Shear-stress mediated nitric oxide production within red blood cells: A dose-response

Abstract

BACKGROUND:

Red blood cells (RBC) are exposed to varying shear stress while traversing the circulatory system; this shear initiates RBC-derived nitric oxide (NO) production.

OBJECTIVE:

The current study investigated the effect of varying shear stress dose on RBC-derived NO production.

METHODS:

Separated RBC were prepared with the molecular probe, diamino-fluoreoscein diacetate, for fluorometric detection of NO. Prepared RBC were exposed to discrete magnitudes of shear stress (1–100 Pa), and intracellular and extracellular fluorescence was quantified via fluorescence microscopy at baseline (0 min) and discrete time-points (1–30 min).

RESULTS:

Intracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 1 min–100 Pa; ii) 5 min–1, 5 Pa; iii) 15 min–1, 5, 35 Pa; iv) 30 min–35 Pa. Extracellular RBC-derived NO fluorescence was significantly increased (p < 0.05) following shear stress exposure when compared to baseline at: i) 5 min – 100 Pa; ii) 15 min–100 Pa; iii) 30 min–40, 100 Pa.

CONCLUSIONS:

These data indicate that: i) a dose-response exists for the RBC-derived production of NO via shear stress; and ii) exposure to supra-physiological shear stress allows for the leakage of RBC intracellular contents (e.g., RBC-derived NO).

Keywords

Haemorheology erythrocyte mechanotransduction vasodilation biocompatibility

1. Introduction

The role of nitric oxide (NO) as a mediator of diverse physiological functions has been established over the last 30 years since Moncada and Ignarro identified the elusive “endothelium derived relaxing factor” as NO in 1987 [1 –3]. The principal source of endogenous NO generation is the nitric oxide synthase (NOS) family of enzymes of which there are three primary isoforms: inducible NOS, neuronal NOS, and endothelial NOS (eNOS) [4]. These isoforms are structurally similar despite being the products of three distinct genes; splice variants of these isoforms have been identified [5]. Indeed, Kleinbongard and colleagues [6] identified a splice variant of the eNOS isotype that is active within red blood cells (RBC-NOS).

All active NOS isoforms, including RBC-NOS, enzymatically produce NO by converting L-arginine to L-citrulline in the presence of co-substrates, nicotinamide adenine dinucleotide phosphate (NADPH), oxygen (O₂) and co-factors that include tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide [7, 8]. The activity of RBC-NOS, however, appears to be highly dependent upon either the extracellular presence of ATP [9], and/or shear stress for activation [10, 11]. As such, the extent of RBC-derived NO production may, in part, be regulated by the magnitude and duration of shear stress to which RBC-NOS is exposed, although the precise relationship is yet to be described.

Red blood cells may be exposed to between approximately 0.1 and 15 Pa [12 –14] as they traverse the human circulatory system, up to 38 Pa in stenotic vessels [15], and up to 350 Pa when exposed to mechanical circulatory support (MCS) devices (e.g., artificial heart valves) [16]. Fischer and colleagues [17] previously demonstrated that RBC-NOS activity is increased during cardiopulmonary bypass and suggested RBC-derived NO may contribute to hypotension. Additionally, NO regulates platelet activity and decreases platelet adhesion, and thus subsequently may minimise thrombus formation [18, 19]. As such, RBC-derived NO released into plasma may inhibit platelet activity and contribute to intravascular bleeding observed during treatment for end-stage cardiac, and kidney failure with MCS (i.e., haemodialysis and ventricular assist devices) [20]. Given the ability of NO to regulate physiological and pathophysiological conditions within the circulatory system, identifying a dose-response of shear stress and NO production within the RBC is of clinical significance.

