or with the addition of chemical platelet agonists. The time course of platelet aggregation is interpreted from alterations in the apparent optical density of the platelet suspension. The rate and extent of platelet dense granule release is estimated from the intensity of the luminescent reaction of platelet released ATP with firefly luciferase and luciferin. Intracellular calcium ion concentration is determined as a function of shear exposure time through the fluorescence intensity of indo-1(5-), a membrane-permeant pentacarboxylate calcium ion chelator.
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.
Mesangial cells serve many functions in the glomerulus, including regulation of glomerular ultrafiltration coefficient, matrix production, and eicosanoid generation. The glomerulus is a vascular bed, and the mesangial cell is continually exposed to rhythmic alterations in intraglomerular pressure. Since increased intraglomerular pressure has been implicated as a potential causative agent in the ultimate development of nephrosclerosis, we sought to determine the effect of continuous stretch-relaxation upon parameters of mesangial cell growth and function. Early passage (2-4) cultured rat mesangial cells were plated onto either rigid-bottom or flexible-bottom culture plates coated with type I collagen. After cell attachment, the cells on flexible supports were exposed to continuous stretch-relaxation for 72 to 96 hours at a rate of 100 cycles/minutes at an applied pressure of 7 to 8 KPa (53 to 61 mm Hg). Cellular morphology was altered by continuous stretch-relaxation, with the majority of mesangial cells presenting stellate or straplike morphology. Fluorescein isothiocyanate-labeled phalloidin staining indicated an increase in density of actin filaments running the long axis of the cell. Stretch-relaxation resulted in an approximately 50% increase in cell number. Prostaglandin production, assessed as irPGE2 production, was increased by stretching in mesangial cells from 28 +/- 1 to 49 +/- 4 pg/10(6) cells (N = 12; p less than 0.005). Mechanical stretch/relaxation increased the percentage of protein representing collagenous proteins from 47 +/- 6% to 70 +/- 4%, as assessed by collagenase susceptibility (p less than 0.025). Analysis of pepsin-resistant proteins synthesized indicated that stretch/relaxation resulted in increases in the relative amounts of types I and III collagens produced/cell. Additionally, stretch/relaxation selectively increased the relative amount of type I-homotrimers produced. Thus, when mesangial cells are exposed to cyclic stretch/relaxation, they exhibit significant alterations in morphology, growth, prostaglandin and collagen production.
Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Small unilamellar liposomes were used in this study of shear stress effects on the trans-bilayer flux of calcium ions (Ca2+). Liposome suspensions were prepared from 99% egg phosphatidylcholine by a microporous filter extrusion technique. The inner aqueous phase of the unilamellar liposomes contained indo-1(5-), a fluorescent indicator of free Ca2+. The external aqueous phase was composed of Hepes-buffered saline containing normal physiological levels of common ionic species. Calcium ion levels were set at 100 nM and 1 mM in the inner and outer aqueous phases, respectively. Liposome suspensions were exposed to graded levels of uniform shear stress in an optically modified rotational viscometer. Intraliposome Ca2+ concentration was estimated from continuous measurement of indo-1(5-) fluorescence. Electronically measured particle size distribution was used to determine liposome surface area for estimation of trans-bilayer Ca2+ flux. Trans-bilayer Ca2+ flux increased linearly with applied shear rate from 27 s-1 to 2700 s-1. Diffusional resistance of the lipid bilayer, not the convective resistance of the surrounding fluid, was the limiting step in the transport of Ca2+. Liposome permeability to Ca2+ increased by nearly two orders of magnitude over the physiologically relevant shear rate range studied. Solute transport in injectable liposome preparations may be dramatically influenced by cardiovascular fluid stress. Solute delivery rates determined in liposomes exposed to static conditions may not accurately predict in vivo, cardiovascular solute transport.
Quiescent rat glomerular mesangial cells were exposed to repeated cycles of stretching and relaxation, and the effects on the rate of collagen production, proliferation, and S6 kinase activity were investigated. Stretch/relaxation induced increases in production of both collagen and non-collagenous proteins. Proliferation of mesangial cells was stimulated by stretch/relaxation and epidermal growth factor, but not by angiotensin II; however, administration of angiotensin II augmented stretch/relaxation-induced cell proliferation. Cytosolic S6 kinase activity was stimulated by stretch/relaxation, angiotensin II, epidermal growth factor, or phorbol 12-myristate 13-acetate. The increased S6 kinase activity was detectable within 30 min after initiation of stretch/relaxation and was blocked by either inhibitors of protein kinase C or prior down-regulation of protein kinase C following prolonged incubation with phorbol 12-myristate 13-acetate. Both translocation of protein kinase C from the cytosolic to the membrane fraction and phosphorylation of an endogenous 80-kDa protein were observed within 5 min of initiation of stretch/relaxation. These results demonstrate that in mesangial cells, mechanical factors alone can induce increases in production of collagen and non-collagenous proteins and in cell proliferation. The observation that stretch/relaxation induced stimulation of S6 kinase activity through protein kinase C-dependent mechanisms suggests that activation of protein kinase C may be a key event in initiating adaptive responses of mesangial cells to increased workload.