Modulation of red blood cell nitric oxide synthase phosphorylation in the quiescent and exercising human forearm.

In vitro investigations demonstrate that human erythrocytes synthesize nitric oxide via a functional isoform of endothelial nitric oxide synthase (NOS) (RBC-NOS). We tested the hypothesis that phosphorylation of RBC-NOS at serine residue 1177 (RBC-NOS1177) would be amplified in blood draining-active skeletal muscle. Furthermore, given hypoxemia modulates local blood flow and thus shear stress, and nitric oxide availability, we performed duplicate experiments under normoxia and hypoxia. Nine healthy volunteers performed rhythmic handgrip exercise at 60% of individualized maximal workload for 3.5 min while breathing room air (normoxia) and after being titrated to an arterial oxygen saturation ≈80% (hypoxemia). We measured brachial artery blood flow by high-resolution duplex ultrasound, while continuously monitoring vascular conductance and mean arterial pressure using finger photoplethysmography. Blood was sampled during the final 30 s of each stage from an indwelling cannula. Blood viscosity was measured to facilitate calculation of accurate shear stresses. Erythrocytes were assessed for levels of phosphorylated RBC-NOS1177 and cellular deformability from blood collected at rest and during exercise. Forearm exercise increased blood flow, vascular conductance, and vascular shear stress, which coincided with a 2.7 ± 0.6-fold increase in RBC-NOS1177 phosphorylation (P < 0.0001) and increased cellular deformability (P < 0.0001) under normoxia. When compared with normoxia, hypoxemia elevated vascular conductance and shear stress (P < 0.05) at rest, while cellular deformability (P < 0.01) and RBC-NOS1177 phosphorylation (P < 0.01) increased. Hypoxemic exercise elicited further increases in vascular conductance, shear stress, and cell deformability (P < 0.0001), although a subject-specific response in RBC-NOS1177 phosphorylation was observed. Our data yield novel insights into the manner that hemodynamic force and oxygen tension modulate RBC-NOS in vivo.

Single-night continuous positive airway pressure treatment improves blood fluid properties in individuals recently diagnosed with obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is a prevalent disorder that causes repetitive, temporary collapses of the upper airways during sleep, resulting in intermittent hypoxaemia and sleep fragmentation. Given those with OSA also exhibit decreased blood fluidity, this clinical population is at heightened risk for cardiovascular disease (CVD) development. Continuous positive airway pressure (CPAP) remains a primary therapy in OSA, which improves sleep quality and limits sleep fragmentation. While CPAP effectively ameliorates nocturnal hypoxic events and associated arousals, it remains unclear whether CVD risk factors are positively impacted. The aim of the present study was thus to assess the effects of an acute CPAP therapy on sleep quality and the physical properties of blood that determine blood fluidity. Sixteen participants with suspected OSA were recruited into the current study. Participants attended the sleep laboratory for two visits: an initial diagnostic visit that included confirmation of OSA severity and comprehensive assessments of blood parameters, followed by a subsequent visit where participants were administered an individualised, acute CPAP therapy session and had their blood assessments repeated. Holistic appraisal of blood rheological properties included assessment of blood and plasma viscosity, red blood cell (RBC) aggregation, deformability, and osmotic gradient ektacytometry. Acute CPAP treatment significantly improved sleep quality parameters, which were associated with decreased nocturnal arousals and improved blood oxygen saturation. Whole blood viscosity was significantly decreased following acute CPAP treatment, which might be explained by the improved RBC aggregation during this visit. Although an acute increase in plasma viscosity was observed, it appears that the alterations in RBC properties that mediate cell-cell aggregation, and thus blood viscosity, overcame the increased plasma viscosity. While deformability of RBC was unaltered, CPAP therapy had mild effects on the osmotic tolerance of RBC. Collectively, novel observations demonstrate that a single CPAP treatment session acutely improved sleep quality, which was accompanied by improved rheological properties.

Piezo1 regulates shear-dependent nitric oxide production in human erythrocytes.

Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine1177 (Ser1177) residue was measured. The contribution of cellular deformation to shear-induced NO production in RBCs was determined by rigidifying RBCs with the thiol-oxidizing agent diamide; rigid RBCs exhibited significantly impaired (up to 80%) capacity to generate NO via RBC-NOS during shear. Standardizing membrane strain of rigid RBCs by applying increased shear did not normalize NO production, or RBC-NOS activation. Calcium imaging with fluo-4 revealed that diamide-treated RBCs exhibited a 42% impairment in Piezo1-mediated calcium movement when compared with untreated RBCs. Pharmacological inhibition of Piezo1 with GsMTx4 during shear inhibited RBC-NOS activation in untreated RBCs, whereas Piezo1 activation with Yoda1 in the absence of shear stimulated RBC-NOS activation. Collectively, a novel, mechanically activated signaling pathway in mature RBCs is described. Opening of Piezo1 and subsequent influx of calcium appear to be required for endogenous production of NO in response to mechanical shear, which is accompanied by phosphorylation of RBC-NOS at Ser1177.NEW & NOTEWORTHY The mechano-sensitive ion channel Piezo1 is expressed in enucleated red blood cells and provides a mechanism of shear-induced red cell nitric oxide production via nitric oxide synthase phosphorylation. Thiol oxidation of red cells decreases Piezo1-dependent calcium movement and thus impairs nitric oxide generation in response to mechanical force. The emerging descriptions of exclusively posttranslational signaling networks in circulating red cells as acute regulators of cell function support that these cells play an important role in cardiovascular physiology that extends beyond passive oxygen transport.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Ex vivo assessment of erythrocyte tolerance to the HeartWare ventricular assist device operated in three discrete configurations.

Despite technological advances in ventricular assist devices (VADs) to treat end-stage heart failure, hemocompatibility remains a constant concern, with supraphysiological shear stresses an unavoidable reality with clinical use. Given that impeller rotational speed is related to the instantaneous shear within the pump housing, it is plausible that the modulation of pump speed may regulate peak mechanical shear stresses and thus ameliorate blood damage. The present study investigated the hemocompatibility of the HeartWare HVAD in three configurations typical of clinical applications: standard systemic support left VAD (LVAD), pediatric support LVAD, and pulmonary support right VAD (RVAD) conditions. Two ex vivo mock circulation blood loops were constructed using explanted HVADs, in which pump speed and external loop resistance were manipulated to reflect the flow rates and differential pressures reported in configurations for standard adult LVAD (at 3150 rev⸱min-1 ), pediatric LVAD (at 2400 rev⸱min-1 ), and adult RVAD (at 1900 rev⸱min-1 ). Using bovine blood, the mock circulation blood loops were tested at 37°C over a period of 6 hours (consistent with ASTM F1841-97) and compared with static control. Hemocompatibility assessments were conducted for each test condition, examining hematology, hemolysis (absolute and normalized index), osmotic fragility, and blood viscosity. Regardless of configuration, continuous exposure of blood to the VAD over the 6-hour period significantly altered hematological and rheological blood parameters, and induced increased hemolysis when compared with a static control sample. Comparison of the three operational VAD configurations identified that the adult LVAD condition-associated with the highest pump speed, flow rate, and differential pressure across the pump-resulted in increased normalized hemolysis index (NIH; 0.07) when compared with the lower pump speed "off-label" counterparts (NIH of 0.04 in pediatric LVAD and 0.01 in adult RVAD configurations). After normalizing blood residence times between configurations, pump speed was identified as the primary determinant of accumulated blood damage; plausibly, blood damage could be limited by restricting pump speed to the minimum required to support matched cardiac output, but not beyond.

Active modulation of human erythrocyte mechanics.

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca2+). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.

Beyond oxygen transport: active role of erythrocytes in the regulation of blood flow.

It was classically thought that the function of mammalian red blood cells (RBCs) was limited to serving as a vehicle for oxygen, given the cells' abundance of cytosolic hemoglobin. Over the past decades, however, accumulating evidence indicates that RBCs have the capacity to sense low-oxygen tensions in hypoxic tissues, and, subsequently, release signaling molecules that influence the distribution of blood flow. The precise mechanisms that facilitate RBC modulation of blood flow are still being elucidated, although recent evidence indicates involvement of 1) adenosine triphosphate, capable of binding to purinergic receptors located on the vascular wall before initiating nitric oxide (NO; a powerful vasodilator) production in endothelial cells, and/or 2) nonvascular NO, which is now known to have several modes of production within RBCs, including an enzymatic process via a unique isoform of NO synthase (i.e., RBC-NOS), which has potential effects on the vascular smooth muscle. The physical properties of RBCs, including their tendency to form three-dimensional structures in low shear flow (i.e., aggregation) and their capacity to elongate in high shear flow (i.e., deformability), are only recently being viewed as mechanotransductive processes, with profound effects on vascular reactivity and tissue perfusion. Recent developments in intracellular signaling in RBCs, and the subsequent effects on the mechanical properties of blood, and blood flow, thus present a vivid expansion on the classic perspective of these abundant cells.

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Accelerated Hemocompatibility Testing of Rotary Blood Pumps.