2. Methods

2.1. Subjects and sampling

Venous blood samples were collected from 15 healthy male donors (age: 27±6 yr). Participants were free of known cardiovascular, metabolic, neurologic, and haematological disorders. Exclusion criteria included: i) cigarette smoking within the previous 12 mo; ii) use of medications known to alter blood fluidity and/or cardiovascular health (no participant was using any pharmacological agent). Blood was collected from a prominent antecubital vein within 90 s of tourniquet application to the upper arm, and anticoagulated with ethylenediaminetetraacetic acid (BD Vacutainer^® REF 368761, Becton Dickinson, USA; EDTA; 1.8 mg/ml^–1). Written and witnessed informed consent was provided by each participant and the use of blood was consistent with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The experimental procedures were reviewed and approved by the Griffith University Human Research Ethics Committee (HREC GU Ref. 2016/712).

2.2. Experimental overview

A subset of 6 aliquots from each blood sample were evaluated per subject in the present study which involved three phases: i) preparation of RBC with the molecular probe, 4,5-diaminofluorescein diacetate (DAF-FM DA; D1946, Sigma-Aldrich, AUS); ii) exposure to specific magnitudes of shear stress (i.e., 1, 5, 10, 35, 40, 100 Pa) for 30 min utilising a counter-oscillating shear generator; and iii) quantification of RBC-NO production via fluorescence during the shear exposures.

2.3. Preparation of red blood cells

Following blood collection, whole blood was centrifuged at 1200× g for 10 min. The plasma and buffy white coat were then removed and the remaining packed RBC were suspended in phosphate buffered saline (0.1 M PBS; pH = 7.4, 287 mOsm/kg). To maximise removal of platelets, the RBC suspension was centrifuged at 1200× g for 5 min with the PBS subsequently removed. The remaining packed RBC were re-suspended in PBS once again before being centrifuged for 5 min at 1200× g. Following removal of PBS, 2μL of packed RBC was suspended in a PBS solution containing DAF-FM DA (final conc. = 4μM). The RBC suspension was incubated at room temperature for 30 min in the dark, then centrifuged for 10 min at 600× g, and the PBS removed. The remaining RBC pellet was re-suspended in PBS and centrifuged for 5 min at 600 g to remove any remaining DAF-FM DA. Finally, the RBC pellet was suspended in polyvinylpropyline diluted in PBS (PVP; pH 7.4±0.05, 290±5 mOsm/kg, 36±1 mPa s; ∼0.2% v/v).

2.4. Shear stress system

The custom-made counter-rotating shear generator [21] was used to expose several levels of uniform shear stresses to RBC in this study. The RBC suspension was loaded into a narrow flow field sandwiched by two transparent surfaces: an upper acrylic cone with an angle of 0.4°, and a lower glass plate with a diameter of 30 mm and thickness of 1.3 mm (Glass # 0050, Lot# 131105, Matsunami Glass Co. Ltd, USA). Shear stress was generated in the fluid by the counter-rotation of the upper cone and lower plate. The counter rotational motion was generated by a gear box integrated motor (BXM6200-A, Oriental motor Co. Ltd, USA) using a timing belt and pulley system (Fig. 1). In this experimental setup, the rotational speed of each cone and cup was accelerated 1.2 times that of the motor shaft. The shear generator was mounted on the microscopic stage of inverted microscope (IX-73, Olympus Corp., Japan) to allow for immediate visualisation of RBC following shear exposure. The shear rate was calculated from the rotational velocity and geometrical features of the counter-oscillating shear generator (Equation 1).

Fig.1

The counter-oscillating shear generating device used for application of shear stress.

Equation 1 . Calculation of shear rate within a custom-made shear generator $\dot{γ} (s^{- 1}) = 1.2 π \times \frac{N}{15} / (\tan 0.4)$

where N is motor rotational speed [ $\frac{rev}{\min}$ ]. From quantification of the shear rate, the shear stress (τ) was calculated (Equation 2).

Equation 2 . Calculation of shear stress applied to RBC from the custom-made shear generator $τ [Pa] = μ \times \dot{γ}$

where μ= viscosity of the suspending medium in pascal second (Pa s), and $\dot{γ}$ is the shear rate in inverse seconds (s^–¹).

The two parameters required for the measurement of shear stress – suspending medium viscosity, and shear rate s^–1 – are presented in Table 1. The mean suspending medium viscosity was measured at 150 and 300 s^–1 and then averaged, using a rotational cone-plate viscometer (0.5 DVII+ with CPE40 spindle, Brookfield Engineering Labs, USA) operated at room temperature (23.3±1.4°C). The mean shear rate values required to obtain the discrete shear stress values of 1, 5, 10, 35, 40, and 100 Pa are also presented in Table 1.

