Biorheology 51 (2014) 171–185 DOI 10.3233/BIR-140665 IOS Press

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Erythrocyte deformability responses to intermittent and continuous subhemolytic shear stress Michael J. Simmonds a , Nazli Atac b , Oguz K. Baskurt b , Herbert J. Meiselman c and Ozlem Yalcin b,∗ a

Heart Foundation Research Centre, Griffith Health Institute, Griffith University, Queensland, Australia b School of Medicine, Koç University, Sariyer, Istanbul, Turkey c Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Received 14 February 2014 Accepted in revised form 5 June 2014 Abstract. BACKGROUND: Previous studies have demonstrated that red blood cells (RBC) either lyse or at least experience mechanical damage following prolonged exposure to high shear stress (100 Pa). Conversely, prolonged shear stress exposure within the physiological range (5–20 Pa, 300 s) was recently reported to improve RBC deformability. This study investigated the relationships between shear stress and RBC deformability to determine the breakpoint between beneficial vs. detrimental exposure to shear stress (i.e., “subhemolytic threshold”). A second aim of the study was to determine whether the frequency of intermittent application of shear stress influenced the subhemolytic threshold. METHODS: RBC were exposed to various levels of shear stress (0–100 Pa) in a Couette type shearing system for 300 s. RBC deformability was then immediately measured via ektacytometry. Parallel experiments were conducted at the same shear stresses, except the application time differed while keeping constant the total exposure time: shear stress was applied either for 30 s and repeated 10 times (10 × 30 s) or applied for 15 s and repeated 20 times (20 × 15 s). RESULTS: For a range of donors, the subhemolytic threshold with constant shear stress application was between 30–40 Pa. When physiological shear stress was applied in an intermittent manner, more frequent applications tended to improve (i.e., increase) RBC deformability. However, when supra-physiological shear stress was applied, both continuous and intermittent protocols damaged RBC. Changes of RBC mechanical behavior occurred without increases of hemoglobin in the suspending media, thus attesting to the absence of hemolysis. CONCLUSION: Shear stress has a biphasic effect on the mechanical properties of RBC, with the duration and rate of exposure appearing to have minimal impact on the subhemolytic threshold when compared with the magnitude of applied shear stress. Keywords: Mechanical damage, red blood cell, deformability, shear stress, exposure time

1. Introduction Red blood cells (RBC) are among the major determinants of blood fluidity and thus flow resistance of the vasculature [2,10]. RBC characteristically deform under conditions of increased shear stress (SS) in *

Address for correspondence: Ozlem Yalcin, School of Medicine, Koç University, Sariyer, Istanbul, Turkey. Tel.: +90 212 3381136; Fax: +90 212 3381168; E-mail: [email protected]. 0006-355X/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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the circulatory system, although this deformation is reversible and RBC return to their initial shape when SS is removed [10]. This shape changing capability in response to SS is a significant characteristic of these cells and contributes importantly to the non-Newtonian flow properties of blood. Classically, it is known that RBC deformability is governed by the geometric and material properties of these cells [2,10, 24], whereas recent findings have introduced a new concept of active regulation of RBC deformability [21,27]. Active regulation is mainly related to altered associations between RBC membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. The additional effects of S-nitrosylation of membrane proteins by nitric oxide (NO) [11] are believed to be involved in the active regulation of RBC deformability [8]. RBC are exposed to varying mechanical stresses throughout their traverse of the circulation without any mechanical damage. However, RBC undergo extremely high SS (>100 Pa) for a short period of time ( 0 represent decreased (i.e., reduced) RBC deformability (n = 10).

Fig. 5. The time constant of the change in RBC deformation, which represents the half time (s) between resting RBC geometry and asymptotic elongation index, following the application of select SS (5–50 Pa) for 300 s. Values are mean ± SEM, n = 10. ∗ p < 0.001 significantly different from all other conditions. a significantly different from 5 Pa. b significantly different from 10 Pa.

open square) had improved RBC deformability at this SS. Moreover, one of these donors (open circles, Fig. 4) maintained ‘normal’ RBC deformability after 40 Pa preconditioning, while all other donors demonstrated damaged RBC at 40 Pa and greater SS. Elongation indexes change during the 300 s preconditioning period and represent the dynamic adjustment of RBC morphology during the application of SS; these data can be fit with an exponential curve to express the rate change of EI during SS exposure. From resting RBC morphology, RBC deform rapidly under SS application and reach a new “steady state” EI in a SS dependent manner; the time constant for this change is presented in Fig. 5. The rate of change of RBC deformability was negatively and non-linearly related to the shear stress utilized during preconditioning, and hence preconditioning had a significant effect on the time constant (F = 105, p < 0.001). The rate of change of RBC deformability was significantly slower when preconditioning was at lower SS: preconditioning at 5 Pa resulted in RBC taking about 2-fold longer to change shape when compared with 10 Pa (5.0 ± 1.1 s vs. 2.3 ± 0.5 s; p < 0.001). A similar relative difference (∼2-fold longer) was observed when comparing 10 Pa and 20 Pa preconditioning (2.3 ± 0.5 s vs. 0.98 ± 0.2 s; p < 0.001). Once RBC were exposed to 20 Pa

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SS, no further significant decrement in RBC deformability was observed, despite a non-significant trend (p = 0.067) being observed when comparing time constants at 20 Pa and 50 Pa. 3.2. Intermittent vs. continuous shear stress application Elongation index-shear stress data for RBC exposed to continuous and intermittent applications of shear stress for 300 s are presented in Fig. 6. Application of 10 Pa for 300 s increased EI at lower shears (i.e., 0.3–2.91 Pa) by 10–20% compared with control (all p < 0.05). This observation was consistent for