Ex vivo hemocompatibility testing is a vital element of preclinical assessment for blood-contacting medical devices. Current approaches are resource intensive; thus, we investigated the feasibility of accelerating hemocompatibility testing by standardizing the number of pump exposures in loops of various sizes. Three identical blood loops were constructed, each with a custom-molded reservoir able to facilitate large-volume expansion. Using the HVAD rotary blood pump operating at 5 L·min -1 and 100 mmHg, three test volumes (80, 160, and 320 ml) were circulated for 4000 pump exposures. Blood sampling was performed at individualized intervals every one-sixth of total duration for the assessment of hemolysis and von Willebrand Factor (vWF) degradation. While steady increases in hemolysis (~24 mg·dl -1 ) were identified in all tests at completion, loop volume was not a primary discriminator. The normalized index of hemolysis did not vary significantly between loops (4.2-4.9 mg·100 L -1 ). vWF degradation progressively occurred with duration of testing to a similar extent under all conditions. These data support an accelerated approach to preclinical assessment of ex vivo blood damage. Adopting this approach enables: enhanced efficiency for rapid prototyping; reduced ex vivo blood aging, and; greater utility of blood, which is presently limited if 450 ml loops are desired.

Bovine erythrocytes are poor surrogates for human when exposed to sublethal shear stress.

Animal blood products are routinely used as surrogates for human tissue in haemocompatibility testing of rotary blood pumps. Bovine blood is particularly attractive due to the animal's large blood volume; however, bovine red blood cells (RBC) differ substantially from those of human, both in biophysical properties and molecular composition. We aimed to determine whether differences also exist in the sensitivity of bovine RBC to a standardised shear stress protocol. Fresh blood from healthy human and bovine donors was exposed to discrete combinations of shear stress using a Couette shearing system, prior to assessment of cellular deformability and mechanical sensitivity. Each sample was exposed to 25 sublethal shear stress combinations (ranging 60-100 Pa × 5-300 s). While bovine RBC exhibited decreased maximal elongation in the absence of conditioning shear, overall deformability at lower shears was ~1.8-fold greater than human. When exposed to any conditioning shear stresses >80 Pa (or 60-70 Pa beyond 5 s), human RBC were significantly rigidified, with greater magnitudes and prolonged exposure compounding this effect. Significantly larger shears were required to rigidify bovine RBC; the most extreme shear condition (100 Pa × 300 s) resulted in approximately three-times more rigidification of human RBC than bovine (137% and 47% respectively). Bovine RBC have superior resilience to mechanical stress when compared with human. Using bovine blood in ex vivo evaluation of rotary blood pumps may thus misrepresent and overestimate device-blood success, and may also have flow-on effects for eventual users. Fresh human blood during early-phase ex vivo testing is thus recommended, given shear-inducing blood pumps are designed for humans - not cattle.

Sub-Fractions of Red Blood Cells Respond Differently to Shear Exposure Following Superoxide Treatment.

Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.

Shear Stress and RBC-NOS Serine1177 Phosphorylation in Humans: A Dose Response.

Red blood cells (RBC) express a nitric oxide synthase isoform (RBC-NOS) that appears dependent on shear stress for Serine1177 phosphorylation. Whether this protein is equally activated by varied shears in the physiological range is less described. Here, we explored RBC-NOS Serine1177 phosphorylation in response to shear stress levels reflective of in vivo conditions. Whole blood samples were exposed to specific magnitudes of shear stress (0.5, 1.5, 4.5, 13.5 Pa) for discrete exposure times (1, 10, 30 min). Thereafter, RBC-NOS Serine1177 phosphorylation was measured utilising immunofluorescence labelling. Shear stress exposure at 0.5, 1.5, and 13.5 Pa significantly increased RBC-NOS Serine1177 phosphorylation following 1 min (p < 0.0001); exposure to 4.5 Pa had no effect after 1 min. RBC-NOS Serine1177 phosphorylation was significantly increased following 10 min at each magnitude of shear stress (0.5, 1.5, 13.5 Pa, p < 0.0001; 4.5 Pa, p = 0.0042). Shear stress exposure for 30 min significantly increased RBC-NOS Serine1177 phosphorylation at 0.5 Pa and 13.5 Pa (p < 0.0001). We found that RBC-NOS phosphorylation via shear stress is non-linear and differs for a given magnitude and duration of exposure. This study provides a new understanding of the discrete relation between RBC-NOS and shear stress.

Calcium dynamically alters erythrocyte mechanical response to shear.

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., 'Gárdos effect'). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.

Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

The cellular deformability of red blood cells (RBC) is exceptional among mammalian cells and facilitates nutrient delivery throughout the microcirculation; however, this physical property is negatively impacted by oxidative stress. It remains unresolved whether the molecular determinants of cellular deformability - which in the contemporary model of RBC are increasingly recognized - are sensitive to free radicals. Moreover, given cellular deformability has recently been demonstrated to increase following exposure to specific doses of mechanical stimulation, the potential for using shear "conditioning" as a novel method to reverse free-radical induced impairment of cell mechanics is of interest. We thus designed a series of experiments that explored the effects of intracellular superoxide (O2 -) generation on the deformability of RBC and also activation of pivotal molecular pathways known to regulate cell mechanics - i.e., PI3K/Akt kinase and RBC nitric oxide synthase (NOS). In addition, RBC exposed to O2 - were conditioned with specific shear stresses, prior to evaluation of cellular deformability and activation of PI3K/Akt kinase and RBC-NOS. Intracellular generation of O2 - decreased phosphorylation of RBC-NOS at its primary activation site (Ser1177) (p < 0.001), while phosphorylation of Akt kinase at its active residue (Ser473) was also diminished (p < 0.001). Inactivation of these enzymes following O2 - exposure occurred in tandem with decreased RBC deformability. Shear conditioning significantly improved cellular deformability, even in RBC previously exposed to O2 -. The improvement in cellular deformability may have been the result of enhanced molecular signaling, given RBC-NOS phosphorylation in RBC exposed to O2 - was restored following shear conditioning. Impaired RBC deformability induced by intracellular O2 - may be due, in part, to impaired activation of PI3K/Akt, and downstream signaling with RBC-NOS. These findings may shed light on improved circulatory health with targeted promotion of blood flow (e.g., exercise training), and may prove fruitful in future development of blood-contacting devices.

Susceptibility of density-fractionated erythrocytes to subhaemolytic mechanical shear stress.

INTRODUCTION:: Accumulating evidence demonstrates that subhaemolytic mechanical stresses, typical of circulatory support, induce physical and biochemical changes to red blood cells. It remains unclear, however, whether cell age affects susceptibility to these mechanical forces. This study thus examined the sensitivity of density-fractionated red blood cells to sublethal mechanical stresses. METHODS:: Red blood cells were isolated and washed twice, with the least and most dense fractions being obtained following centrifugation (1500 g × 5 min). Red blood cell deformability was determined across an osmotic gradient and a range of shear stresses (0.3-50 Pa). Cell deformability was also quantified before and after 300 s exposure to shear stresses known to decrease (64 Pa) or increase (10 Pa) red blood cell deformability. The time course of accumulated sublethal damage that occurred during exposure to 64 Pa was also examined. RESULTS:: Dense red blood cells exhibited decreased capacity to deform when compared with less dense cells. Cellular response to mechanical stimuli was similar in trend for all red blood cells, independent of density; however, the magnitude of impairment in cell deformability was exacerbated in dense cells. Moreover, the rate of impairment in cellular deformability, induced by 64 Pa, was more rapid for dense cells. Relative improvement in red blood cell deformability, due to low-shear conditioning (10 Pa), was consistent for both cell populations. CONCLUSION:: Red blood cell populations respond differently to mechanical stimuli: older (more dense) cells are highly susceptible to sublethal mechanical trauma, while cell age (density) does not appear to alter the magnitude of improved cell deformability following low-shear conditioning.

Recovery time course of erythrocyte deformability following exposure to shear is dependent upon conditioning shear stress.

BACKGROUND: Red blood cell (RBC) deformability may increase, or decrease, following application of shear stress ("shear conditioning"), depending upon the specific magnitude and duration of exposure. However, the time course of altered RBC deformability following shear remains unresolved. OBJECTIVE: We utilised shear conditioning known to increase (10 Pa) or decrease (64 Pa) RBC deformability and subsequently rested the cells; serial measurements of deformability during the rest period facilitated defining the time course of recoverability. A second experiment repeated the shear conditioning and recovery period to explore whether multiple duty-cycles augmented the response following the initial exposure. METHODS: Shear conditioning was performed for 300 s at the desired shear stress. Ektacytometry was used to quantify human RBC deformability immediately and during rest (3, 5, 60, 120, 240, 300 s) using discrete samples. RBC were shear conditioned twice in a separate experiment, with 300 s rest separating the conditioning. RESULTS: Shear conditioning at 10 Pa induced increased cell deformability by 19.5 ± 0.3%, which reduced to 7.2 ± 0.4% after 300 s of rest. Shear conditioning at 64 Pa decreased cell deformability by 30.5 ± 13.9%, and after 300 s rest, remained decreased (19.3 ± 9.4%) compared with baseline. The second duty-cycle augmented initial responses induced by shear conditioning. CONCLUSION: Specific shear conditioning results in either temporarily increased cell deformability, or a less reversible decrease of RBC deformability.