Table 1

Average viscosity and shear data utilised for the experimental protocol

	Mean	Std. error	95% Confidence interval
	Mean	Std. error	Lower	Upper
PVP viscosity (mPa · s)	36	1	34	38
1 Pa
Shear stress (Pa)	1	0.04	1	1
Shear rate (s^–1)	36	0.0	36	36
5 Pa
Shear stress (Pa)	5	0.1	5	5
Shear rate (s^–1)	144	0.0	144	144
10 Pa
Shear stress (Pa)	10	0.09	10	10
Shear rate (s^–1)	288	9	268	308
35 Pa
Shear stress (Pa)	35	0.07	35	35
Shear rate (s^–1)	998	26	943	1053
40 Pa
Shear stress (Pa)	40	0.1	40	40
Shear rate (s^–1)	1144	28	1085	1204
100 Pa
Shear stress (Pa)	100	0.08	100	100
Shear rate (s^–1)	2850	75	2690	3010

PVP – polyvinylpyrrolidone, mPa · s – millipascal second, Pa – pascal, s^–1 – inverse second.

2.5. Shear stress application to red blood cell suspensions

An aliquot (100μL) of the previously incubated RBC-PVP suspension was loaded into the counter-oscillating shear generator for the application of shear stress. A separate and fresh aliquot of the RBC suspension was utilised for each discrete magnitude of shear stress (i.e., 1, 5, 10, 35, 40, 100 Pa) with the order of magnitude randomised. Prior to starting the counter-oscillating shear generator, the RBC suspension was visualised with a fluorescence objective (LUCPLFLN 40X/0.60) attached to the previously described inverted microscope coupled to a CMOS camera (optiMOS™ sCMOS, QImaging, AUS); digital images were thus obtained for each sample. Afterward, the rotational velocity was adjusted to obtain the desired discrete magnitude of shear stress to be applied to the RBC suspension (e.g., 1, 5, … , 100 Pa). The counter-oscillating shear generator was paused following 1, 5, and 15 min, and stopped after 30 min, for examination of the RBC suspension and acquisition of digital images.

2.6. Detection of red blood cell nitric oxide production

The molecular probe, DAF-FM DA, was utilised for the semi-quantification of RBC-derived NO production. Incubation of RBC with DAF-FM DA allowed for the molecular probe to permeate into the RBC, with excess probe in the suspending medium removed via centrifugal wash steps. The 30-min incubation in PVP thereafter, allowed for the cleavage of DAF-FM DA by intracellular esterases to form the cell impermeable DAF-FM. The succeeding reaction of DAF-FM DA with NO and/or reactive nitrosating species provided formation of a fluorescent triazole, DAF-FM-T (λ_max excitation 500 nm;λ_max emission 515 nm). Subsequently, the RBC suspensions were excited (λ 494 nm) at discrete time-points (i.e., baseline, 1, 5, 15, and 30 min) via a mercury vapor short arc lamp (X-Cite 120Q, Excelitas Inc., USA) attached to the aforementioned microscope, and the emitted fluorescence (λ 518 nm) captured via the attached digital CMOS camera. As such, the extent of fluorescence intensity was considered indicative of the quantity of NO formed.

2.7. Data analysis

Initially, analysis of the digitally captured images were performed with the open-source, Java-based image processing program, F.I.J.I (National Institutes of Health, Bethesda, Maryland, USA) [22, 23]. Greyscale images were uploaded into the program and regions of interest were identified for each RBC, and also for cell-free areas of each image. The fluorescence intensity of each ROI was then measured, and the cellular fluorescence was quantified and corrected for the mean background fluorescence intensity. For each image taken from a participant at each time-point and magnitude of shear stress, the individual RBC’s fluorescence intensity and background intensity was evaluated by the ROUT method [24] (Q = 1%), with any detected outliers removed. Thereafter, the mean fluorescence intensity of RBC and background ROIs were calculated for each participant at each time-point and magnitude of shear stress. The influence of inter-subject variability was subsequently negated by normalising each participant’s data via calculation of the fold-change in mean fluorescence intensity of RBC and background ROIs (Equation 3).