Fig. 6. Elongation indexes measured at SS between 0.3 and 50 Pa for RBC exposed to 300 s of SS applied either continuously (1 × 300 s) or intermittently (20 times for 15 s each, 20 × 15; 10 times for 30 s each, 10 × 30). Experiments were conducted at 10 Pa (A), 30 Pa (B) and 50 Pa (C). Control (Con) data were obtained for RBC that were not preconditioned. Data points are mean ± standard error, except Con is presented without symbols or error bars for clarity, n = 10. ∗∗∗ p < 0.001 all conditions different from Con. ∗∗ p < 0.01 all conditions different from Con. ∗ p < 0.05 all conditions different from Con. † p < 0.05 20 × 15 increased compared with Con.

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all three conditions of SS application (1 × 300 s, 10 × 30 s, 20 × 15 s), and thus the 300 s application of 10 Pa increased the EI irrespective of whether SS was continuously or intermittently applied (Fig. 6(A)). When preconditioning was at 30 Pa (Fig. 6(B)), EI values were significantly increased at 0.3 Pa for all conditions (p < 0.001) and the 20 × 15 s preconditioning resulted in a significant increase in EI at 0.53 Pa (p < 0.05); no other differences were observed for 30 Pa preconditioning. The 50 Pa preconditioning markedly altered RBC deformability (Fig. 6(C)): EI was significantly increased at 0.3 and 0.53 Pa (p < 0.001) irrespective of whether preconditioning was applied continuously or intermittently. No differences in EI were observed at 0.91 and 1.65 Pa, but decreases were observed at 2.91–9.09 Pa inclusive, for both continuous and intermittent applications of SS (p range: 3 Pa, Fig. 6(A)).

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The present findings indicate that intermittent SS within the physiological range tends to increase RBC deformability, but no more so than a single application of SS when the application duration is constant. This finding may indicate that the mechanosensitive pathways responsible for actively regulating RBC deformability following SS exposure are: (a) activated within 15 s; (b) do not reverse completely after 6–8 s of stasis prior to subsequent exposure; (c) are not diminished/reversed during 300 s total exposure to SS. These outcomes are curious, given that the RBC in vivo is not exposed to a constant SS over a prolonged (i.e., 5 min) period. Moreover, it appears that the various biochemical factors responsible for active regulation of RBC deformability are not depleted within 300 s. It is thus suggested that further studies should investigate whether products involved in facilitating RBC deformation are most active during the initial exposure to SS (i.e., seconds) and/or are involved in maintaining steady state deformation during prolonged exposure to mechanical stress. The decreased RBC deformability observed for suspensions exposed to 30–100 Pa for both intermittent and continuous SS in the present study may reflect alterations in intracellular ion concentration and/or physical changes to the plasma membrane. Classically, increased intracellular calcium concentration has been associated with decreased RBC deformability [10], which may explain the rigidification of RBC preconditioned at higher SS in the present study. Indeed, Oonishi et al reported intracellular Ca concentration increased up to 50% when cells were exposed to 130 Pa for 2 min [29] and the resultant mechanical trauma was subsequently prevented with the use of calcium-channel blockers. On the other hand, Baskurt et al. [5] elegantly demonstrated that RBC rigidification following exposure to SS (120 Pa for 15–120 s) was diminished in the presence of a NO-donor. It was also demonstrated that a potassium-channel blocker, in the absence of NO-donors, could achieve similar protection against mechanical trauma. Thus it now appears that intracellular ion concentration, particularly potassium, is critically involved in maintaining RBC integrity. The mechanical trauma to RBC membranes is also likely to explain, in part, the SS-associated reduction in RBC deformability, given that fragmentation of RBC is observed when some “critical” shear is exceeded [22,23,34]. Indeed, membrane elasticity diminishes when broken cytoskeletal attachments bind to Band 3, ultimately reducing plasma membrane surface area [22]. However, since free hemoglobin did not increase for any sheared sample, it appears that these mechanical changes in RBC were not associated with overt hemolysis, supporting the concept of “subhemolytic damage” occurring during 300 s exposure to SS less than 100 Pa. The physiological consequences of the findings of the present study are not straightforward given that the duration and frequency of SS exposure are not directly transferable to the in vivo circulatory system. The phenomena observed in the present study indicate, however, that RBC deformability is significantly improved following exposure to SS levels typical of those experienced in the human arterial circuit. It is therefore attractive to speculate that the pathways involved in the active regulation of RBC deformability may be “primed” within larger vessels (e.g., arteries) for improved microcirculatory transit. This hypothesis is indirectly supported by experimental models using suspensions with mildly decreased RBC deformability that indicated significantly increased flow resistance [6], longer RBC transit time [18], and redistribution of tissue hematocrit that ultimately disrupted tissue oxygen delivery [31]. If the converse is true, that improved RBC deformability following exposure to SS results in improved tissue transit by RBC, models designed to stimulate the active regulation of RBC deformability could be of therapeutic value. Such an improvement would have obvious benefits in clinical situations where RBC are exposed to alternating high- and lower-SS conditions over time (e.g., extracorporeal membrane oxygenation). Thus, it appears that the improvement of RBC deformability following preconditioning with SS may represent a physiological adaptation for improved tissue transit and oxygen/nutrient delivery.

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Erythrocyte deformability responses to intermittent and continuous subhemolytic shear stress.

Previous studies have demonstrated that red blood cells (RBC) either lyse or at least experience mechanical damage following prolonged exposure to hig...
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