title: The effects of shear stress on blood
Creators: name: Horobin, Jarod
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Stress
subject: Haemolysis
subject: Impaired tissue perfusion
subject: Platelet activity
subject: Thrombus formation
subject: Intravascular bleeding

description: The scientist’s advancement in human biological and physiological understanding, and the engineer’s increasing capabilities has allowed for development of mechanical circulatory support (MCS) devices with improved biocompatibility. Indeed, investigations into the mechanical responses of red blood cells (RBC) to supra-physiological shear stress and the underlying biochemical processes have provided an outline of the RBC mechanical sensitivity (MS) and allowed for identification of the functional tolerance of RBC (i.e., the ability of the RBC to deform) to shear stress. Nevertheless, the adaptations of RBC to supra-physiological shear stress are complex, being influenced by shear stress magnitude, duration, frequency and relaxation time, as well as underlying biochemical processes. The principal aim of this thesis was therefore to investigate the effects of shear stress, within the physiological and supra-physiological domains (viz., shear stress reflective of MCS devices), on blood biochemical and mechanical properties. The subjects of the present studies were all 26 ± 7 yr old males free of known cardiovascular, metabolic, neurologic, and haematological disorders. The purpose of Study One was to examine RBC tolerance to the upper limits of supra-physiological shear stress within the subhaemolytic domain. RBC suspensions were exposed to discrete magnitudes of shear stress (5 - 100 Pa) for specific durations (1 - 16 s). RBC deformability was subsequently measured via ektacytometry and to identify whether haemolysis occurred during exposure to shear stress, free haemoglobin in plasma (pfHb) was quantified via spectrophotometry. To evaluate the susceptibility of RBC to subhaemolytic damage, the MS index was calculated from RBC deformability data. Study One found RBC MS was significantly improved following exposure to a pre-conditioning shear stress of 5 Pa for 16 s, 25 Pa for 2 – 16 s, 50 Pa for 1 – 8 s, and 75 Pa for 1 s (p &lt; 0.05). In contrast, RBC MS was significantly impaired following exposure to a pre-conditioning shear stress of 75 Pa for 8 – 16 s, and 100 Pa for 4 – 16 s (p &lt; 0.05). For all shear conditions, there was no significant increase in pfHb.. As such, Study One, utilising a predictive model recently developed for the assessment of RBC MS, found shear stress at the upper limits of the subhaemolytic domain impairs the RBC MS. Use of this model for haemocompatibility testing of MCS devices can provide insight for the design of future devices. Study Two aimed to validate the MS index via investigation of RBC MS exposed to supra-physiological shear above, and below the subhaemolytic threshold across different durations of exposure. The main finding being that whilst a pre-conditioning shear stress above the subhaemolytic threshold can impair RBC deformability, as indicated by the MS index, a pre-conditioning shear stress below the subhaemolytic threshold can improve RBC deformability (p &lt; 0.001). The purpose of Study Three was to investigate changes in RBC deformability following exposure to shear stress below the reported “haemolytic threshold” using shear exposure durations per minute (i.e., duty-cycles) reflective of that employed by MCS devices. Blood samples were exposed to an intermittent shear stress protocol of 1 s at 100 Pa, every 60 s for 60 duty-cycles. During the remaining 59 s per min, the cells were left at stasis until the subsequent duty-cycle commenced. At discrete time points (15/30/45/60 duty-cycles), an ektacytometer measured RBC deformability immediately after shear exposure at 100 Pa. Additionally, the pfHb, a measurement of haemolysis, was quantified via spectrophotometry, following 30, and 60 duty cycles. As such, Study Three found supra-physiological shear stress impaired RBC properties, as indicated by: 1) decreased maximal elongation of RBC at infinite shear stress following 15 duty-cycles (p &lt;0.05); 2) increased real-time RBC deformability during application of the supra-physiological shear stress protocol (100 Pa) following exposure to 1 duty-cycle (F (1.891, 32.15) = 12.21, p = 0.0001); and 3) increased pfHb following 60 duty-cycles (p &lt;0.01). The findings from Study Three, therefore, indicate that exposure of RBC to short-term, repeated supra-physiological shear stress, impairs RBC deformability, with the extent of impairment exacerbated with each duty-cycle, and ultimately precipitates haemolysis. The intention of Study Four was to begin investigating the underlying biochemical processes that contribute to RBC function following exposure to supra-physiological shear stress. As RBC traverse the circulatory system, they are exposed to varying degrees of shear stress that initiates RBC-derived nitric oxide (NO) production that can enhance RBC deformability, and contribute to vasodilation. Thus, Study Four began an investigation into the dose-response of shear-mediated activation of RBC-nitric oxide synthase (NOS) given that RBC-NOS appears to be highly dependent upon shear stress for activation. Whole blood samples were exposed to a specific “conditioning” magnitude of shear stress (0.5, 1.5, 4.5, 13.5 Pa) for discrete exposure times (1, 10, 30 min). Immediately following the conditioning period, the blood sample was