Equation 3 . Normalisation of mean fluorescence intensity $Fold change = MF I^{n} / MF I^{0}$

where n = mean fluorescence intensity (MFI) for a given time-point, and 0 = MFI at baseline.

2.8. Statistical analysis

The data are reported as mean±standard error, unless otherwise stated. For all measurements, repeated measures two-way ANOVA were used to determine whether significant differences in the means existed, and Bonferroni post-hoc tests subsequently applied when examining multiple comparisons (Prism, GraphPad Software Inc, Release 7.02, USA). Significance was considered at alpha 0.05.

3. Results

Figure 2 depicts example fluorescence images of RBC following exposure to each magnitude and duration of shear stress, and demonstrates the changes in intracellular, and extracellular, NO-bound DAF-FM.

Fig.2

Fluorescence images of red blood cell-derived nitric oxide-bound diaminofluoroscein following exposure to shear stress exposed to discrete magnitudes (A – 1 Pa, B – 5 Pa, C – 10 Pa, D – 35 Pa, E – 40 Pa, F – 100 Pa) and time-points (1 – 1 min, 2 – 5 min, 3 – 15 min, 4 – 30 min).

3.1. Intracellular nitric oxide-bound diaminofluoroscein

The fold-change in NO-bound DAF-FM fluorescence following exposure to discrete magnitudes (1, 5, 10, 35, 40, and 100 Pa), and time-points (0, 1, 5, 15, and 30 min) of shear stress are presented in Fig. 3. Exposure to 1 Pa significantly increased intracellular fluorescence after 15, and 30 min when compared to baseline (p < 0.05, p < 0.0001). Similarly, intracellular fluorescence was significantly increased with exposure to 5 Pa only after 15, and 30 min when compared to baseline (p < 0.001, p < 0.05). Exposure to 10 Pa did not significantly increase intracellular fluorescence at any time-point. In contrast, exposure to 35 Pa significantly increased intracellular fluorescence after 5, 15, and 30 min when compared to baseline (p < 0.0001, p < 0.01, p < 0.0001). The exposure of RBC to 40 Pa induced a significant increase of intracellular fluorescence following 5 min (p < 0.05), whilst all other time-points were not significantly different when compared to baseline. Likewise, intracellular fluorescence was significantly increased after 1 min of exposure to 100 Pa (p < 0.01), whilst all other time-points were not significantly different when compared to baseline.

Fig.3

Red blood cell intracellular fluorescence as an indicator of nitric oxide-bound diaminofluoroscein fluorescencefollowing exposure to shear stress. Data are clustered according to exposure magnitude (1, 5, 10, 35, 40, and 100 Pa) and presented as fold-change from baseline±standard error. ^*, p < 0.05 significantly different from baseline (t = 0 min). ^**, p < 0.01 significantly different from baseline. ^***, p < 0.001 significantly different from baseline. ^****, p < 0.0001 significantly different from baseline.

3.2. Extracellular nitric oxide-bound diaminofluoroscein

The fold-change in extracellular fluorescence (i.e., the background ROI) was analysed and is depicted in Fig. 4. There was no significant difference in extracellular fluorescence between any shear stress magnitude at baseline (t = 0 min) and following 1 min of exposure to shear stress. After 5 and 15 min of exposure to shear stress, the extracellular fluorescence was significantly increased at 100 Pa when compared to baseline (p < 0.0001). Moreover, exposure to 30 min of shear stress significantly increased the extracellular fluorescence at 40 and 100 Pa when compared to baseline (p < 0.01, p < 0.0001).

Fig.4

Extracellular fluorescence as an indicator of nitric oxide-bound DAF-FM leaked from red blood cells following exposure to shear stress. Data are clustered according to exposure magnitude (1, 5, 10, 35, 40, and 100 Pa) and presented as fold-change from baseline±standard error. ^**, p < 0.01 significantly different from baseline (t = 0 min). ^****, p < 0.0001 significantly different from baseline.

4. Discussion

This study is the first to report the dose-dependent production of RBC-derived NO in response to discrete magnitudes and durations of shear stress exposure. The principle outcome observed in the current study was that the amount of RBC-derived NO produced via shear stress is dependent upon both shear stress magnitude and duration. Moreover, the fate of RBC-derived NO may depend on the magnitude and duration of shear stress exposure, given that: i) intracellular fluorescence was significantly increased at lower magnitudes of shear stress; and ii) extracellular fluorescence was significantly increased at higher magnitudes of shear stress, for given time-points. Overall, the current study demonstrates the capacity of RBC to produce NO when exposed to various magnitudes and durations of shear stress.

The present study found that application of shear stresses at 1 and 5 Pa, typical of that observed within the human circulatory system [13], significantly increased RBC-derived NO. These data are incongruent with previous work by Ulker et al. [25] who demonstrated NO did not significantly increase following exposure to 1 Pa for 30 min; albeit, under hypoxic conditions and utilising an electrochemical probe for the detection of NO. Given that O₂ is a primary substrate for the NOS-derived production of NO [7, 8], it is plausible that a shear stress magnitude of 1 and 5 Pa under oxygenated conditions may increase RBC-NOS activity and subsequently NO production.

The current study observed that intracellular fluorescence was not significantly increased with exposure to 10 Pa at any time-point (Fig. 3). Likewise, no significant change in extracellular fluorescence was observed following exposure to 10 Pa at any time-point (Fig. 4). The upper boundary of physiological shear stress observed within the microcirculation is approximated to be between 10 – 15 Pa [12, 13]. As no previous studies, to our knowledge, have examined NO production within any cell type after exposure to shear stress of this magnitude (i.e., 10 Pa), the mechanistic link for the current finding is unclear. It is possible that co-factors and/or co-substrates (e.g., L-arginine, NADPH) required for RBC-NOS dependent production of NO were depleted during exposure to a shear stress magnitude of 10 Pa. It is also quite plausible that any amount of NO produced at this specific shear may have had some intracellular fate that limited its detection.

The present study also investigated RBC-derived NO production at shear magnitudes below (35 Pa) and above (40 Pa) the subhaemolytic threshold (i.e., the shear stress magnitude/duration at which RBC deformability is impaired), as identified by Simmonds and Meiselman [26]. Intracellular NO fluorescence was significantly increased following exposure to 35 Pa after 5, 15, and 30 min (Fig. 3; p < 0.01) with no significant difference in extracellular fluorescence (Fig. 4). When RBC were exposed to a shear stress magnitude just above the subhaemolytic threshold (i.e., 40 Pa), intracellular fluorescence was significantly increased after 5 min (Fig. 3; p < 0.05); however, intracellular fluorescence was not significantly different after 15 and 30 min at 40 Pa. Additionally, the exposure of RBC to 40 Pa for 30 min significantly increased extracellular fluorescence (Fig. 4). Given the ability of NO to enhance RBC deformability [27, 28], the current study indicates that exposure to supra-physiological, but not subhaemolytic, shear stress may improve RBC deformability via shear stress-mediated production of NO. The increased extracellular fluorescence following exposure to 40 Pa for 30 min indicates that RBC membrane pores have begun to develop that allow for the leakage of intracellular contents (e.g., RBC-derived NO) into the extracellular environment. This is consistent with previous studies that have indicated RBC may develop pores following exposure to supra-physiological shear through which cellular contents may be leaked [29 –31].

When RBC were exposed to a supra-physiological shear well-beyond the subhaemolytic threshold (i.e., 100 Pa), intracellular fluorescence was significantly increased after 1 min (Fig. 3; p < 0.01) with no change at any other time-point, whereas extracellular fluorescence was significantly increased after 5, 15, and 30 min (Fig. 4; p < 0.0001). As such, it is plausible that the pores known to develop in the RBC membrane due to an ‘over-stretch’ by supra-physiological shear stress may have facilitated leakage of intracellular contents, including NO [32]. Current MCS devices expose blood to a supra-physiological shear stress magnitude of ∼100–150 Pa [33]; thus, patients with circulatory assist devices may be prone to RBC releasing a substantial amount of NO into the extracellular environment. As NO mediates platelet activity via inhibition of calcium mobilisation [18 , 35], it is possible that RBC-derived NO is leaked into the circulatory system following exposure to supra-physiological shear stress and may contribute to the increased incidence of intravascular bleeding observed in individuals with continuous-flow MCS devices [36].