fixed for measurement of RBC-NOS activation utilising immunofluorescence labelling. RBC-NOS activation without prior exposure to shear stress was also taken for quantification of baseline RBC-NOS activation. Shear stress at 0.5, 1.5, and 13.5 Pa significantly increased RBC-NOS activation following 1 min (p &lt; 0.05). No significant increase in RBC-NOS activation was observed after 10 min for any magnitude of shear stress; however, RBC-NOS activation at 0.5, 4.5, and 13.5 Pa was significantly increased after 30 min (p &lt; 0.05). Study Four found Shear stress at 0.5, 1.5, and 13.5 Pa significantly increased RBC-NOS activation following 1 min (p &lt; 0.05). No significant increase in RBC-NOS activation was observed after 10 min for any magnitude of shear stress; however, RBC-NOS activation at 0.5, 4.5, and 13.5 Pa was significantly increased after 30 min (p &lt; 0.05). The present study found that the enzymatic response of RBC-NOS to shear stress is non-linear and differs for a given magnitude and duration of shear stress exposure To further investigate these processes, Study Five investigated a dose-response of shear stress and RBC-derived NO production. Human RBC, separated from whole blood, were prepared with the molecular probe, diamino-fluorescein diacetate for fluorometric detection of NO. Subsequently, prepared RBC were exposed to discrete magnitudes of shear stress (1, 5, 10, 35, 40, 100 Pa) for 30 min. The primary findings from Study Five being that intracellular RBC-derived NO fluorescence was significantly increased (p &lt; 0.05) at the following timepoints and magnitudes of shear stress exposure when compared to baseline: (a) 1 min - 100 Pa; (b) 5 min - 1, 5 Pa; (c) 15 min – 1, 5, 35 Pa; (d) 30 min – 35 Pa. Extracellular RBC-derived NO fluorescence was significantly increased (p &lt; 0.05) at the following timepoints and magnitudes of shear stress exposure when compared to baseline: (a) 1 min – nil; (b) 5 min – 100 Pa; (c) 15 min – 100 Pa; (d) 30 min – 40, 100 Pa. These data indicate that: 1) a dose-response exists for the RBC-derived production of NO via shear stress; and 2) exposure to supra-physiological shear stress induces the release/leakage of RBC-derived NO into the extracellular milieu. To complete the current Thesis, Study Six directly investigated the effects of supra-physiological shear stress from a MCS device on RBC function, whilst also measuring routine biomarkers of haemocompatibility. Study Six introduced pulsatile flow to the HeartWare HVAD using a custom-built controller and compared haemocompatibility biomarkers (i.e., platelet aggregation, concentrations for ADAMTS13, von Willebrand factor (vWf), and pfHb, RBC deformability, and RBC-NOS activity between continuous and pulsatile flow in a blood circulation loop over 5 hours. The HeartWare HVAD was operated using a custom-built controller, at continuous speed (3282 rev/min) or in a pulsatile mode (mean speed = 3273 rev/min, amplitude = 430 rev/min, frequency = 1 Hz) to generate a blood flow rate of 5.0 L/min, HVAD differential pressure of 90 mmHg for continuous flow and 92 mmHg for pulsatile flow, and systolic and diastolic pressures of 121/80 mmHg. For both flow regimes, Study Six found; 1) ADP- and Collagen-induced platelet aggregation, and ADAMTS13 concentration significantly decreased after 5 hours (p &lt; 0.01; p &lt; 0.05), 2) Ristocetin-induced platelet aggregation significantly increased after 45 min (p &lt; 0.05), 3) vWf concentration did not significantly differ at any time point, 4) pfHb significantly increased after 5 hours (p &lt; 0.01), 5) RBC deformability improved during the continuous flow regime (p &lt; 0.05) but not during pulsatile flow, and 6) RBC-NOS activity significantly increased during continuous flow (15 min), and pulsatile flow (5 hours; p &lt; 0.05). Subsequently, Study Six demonstrated: 1) speed modulation does not improve haemocompatibility of the HeartWare HVAD based on no observable differences being detected for routine biomarkers, and 2) the time-course for increased RBC-NOS activity observed during continuous flow may have improved RBC deformability. The findings presented in this thesis confirm that the supra-physiological shear stress blood is exposed to whilst traversing MCS devices is detrimental to RBC function and is likely contributing to ‘downstream’ complications such as haemolysis, impaired tissue perfusion, increased platelet activity, thrombus formation, and intravascular bleeding. Important findings of the present thesis, however, suggest that applications of these data may be beneficial in clinical populations that require life-support and/or have tissue perfusion limitations. Indeed, the potential of NO to mediate multiple processes within the vasculature, including RBC deformability, platelet activation, immune defence and vasodilation, are clear indicators of the therapeutic potential of NO. Understandably, research endeavours into the therapeutic benefits of NO for MCS range from an anticoagulant, an inhalant during extracorporeal membrane oxygenation, and a biopolymer coating of vascular grafts. And yet, research into the therapeutic benefits of NO on RBC function is, to date, limited. Nevertheless, the potential of NO as a ‘killer’ molecule should not be understated. Further investigations are thus required into the actions of RBC-derived NO are required, especially considering the current thesis confirms RBC may release vast quantities of NO into the extracellular milieu when exposed to supra-physiological shear stress.