5. Conclusion

The primary findings from the present study include: i) the dose-response of RBC-derived NO production to shear stress; and ii) the leakage of intracellular contents (e.g., RBC-derived NO) into the extracellular milieu following exposure to supra-physiological shear stress (40 and 100 Pa). A limitation of the current study was that an analytical technique (e.g., liquid chromatography coupled mass spectrometry) to confirm NO as the nitrosative species in the extracellular environment was not employed. Nevertheless, the clinical implication of these data are that some of the complications associated with MCS (e.g., internal bleeding) may be induced by the supraphysiological shear stress and associated extracellular NO accumulation.

Disclosure

The authors declare no conflict of interest.

Footnotes

Acknowledgments

The authors would like to recognise the financial assistance provided by the Japan Society for Promotion of Science (JSPS; KAKENHI Grant Number JP17K01370).

References

Ignarro

, Buga

, Wood

, Byrns

, Chaudhuri

. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Sciences of the United States of America. 1987;84(24):9265–9.

Ignarro

, Byrns

, Buga

, Wood

. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circulation Research. 1987;61(6):866–79.

Palmer

, Ferrige

, Moncada

. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327(6122):524–6.

Forstermann

, Kleinert

. Nitric oxide synthase: Expression and expressional control of the three isoforms. Naunyn-Schmiedeberg's Archives of Pharmacology. 1995;352(4):351–64.

Alderton

, Cooper

, Knowles

. Nitric oxide synthases: Structure, function and inhibition. The Biochemical Journal. 2001;357(Pt 3):593–615.

Kleinbongard

, Schulz

, Rassaf

, Lauer

, Dejam

, Jax

, et al.Red blood cells express a functional endothelial nitric oxide synthase. Blood. 2006;107(7):2943–51.

Eligini

, Porro

, Lualdi

, Squellerio

, Veglia

, Chiorino

, et al.Nitric oxide synthetic pathway in red blood cells is impaired in coronary artery disease. PloS One. 2013;8(8):e66945.

Forstermann

, Sessa

. Nitric oxide synthases: Regulation and function. European Heart Journal. 2012;33(7):829–37, 37a-37d.

Ulker

, Ozen

, Abdullayeva

, Koksoy

, Yaras

, Basrali

. Extracellular ATP activates eNOS and increases intracellular NO generation in Red Blood Cells. Clinical Hemorheology and Microcirculation. 2018;68(1):89–101.

10.

Bohmer

, Beckmann

, Sandmann

, Tsikas

. Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes. Blood. 2012;119(5):1322–3.

11.

Ulker

, Yaras

, Yalcin

, Celik-Ozenci

, Johnson

, Meiselman

, et al.Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells. Nitric Oxide: Biology and Chemistry / Official Journal of the Nitric Oxide Society. 2011;24(4):184–91.

12.

Koutsiaris

, Tachmitzi

, Batis

. Wall shear stress quantification in the human conjunctival pre-capillary arterioles in vivo. Microvascular Research. 2013;85:34–9.

13.

Papaioannou

, Stefanadis

. Vascular wall shear stress: Basic principles and methods. Hellenic Journal of Cardiology: HJC = Hellenike Kardiologike Epitheorese. 2005;46(1):9–15.

14.

Lipowsky

HH.

Shear stress in the circulation. In: Bevan

, Kaley

, Rubanyi

, editors. Flow-Dependent Regulation of Vascular Function. New York: Oxford University Press; 1995.

15.

Loscalzo

, Schafer

AI.

Thrombosis and hemorrhage. 3rd ed.Philadelphia: Lippincott Williams & Wilkins; 2003. xviii, 1142 p., 16 p. of plates p.

16.

Dasi

, Simon

, Sucosky

, Yoganathan

. Fluid mechanics of artificial heart valves. Clin Exp Pharmacol Physiol. 2009;36(2):225–37.

17.

Fischer

, Schindler

, Brixius

, Mehlhorn

, Bloch

. Extracorporeal circulation activates endothelial nitric oxide synthase in erythrocytes. The Annals of Thoracic Surgery. 2007;84(6):2000–3.

18.

Buechler

, Ivanova

, Wolfram

, Drummer

, Heim

, Gerzer

. Soluble guanylyl cyclase and platelet function. Annals of the New York Academy of Sciences. 1994;714:151–7.

19.

Franke

, Reuter

, Mrowietz

, Bach

, Jung

. Light and scanning-electron microscopic examination of coronary stents in aperfusion-model. Effects of an NO-donor/quantitative analysis. Clinical Hemorheology and Microcirculation. 2001;24(2):101–9.

20.

Jahann

, Shami

. Gastrointestinal bleeding during index hospitalization for mechanical circulatory support devices implantation: Is the squeeze worth the ooze?Digestive Diseases and Sciences. 2017;62(1):7–9.

21.

Watanabe

, Shimada

, Hakozaki

, Hara

. Visualization of erythrocyte deformation induced by supraphysiological shear stress. The International Journal of Artificial Organs. 2018:391398818793387.

22.

Schindelin

, Arganda-Carreras

, Frise

, Kaynig

, Longair

, Pietzsch

, et al.Fiji: An open-source platform for biological-image analysis. Nature Methods. 2012;9(7):676–82.

23.

Schindelin

, Rueden

, Hiner

, Eliceiri

. The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev. 2015;82(7-8):518–29.

24.

Motulsky

, Brown

. Detecting outliers when fitting data with nonlinear regression - a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics. 2006;7:123.

25.

Ulker

, Sati

, Celik-Ozenci

, Meiselman

, Baskurt

. Mechanical stimulation of nitric oxide synthesizing mechanisms in erythrocytes. Biorheology. 2009;46(2):121–32.

26.

Simmonds

, Meiselman

. Prediction of the level and duration of shear stress exposure that induces subhemolytic damage to erythrocytes. Biorheology. 2016;53(5-6):237–49.

27.

Bor-Kucukatay

, Wenby

, Meiselman

, Baskurt

. Effects of nitric oxide on red blood cell deformability. American Journal of Physiology Heart and Circulatory Physiology. 2003;284(5):H1577–84.

28.

Grau

, Pauly

, Ali

, Walpurgis

, Thevis

, Bloch

, et al.RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability. PloS One. 2013;8(2):e56759.

29.

Lubowitz

, Harris

, Mehrjardi

, Sutera

. Shear-induced changes in permeability of human RBC to sodium. Transactions - American Society for Artificial Internal Organs. 1974;20B:470–3.

30.

Sutera

. Flow-induced trauma to blood cells. Circulation Research. 1977;41(1):2–8.

31.

Watanabe

, Sakota

, Ohuchi

, Takatani

. Deformability of red blood cells and its relation to blood trauma in rotary blood pumps. Artificial Organs. 2007;31(5):352–8.

32.

Sutera

, Mehrjardi

. Deformation and fragmentation of human red blood cells in turbulent shear flow. Biophys J. 1975;15(1):1–10.

33.

Selgrade

, Truskey

. Computational fluid dynamics analysis to determine shear stresses and rates in a centrifugal left ventricular assist device. Artificial Organs. 2012;36(4):E89–96.

34.

Fitzgerald

, Phillips

. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. The Journal of Biological Chemistry. 1985;260(20):11366–74.

35.

Geiger

, Nolte

, Walter

. Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors. The American Journal of Physiology. 1994;267(1 Pt 1):C236–44.

36.

Draper

, Huang

, Gerson

. GI bleeding in patients with continuous-flow left ventricular assist devices: A systematic review and meta-analysis. Gastrointest Endosc. 2014;80(3):435–46e1.